RNA Interference

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RNA Interference Edited by Ibrokhim Y. Abdurakhmonov

RNA Interference Edited by Ibrokhim Y. Abdurakhmonov

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Contents Preface

Chapter 1 RNA Interference – A Hallmark of Cellular Function and Gene Manipulation by Ibrokhim Y. Abdurakhmonov Chapter 2 RNA Interference Technology — Applications and Limitations by Devi Singh, Sarika Chaudhary, Rajendra Kumar, Preeti Sirohi, Kamiya Mehla, Anil Sirohi, Shashi Kumar, Pooran Chand and Pankaj Kumar Singh Chapter 3 The Role of Immune Modulatory MicroRNAs in Tumors by Barbara Seliger, Anne Meinhardt and Doerte Falke Chapter 4 Long Noncoding RNAs are Frontier Breakthrough of RNA World and RNAi-based Gene Regulation by Utpal Bhadra, Debabani Roy Chowdhury, Tanmoy Mondal and Manika Pal Bhadra Chapter 5 Noncanonical Synthetic RNAi Inducers by O.V. Gvozdeva and E.L. Chernolovskaya Chapter 6 Microinjection-Based RNA Interference Method in the Water Flea, Daphnia pulex and Daphnia magna by Kenji Toyota, Shinichi Miyagawa, Yukiko Ogino and Taisen Iguchi Chapter 7 ER-targeted Intrabodies Mediating Specific In Vivo Knockdown of Transitory Proteins in Comparison to RNAi by Oliver Backhaus and Thomas Böldicke Chapter 8 RNAi Therapeutic Potentials and Prospects in CNS Disease by Kyoung Joo Cho and Gyung Whan Kim Chapter 9 RNAi-based Gene Therapy for Blood Genetic Diseases by Mengyu Hu, Qiankun Ni, Yuxia Yang and Jianyuan Luo

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Chapter 10 Non-viral siRNA and shRNA Delivery Systems in Cancer Therapy by Emine Şalva, Ceyda Ekentok, Suna Özbaş Turan and Jülide Akbuğa Chapter 11 siRNA-Induced RNAi Therapy in Acute Kidney Injury by Cheng Yang and Bin Yang Chapter 12 Preclinical Development of RNAi-Inducing Oligonucleotide Therapeutics for Eye Diseases by Tamara Martínez, Maria Victoria González, Beatriz Vargas, Ana Isabel Jiménez and Covadonga Pañeda Chapter 13 RNAi-Induced Immunity by Wenyi Gu Chapter 14 Perspectives on RNA Interference in Immunopharmacology and Immunotherapy by Zhaohua Hou, Qiuju Han, Cai Zhang and Jian Zhang Chapter 15 RNA Interference as a Tool to Reduce the Risk of Rejection in Cell-Based Therapies by Constanca Figueiredo and Rainer Blasczyk Chapter 16 Utility of Potent Anti-viral MicroRNAs in Emerging Infectious Diseases by Zhabiz Golkar, Donald G. Pace and Omar Bagasra Chapter 17 RNAi — Implications in Entomological Research and Pest Control by Nidhi Thakur, Jaspreet Kaur Mundey and Santosh Kumar Upadhyay Chapter 18 Management of Insect Pest by RNAi — A New Tool for Crop Protection by Thais Barros Rodrigues and Antonio Figueira Chapter 19 RNA Interference – Natural Gene-Based Technology for Highly Specific Pest Control (HiSPeC) by Eduardo C. de Andrade and Wayne B. Hunter Chapter 20 RNA Interference – A Powerful Functional Analysis Tool for Studying Tick Biology and its Control by Remil Linggatong Galay, Rika Umemiya-Shirafuji, Masami Mochizuki, Kozo Fujisaki and Tetsuya Tanaka

Preface

RNA interference (RNAi), a hallmark of all biological sciences of twenty-first century, is an evolutionarily conserved and doublestranded RNA-dependent eukaryotic cell defense process. Opportunity to utilize an organisms own gene and to systematically induce and trigger RNAi for any desired sequence made RNAi an efficient approach for functional genomics, providing a solution for conventional longstanding obstacles in life sciences. RNAi research and application have significantly advanced during past two decades. This book RNA interference provides an updated knowledge and progress on RNAi in various organisms, explaining basic principles, types, and property of inducers, structural modifications, delivery systems/methodologies, and various successful bench-to-field or clinic applications and disease therapies with some aspects of limitations, alternative tools, safety, and risk assessment.

Chapter 1

RNA Interference – A Hallmark of Cellular Function and Gene Manipulation Ibrokhim Y. Abdurakhmonov Additional information is available at the end of the chapter http://dx.doi.org/10.5772/62038

Abstract The discovery of RN“ interference RN“i and its utilization in downregulation of spe‐ cific target transcripts have revolutionized gene function analysis and elucidation of many key biochemical/genetic pathways. The insights into gene function, combined with a technology that made silencing of gene function possible using the potent, highly spe‐ cific and selective RN“i approaches, provided the solution to longstanding complex ob‐ stacles in targeted crop improvementsfor agriculture, and disease therapies for medicine. In this introductory chapter, I aim briefly to cover the basics and peculiarities of RN“i and the advances made in understanding the mechanisms, components, function, evolu‐ tion, application, safety and risk assessment of RN“i, while at the same time highlighting the related chapters of this book. Keywords: Gene silencing, RN“ interference, RN“i inducers and delivery, RN“i-based disease therapy, biosafety

. Introduction The central dogma of genetics as first presented by Francis Crick is that genes, packed inside the cell as the deoxyribonucleic acid DN“ molecule, are transcribed into messenger ribonu‐ cleic acids mRN“ , which are subsequently translated into proteins or enzymes . These final protein products provide all life functions, and together with DN“ and RN“, constitute the molecules of life. Therefore, if there is a disruption interference of a gene function, messenger RN“ synthesis, or protein translation, normal life processes get altered or even stopped. No gene-no messenger , or no messenger-no protein , has been the basis of understanding biological processes. One of the easy-to-access points in cellular processes is messenger RN“ due to its cytoplasmic location, naked structure, comparatively short half-life, and temporal existence between transcription and translation. Further, mRN“ is in between the chain of

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events from DN“ to protein it has the universal chemical structure, consisting of only four nucleotides, regardless of the encoded message. In contrast, proteins are chemically much more variable, consisting of combinations of different amino acids, with side chains that vary from very hydrophilic to highly hydrophobic. If mRN“ is altered or eliminated before translation, there is no functional gene product, which results in changing the cellular process from the native state. This is the entire rationale of RN“ interference RN“i . RN“ interference is a process in eukaryotic cells in which double stranded endogenous or exogenous RN“ molecules trigger a cytoplasmic response, which involves sequence specific target identification and destruction. This may include native messenger RN“s mRN“s that code vitally important proteins [ ]. “ny type of double-stranded RN“ dsRN“ molecules can activate RN“i where long dsRN“s, microRN“s miRN“s and small interfering RN“s siRN“s and their various forms and modifications are considered the main players/inducers [ ]. Let us take a look at RN“i discovery history. Plant scientists in the s first used targeted gene silencing by introducing an antisense gene into plants. The first example was silencing of a nopaline synthase NOS gene, for which the silencing was only visible by loss of a band on a Norther blot and loss of NOS activity [ ]. The second antisense gene used in plants targeted the petunia chalcone synthase CHS gene, encoding the first step in floral pigment production, and the result was visible in the loss of petal pigmentation [ ]. Curiously, attempts to create dark pigmented petunia flowers by overexpression of the same CHS gene resulted in similar colorless petunia petals [ , ]. It was thought that such a phenotype was due to post-transcriptional inhibition of gene expression via an increased rate of mRNA degradation [ ]. The observed phenomenon was named as co-suppres‐ sion of gene expression and the molecular mechanism behind co-suppression remained unknown for many years [ ]. Later, a transient gene inactivation of the carotenogenic albino-3 AL-3 and albino-1 AL-1 genes was reported after transformation with homologous sequen‐ ces in Neurospora crassa [ ]. This phenomenon, named as gene quelling , was observed to be severely destructive but spontaneously and progressively reversible and monodirectional, resulting in mutant, intermediate, and wild-type phenotypes [ ]. In the years to follow, the cosuppression phenomenon were attributed to inverted repeat T-DN“ insertions, which result in RN“ transcripts with internal complementary sequences that can fold back on themselves, generating double-stranded RN“ and can seed the now well-known “rgonaute/dicer silencing system. Following these seminal discoveries, similar phenomena were discovered in other organisms including the nematode Caenorhabditis elegans and insects Drosophila melanogaster from studying the function of a PAR-1 gene required for establishing embryo polarity in the former and alcohol dehydrogenase in the latter ADH [ , ]. These studies not only demonstrated a wide range of functionality of co-suppression phenomenon but also prompted an intense effort to understand the exact mechanism causing this process. In one experiment, injection of dsRN“s associated with muscle protein production into nematodes successfully silenced the targeted gene. The effect on muscle production was not observed using either mRN“ or antisense RN“ [ ]. With this work, for the first time, the agent directly responsible for cosuppression was identified and formally named as RNA interference or RN“i. This work was later recognized with the Nobel Prize.

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In plants, the suppression of targeted genes during viral infections was discovered [ ] and subsequently developed into a system by which plant gene function may be studied through inhibition by infection with viruses bearing a short sequence targeted against plant mRN“s [ ]. This phenomenon was termed as "virus-induced gene silencing" VIGS and is often used to study gene function in plant species that are recalcitrant to transformation or just take a very long time to regenerate. Over the past decade, RN“i has been demonstrated in many eukaryotes including humans as well as some prokaryotic life forms [ ] and has been recognized to form an integral part of many gene regulatory networks during development. This revolutionary breakthrough in biological science has become a valuable in vitro, in vivo, and ex vivo manipulation of gene expression, allowing for large-scale studies of gene function. It is now a routine laboratory practice to introduce the desired gene-specific dsRN“ inducers into cells and selectively, robustly, and systematically silence the targeted sequence signature revealing its cellular function. In addition, RN“i has become an efficient tool for agricultural biotechnology to improve production [ ] and combat disease pests as well as for medicine and molecular pharmacology to cure complex infectious, inflammatory, and hereditary diseases [ ].

Figure . Dynamics of scientific publications devoted to the RN“ interference for the past three decades. Source: PubMed [ ] data sorted by the year of publications, which were retrieved by the search with the unquoted keyword RNA interference ]

RN“i research has rapidly advanced and expanded over the past decade, evidenced by increasing numbers of publications, research projects, and practical applications in both agriculture and medicine. For example, searching Google Scholar [ ] with the unquoted keyword RNA interference retrieved over million , , documents. Repeating the same search with organism-specified RN“ interference in PubMed database [ ] on the same date returned a total of , indexed scientific documents with a major pick after reaching to over , scientific publications per year Figure . The distribution of specified search results revealed a number of PubMed-indexed, RN“i-related publications for human , , plant , , animal , , insect , , fungal , and prokaryotic

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organisms. Moreover, the therapeutic application of RN“i is also expanding rapidly with , articles related to this topic and found in PubMed searching with RN“ interference therapy keyword. In this brief introductory chapter, I aim to cover the basic understanding behind RN“i and an update knowledge on its applications, limitations, safety, and risks, highlighting and discussing some of the key points presented in this book.

. Components, mechanism, and function The principle mechanism of RN“i is complex, but very straightforward and easy to under‐ stand. RN“i is induced by the introduction of specific exogenous dsRN“ either by virus genome RN“s, injection of synthetic dsRN“s or, in plants, is mediated by “grobacterium. RN“i is also part of the normal development and dsRN“s are produced by endogenous genes encoding miRN“ precursors or other long dsRN“ molecules. In either case, the dsRN“s are recognized by the enzyme dicer and cleaved into short, double-stranded fragments of ~ base pair long siRN“s [ ]. These siRN“s are separated into two single-stranded RN“s ssRN“s , which are referred to as the passenger and the guide strands. The passenger strand is degraded, while the guide strand is picked up by the RN“-induced silencing complex RISC that has enzymatic digestion activity and contains the key components of “rgonaute “GO and P-element induced wimpy testis PIWI proteins [ ]. The RISC proteins perform the unwinding of the guide and passenger strands in “TP-independent manner [ , ] however, “TP is required to unwind and remove the cleaved mRN“ strand from the RISC complex after catalysis [ ]. There are effector proteins such as RDE- nematodes and R D insects that recognize exogenous dsRN“s and stimulate dicer activity. R D also has a differentiating function for siRN“ strands by stably binding to ' end of the passenger strand, thus directing the guide strand to the RISC [ ]. Here, it should be noted that the ′ end of the guide strand is involved in matching and binding the target mRN“ while the ′ end physically arranges target mRN“s into the cleavage-favorable site of the RISC complex [ ]. “GO/PIWI proteins localize within the specific P-body regions in the cytoplasm, considered to be a critical site for RN“i [ – ]. It is not clear as yet how the guide strand-bound active RISC complex finds mRN“ targets within the cell, but it is known that this process is sequence-specific. Once the target mRN“ is identified and captured though RN“i machinery, RISC cleaves the target mRN“ rendering it untranslatable [ ]. In most cases, the entire process is triggered by amplification of the cleavage process through synthesis of additional dsRN“s from primarily digested fragments of mRN“. Upon annealing to the mRN“ target, the guide RN“ may also be extended by RN“-dependent RN“ polymerase RdRP , resulting in extended secondary dsRN“s which in turn may lead to the formation of new siRN“s that enhance and further systematically spread the degrada‐ tion of the target mRN“ in cytoplasm [ , ]. “lthough the pathways toward RN“i from exogenous and endogenous dsRN“ converge at the RISC and use the same downstream RN“i machinery, there are also some clear differences in their processing and handling [ ]. Endogenous dsRN“s cleaved by dicer produce – bp fragments with a two-nucleotide overhang at the ′end of siRN“ duplex [ ], while the length of exogenous dsRN“s-derived siRN“s, required for specificity, is unknown. Exoge‐

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nous dsRN“s are distinctly handled by the above-mentioned effector proteins, RDE- or R D [ , ], whereas siRN“ derived from endogenous dsRN“s i.e., miRN“ precursors are handled by double-stranded miRN“ precursor-binding DGCR and Drosha proteins with RN“se III enzyme activity. Plants do not have Drosha homologs, instead, processing of miRN“ to siRN“s is carried out by one of four dicer-like proteins. Endogenous miRN“s , except some plant miRN“s, typically have several mismatches to the target sequence, while siRN“s derived from exogenous dsRN“s usually are designed to have a perfect match to the target. Most importantly, endogenous dsRN“-derived miRN“s are capable of mildly inhibiting the translation of hundreds of mRN“s [ – ], while exogenous dsRN“-derived ones usually silence only single specific target [ ]. Depending on organisms, for instance in C. elegans and D. melanogaster, distinct “rgonaute proteins and dicer enzymes [ , ] process miRN“s and exogenous siRN“s. Furthermore, endogenously processed miRN“s prevalently interact with miRN“ response elements MREs located within the '-UTRs region of target mRN“s. Upon binding to MREs, miRN“s can decrease the gene expression of various mRN“s by either inhibiting translation in animals or directly causing degradation of the transcript in plants . In contrast, exogenous dsRN“-derived siRN“s may interact with any complementary sequence region of the target mRN“, causing direct cleavage of the transcript [ ]. miRN“s may actually regulate translation of target mRN“s in dual ways, as translation regulation by miRN“s oscillates between repression and activation during the cell cycle through a yet unknown mechanism [ ]. The main biological function of RN“i is regulation of gene activity of cells at the posttranscriptional level PTGS either by the inhibition of translation of mRN“ or by direct degradation of the mRN“. In addition to PTGS, RN“i pathway components may contribute to maintenance of genome organization and structure, mediated by RN“-induced histone modification. Histone modification in turn affects heterochromatin formation and may silence gene activity at the pre-transcriptional level [ ]. This process is referred to as RN“-induced gene silencing RITS and requires dicer, siRN“ and RISC component proteins such as “GO and R D [ ]. In addition, RN“i components and inducers siRN“/dicer/“GO may also possibly upregulate expression of genes in binding into a promoter region and through histone demethylation, a process dubbed RN“ activation [ , ]. ”ecause of sequence-specific recognition, regulatory properties, and the possibility of systemic spreading of dsRN“s, RN“i is the key sterilizing agent of cells and tissues, and it functions as potent immune response against foreign nucleic acids from viruses, transposons, or transformation events which can invade and harm the genome and its stability [ ]. The chapters presented in Section of this book have a more detailed coverage of the history of the RN“i discovery, mechanism, and functional components and on the biological role of RN“i including natural small RN“s/microRN“s as well as long noncoding RN“s in gene regulations.

. Differences among organisms “lthough the RN“i pathway is a universal process in eukaryotic cells, and it consists of similar component s , mechanisms, and functions as described above, there are some variations

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among organisms in both up-take of exogenous dsRN“s and induction of RN“i. First, RN“i is systemic and heritable in plants and C. elegans. The systemic spreading of RN“i in plants occurs because of transfer of siRN“s between cells through plasmodesmata and the phloem [ ]. Second, in plants, RN“i induces epigenetic silencing of genes through methylation of promoters of targeted genes which may be passed to the next generation [ ], while in Drosophila and mammals this is not the case. Third, plant miRN“s have perfect or nearly perfect complementary to their target genes and directly cleave and degrade targeted mRN“. In contrast, animal miRN“s have one or more mismatches to target sequence and halt the translation process [ ]. RN“i is not found in some eukaryotic protozoa e.g., Leishmania major and Trypanosoma cruzi [ , ]. Some fungi e.g., Saccharomyces cerevisiae lack specific RN“i component s and the reintroduction of these missing components can recover RN“i [ , ]. Further, prokaryotic organisms have distinctive RN“-dependent gene regulation system controlled by RN“ products of translation-inhibiting genes. These regulatory RN“s are not processed by dicer enzymes, differentiating them from eukaryotic RN“i [ ]. However, recently, the clustered regularly interspaced short palindromic repeats CRISPR/Cas interference system has been characterized in prokaryotes, which is a gene silencing pathway analogous to eukaryotic RN“i systems [ ]. The CRISPR interference system has its specific components, advantages, and limitations that are well described in the literature [ , ], but will not be presented here. The chapter by Dr. Devi Singh and his colleagues in Section of this book has a detailed coverage of RN“i in various organisms. RN“i in various organisms is also discussed in the chapter by Galay et al. presented in Section of this book, highlighting the specifics of RN“i in ticks while Dr. Tayota and his colleagues present an interesting methodological paper on RN“i in the water flea in Section .

. Evolution Studies on components, mechanisms, and functions of RN“i have demonstrated variations among organisms, differences in eukaryotes and prokaryotes, and indicate that RN“i is derived from an ancestral immune defense function against transcripts of transposons and viruses [ , ]. “lthough some eukaryotes might have lost RN“i components or, even, the entire pathway following the emergence of the Eukaryota, parsimony-based phylogenetic analyses suggest that an ancestral lineage of all eukaryotes possibly had a primitive RN“i capability including relevant components for some key functions such as histone modification [ ]. Phylogenetic studies also indicate that miRN“s of plants and animals may have evolved independently, but the conservation of some key proteins involved in RN“i also indicate that the last common ancestor of modern eukaryotes already possessed an siRN“-based gene silencing system. The RN“i-like defense system of prokaryotes is functionally similar, but structurally distinct from the eukaryotic RN“i system [ ]. It seems likely that a proto-RN“i system possessed at least some form of dicer-like, “GO, PIWI, and RdRP proteins. These basic components were shared by major eukaryotic lineages and functioned within an RN“ degradation exosome complex [ ].

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”eing an important component of an antiviral innate immune defense system in eukaryotes, RN“i components and various interaction/regulatory mechanisms, including the miRN“ pathway, evolved later but at faster rates under strong directional selection [ ]. This could have been a means of generating an improved response to the evolutionary arms race with viral genes. Correspondingly, some plant viruses have evolved the means to suppress the RN“i response in their host cells [ ]. Extensive studies reported that an ancient duplication of RN“i components followed by species-specific gene duplications and losses provided evolutionary diversification, specificity and adaptation of the RN“i system in many organisms [ ]. Chapter s presented in Section has covered some evolutionary aspects of RN“i.

. Applications Since its first discovery as anti-sense gene suppression, co-suppression or quelling phenomenon, the sequence specificity, efficiency, and systemic spreading in some organisms characteristics of RN“i to suppress target gene expression have caught researchers’ attention and soon became an attractive and powerful tool for gene function discovery in life sciences [ ]. ”y full or partial suppression of target gene expression using RN“i, the change in cell physiology and/or developmental phenotype helps to reveal the function of the target gene. Therefore, a utilization of RN“i has revolutionized the annotation of cellular functions of many unknown and unique genes, adding to our understanding the complex genetic/biochemical pathways and their interactions. Thanks to its partial silencing effect, RN“i also helped to discover the function of genes when complete knockout would cause lethality [ ]. Moreover, by targeting homologous sequences within a gene family, a single RN“i construct can suppress the expression of multiple members of a gene family, and thus reveal phenotypes that would have been missed in a single mutant due to redundancy in gene function. The results of the functional genomics studies, advances in the understanding of the RN“i mechanism, improved design of trait-specific RN“i inducers such as miRN“s , selection of target gene sequences combined with the development of proper delivery systems, as well as screens for off-target and cross reactivity have brought the practical applications of RN“i far beyond its initial experimental reach. “gricultural application of RN“i through tissue culture-derived genetic modifications and transgenic research in a wide range of technical, food, and horticulture crops have been particularly successful and have solved many problems. Examples include, but are not limited to, crop yield and quality improvements [ , ], food/nutrient quality improvements and fortification [ – ], decreasing the harmful precursors and carcinogens [ , ], and im‐ provement of plant pest and disease resistance [ – ]. Many of these applications are now evaluated for commercialization or are already in commercial production [ ]. In this context, targeting far red FR photoreceptor gene PHYA1 using RN“i approach [ ], our team succeed to develop the world’s first RN“i cotton cultivars with improved fiber quality and other key agronomic traits without adversely affecting the yield, which successfully passed multi-environmental large field trials and have been approved for cotton farming in Uzbeki‐ stan.

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Therapeutic application of RN“i has also been successful in medicine and molecular phar‐ macology with examples in inflammatory and infectious disease [ - ], cancer [ - ], as well as hereditary and neurodegenerative diseases [ ]. Indeed, for many other disorders RN“i may have great potential. To highlight advances made on this field, in Section of this book, we present several relevant chapters on advances of RN“i application in key human diseases of blood, ocular, nervous, kidney, and oncogenic origin. In addition, Section chapters discuss RN“i utilization in various immune and infectious diseases. Section chapters present the latest advances of RN“i application in studies of insects and parasitic pests such as ticks. “ll of these chapters highlight various aspects of RN“i and add interesting insights to the present RN“i discussions.

. Safety and risk assessment Manipulation of the organisms’ own genetic sequence signature s cis-genesis is usually considered safer compared to trans-genesis that utilizes foreign genetic material to create genetically modified GM crops and its products [ ]. However, for RN“i, when broken down to ~ nucleotides this quickly may lose its meaning, as a trans-RN“i will only work if it has sufficient homology to an endogenous target transcript. Chemically, RN“ is generally recognized as safe GRAS or it is rarely formally considered in risk assessment [ ]. Despite this and many other examples of successful application of RN“i technology in agriculture and medicine, there may be risks associated with high or repeated dosages of dsRN“, which inadvertently may interfere with unintended target sequences. “ growing body of evidences suggests that testing for the safety and assessing possible risks associated with the use of RN“iderived products sound practical, in particular, evidence of the remarkable stability of dsRN“s in the environment, their survival and resistance in the acidic conditions of the digestive tracts of higher organisms, and consequent transmissibility of dsRN“ from foods to humans/ animals. Further, production of possibly harmful secondary dsRN“s [ ] by primary RN“i inducers raised an early warning signal regarding the GR“S signature of any RN“ molecule and the possibility of risks for human health and environment. Safety concerns about RN“i-based drugs are exemplified by the lethality of out of distinct RN“i therapy experiments in mice because of potential "off-target" effects that could shut down non-targeted gene s with sequence similarity to therapeutic RN“i inducer [ ]. This observed lethality, however, could be due to oversaturation" of the dsRN“ pathway and delivery issues of short hairpin RN“s [ ] that needs to be optimized for harmless therapeutic applications. There are several suggested approaches to minimize or eliminate such offtarget , oversaturation or delivery issues, in particular through the use of comprehensive in silico target and off-target analyses [ ], modified designing of RN“i inducers with improved target selectivity, and efficient delivery systems. There may also be concerns about the uptake of intact plant miRN“ by consumers through plant diet. Plant microRN“s and some long dsRN“ molecules, with sequence complimentary and perfect matches to endogenous human genes, were demonstrated to survive the digestive

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tract of humans and can freely and routinely enter the blood system [ , ]. In vitro human cell culture experiments further showed that such plant siRN“ entered into human blood system could silence endogenous human genes due to sequence complementarity. While this may require attention of regulatory systems on one hand, on the other hand, human con‐ sumption of food crops with natural occurring siRN“s is considered safe and so far has not caused any dramatic biohazards or risk [ ]. The chapters in Section of this book also present updated information on RN“i delivery methods e.g. Tayota et al. synthesis, chemical and structural modifications, and designing for high specificity and selectivity of RN“i inducers see Gvozdeva and Chernolovskaya , and limitations of RN“i and possible alternative technology such as ER-targeted intrabodies for gene silencing see chapter by ”ackhaus and ”öldicke . Risk assessment and available protocols/guidelines are in the early stages of development. Some suggest that dsRN“-derived products must be subject to risk assessment studies [ ]. Other findings indirectly support the safety of RN“i [ , ], provided its use is within specific dosage ranges, the correct delivery system is in place and RN“i inducers without possible offtarget effects, unintended gene silencing and secondary dsRN“ production can be designed. However, it is always advisable to admit to possible risks of any novel genomic technology, including RN“i, and consider potential biohazards and evaluate risks for environmental health, before release of a new product [ , – ]. To accomplish this, Heinemann et al. [ ] proposed the following five-step guidance: to perform detailed in silico comparative bioinformatics analyses for targets of designed dsRN“ and identify possible off-targets in key consumers to experimentally quantify designed dsRN“s, and the processing of any other unknown sequence signatures or secondary dsRN“ as a result of introducing intended RN“i inducer into recipient or its product to test possible biohazards and risks due to exposure of RN“i product in animal and human cell/tissue culture to conduct animal feeding experiments for the long-term physiological and toxicological patterns and possible chronic effects and to perform clinical trials of RN“i-derived products in humans.

. Conclusions and future perspectives Thus, being a revolutionizing discovery in genome biology to characterize functions of any desired unknown genetic sequences, the discovery of RN“i has significantly widened our knowledge on core cellular processes. This knowledge has created opportunities and solutions to longstanding obstacles in conventional agriculture and medicine, offering a bright future to curing complex human and animal diseases, improve crop production and protection, and a sustained global food security through proper manipulation of key genes with agricultural or medicinal importance. “lthough key issues on specificity, selectivity, and delivery of RN“i inducing structures still exist, and some safety risks associated with the use of RN“i products have been recognized, the general believe is that RN“i is a safer technology than transgenomics utilizing foreign genetic information. Safe applications, however, require proper designing, dosage and delivery of RN“i inducers, and before its delivery for wide consumer market, the safety risks should be assessed. “ddressing the advances made over the past three decades in RN“i research and commercialization, in this book, we have compiled and

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presented a diverse collection of chapters contributed by the science research communities. We all believe that RN“i, in combination with the rapidly expanding genomic information in key organisms and novel genome editing tools, will become even more powerful and efficient, and that we will all enjoy its benefits far into the future.

Acknowledgements I thank the “cademy of Sciences of Uzbekistan and Committee for Coordination Science and Technology Development of Uzbekistan for basic science F“-F -T and several applied F“-“ -T and F“-“ -T and innovation I- - / and I -FQ- - research grants. I am particularly grateful and thank the Office of International Research Programs OIRP of the United States Department of “griculture USD“ – “gricultural Research Service “RS and U.S. Civilian Research & Development Foundation CRDF for international cooperative grants P ,P ”, and UZ”-T“, which are devoted to study, development, application, risk assessment, and commercialization of RN“i cotton cultivars and their products. I sincerely thank Dr. “lexander R. van der Krol, Waginengen University, Nether‐ lands, and Dr. Eric J. Devor, Iowa State University, the US“, for their critical reading and suggestions to this chapter manuscript.

Author details Ibrokhim Y. “bdurakhmonov* “ddress all correspondence to: [email protected] Center of Genomics and ”ioinformatics, “cademy of Science, Ministry of “griculture and Water Resources, and UzCottonIndustry “ssociation of the Republic of Uzbekistan, Tashkent, Uzbekistan

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RNA Interference – A Hallmark of Cellular Function and Gene Manipulation http://dx.doi.org/10.5772/62038

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] Izquierdo M. Short interfering RN“s as a tool for cancer gene therapy". Cancer Gene Ther. : – . DOI: . /sj.cgt.

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DOI:

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/news

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Chapter 2

RNA Interference Technology — Applications and Limitations Devi Singh, Sarika Chaudhary, Rajendra Kumar, Preeti Sirohi, Kamiya Mehla, Anil Sirohi, Shashi Kumar, Pooran Chand and Pankaj Kumar Singh Additional information is available at the end of the chapter http://dx.doi.org/10.5772/61760

Abstract RN“ interference RN“i , an evolutionarily conserved mechanism triggered by doublestranded RN“ dsRN“ , causes gene silencing in a sequence-specific manner. RN“i evolved naturally to mediate protection from both endogenous and exogenous pathogen‐ ic nucleic acids and to modulate gene expression. Multiple technological advancements and precision in gene targeting have allowed a plethora of potential applications, ranging from targeting infections in crop plants to improving health in human patients, which have been reviewed in this chapter. Keywords: RN“ interference, miRN“, RN“i mediated gene silencing, RN“-induced si‐ lencing complex

. Introduction “scribing the structure and function relationship to a gene and modulating its expression to manifest the desired phenotype have been major challenges for scientists. [ ] In order to elucidate the phenotype s associated with a given gene, various gene-targeting techniques have been tried with mixed success. Gene silencing can be executed at transcriptional gene silencing TGS and posttranscriptional gene silencing PTGS levels. [ ] The TGS involves targeting genes at DN“ level by altering promoter and enhancer efficiencies, methylation status of genes, and deleting parts of genes by homologous recombination, transcription activator-like effector nucleases T“LENs , and clustered regularly interspaced short palin‐ dromic repeats CRISPR /Cas systems. [ ] The PTGS techniques rely upon the breakdown of mRN“ by various technologies, including antisense RN“, ribozymes, DN“zymes, micro‐

22

RNA Interference

RN“s, and RN“ interference RN“i . [ ] “mong all these techniques, RN“i is the most efficient tool for targeted gene silencing. RN“i is now routinely utilized across multiple biological disciplines to determine gene function. RN“i is also being utilized for therapeutic interventions to downregulate the expression of genes involved in disease pathogenesis. The current review is focused on recent advancements in the biology and applications of RN“i.

. RNAi-mediated gene silencing: A historical perspective across multiple species . . Discovery of RNAi in plants and fungi R. Jorgensen and his colleagues identified a novel mechanism of post-transcriptional gene silencing in Petunia. [ ] They were attempting to introduce a chalcone synthase gene under a strong promoter to deepen the purple color of Petunia flowers however, instead of getting a stronger purple color flower they observed that most flowers lost their color. Thus, they observed diminished expression of both the homologous endogenous gene and the exoge‐ nously introduced transgenic copy of the gene and termed the phenomenon as co-suppression. [ ] “lthough the exact mechanism of this phenomenon remained undeciphered at that time, the posttranscriptional nature of gene silencing was still appreciated. [ - ]The suppression of endogenous gene expression by transformation of exogenous homologous sequences was later termed as quelling in Neurospora crassa. [ , ] . . RNAi in Ceanorhabdites elegans In , Guo and Kempheus attempted to knock down the expression of P“R- gene by antisense RN“ in C. elegans they observed a similar loss of gene expression with sense RN“ controls as well [ ]. “t that time, they could not explain the mechanistic basis of such an observation. In , “ndrew Fire, Craig C. Mello, and their colleagues demonstrated efficient and specific interference of gene expression by introducing double-stranded RN“ in the nematode C. elegans [ ]. The genetic interference was genetically heritable and was stronger than the antisense strategy. This novel phenomenon was termed as RN“ interference or RN“i by Fire and colleagues [ ]. Subsequently, Lisa Timmons and “ndrew Fire demonstrated that C. elegans, when fed on bacteria genetically engineered to express dsRN“ for unc- and fem- genes, showed specific and reversible silencing of unc- and fem- genes in the worm [ , ]. High-throughput genetic screens have been developed by either feeding the worms on genetically engineered bacteria expressing dsRN“ or soaking or injecting the nematode with dsRN“. Functional genomic analysis of chromosomes I and III in C. elegans have been performed by Fraser and Gonczy, respectively, utilizing the RN“ interference strategy [ , ]. . . RNAi technology in Drosophila Specific gene silencing has been achieved in the embryo extracts and cultured cells of Drosophila flies by utilizing the RN“i tool [ ]. Zamore and colleagues utilized Drosophila melanogaster

RNA Interference Technology — Applications and Limitations http://dx.doi.org/10.5772/61760

embryo lysates to demonstrate the cleavage of long dsRN“ strands into short interfering dsRN“ fragments siRN“ of ~ nucleotides nt [ ]. Later Elbashir and colleagues demon‐ strated that chemically synthesized - or -nt-long dsRN“ carrying ′ overhangs could induce efficient RN“ cleavage in embryo extracts from Drosophila [ ]. . . RNAi in mammalian systems “ global nonspecific inhibition of protein synthesis was observed in mammalian cells by exposing them to dsRN“s that were greater than base pairs bp in length [ ]. RN“dependent protein kinase PKR , and ′, ′ oligoadenylate synthetase ′, ′-O“S were responsible for the nonspecific silencing. PKR phosphorylates eIF- α, a translation initiation factor, to shut down global protein synthesis. “ synthesis product of enzyme ′, ′-O“S activates RNase L, which induces nonspecific degradation of all mRN“s in a mammalian cell [ ]. Long dsRN“s induce interferon response that activates both of these enzymes in mammalian cells [ ]. The nonspecific interference pathways represent the mammalian cell response to viral infection or other stress [ ]. Tuschl and colleagues demonstrated that RN“ interference could be directly mediated by small interference RN“ siRN“ in cultured mammalian cells [ ]. However, because siRN“ does not integrate into the genome, the RN“i response from siRN“ is only transient. In order to induce stable gene suppression in mammalian cells, Hannon and his colleagues utilized RN“ Pol III promoter-driven e.g., U or H expression of short hairpin RN“s shRN“s [ ]. Various approaches have since been developed for mammalian cells to obtain successful gene silencing. Some of the successful gene silencing approaches are listed in Table . Kingdom

Species

Silencing process

Induction stimulus

Fungi

Neurospora

Quelling

Transgene s

Saccharomyces pombe

RN“i

dsRN“

Arabidopsis, Coffea canéfora, Nicotiana,

Transcriptional or Post-

Transgene s and

Petunia

transcriptional gene silencing, viruses

Plants

co-suppression Invertebrates

Paramecium

Homology-dependent gene

Transgene s

silencing Amblyomma americanum, Anopheles,

RN“i

dsRN“

RN“i,

dsRN“,

TGS

Transgene s

Co-suppression, RN“i,

dsRN“

Brugia malayi, Dugesia japonica, Hydra, Leishmania donovani, Schistosoma mansoni, Tribolium castaneum, Trypanosoma brucei, etc. Ceanorhabditis elegans Drosophila melanogaster

Transcriptional gene silencing Transgene s Vertebrates

Human, Mouse, Zebrafish,

Table . Gene silencing approaches

RN“i

dsRN“

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RNA Interference

. The mechanism of silencing RN“i-mediated gene silencing is executed by siRN“s. The process of silencing begins with the cleavage of long dsRN“s into – -nt fragments of siRN“s in cytoplasm [ , ]. The process is catalyzed by Dicer enzyme [ ]. These siRN“s are inserted into multiprotein silencing complex, which is known as RN“-induced silencing complex RISC . Subsequent unwinding of siRN“ duplex, in turn, leads to active confirmation of RISC complex RISC* . Next, target mRN“ mRN“ to be degraded is recognized by antisense RN“, which signals RISC complex for the endonucleolytic degradation of the homologous mRN“. Tuschl and his colleagues have defined the directionality of dsRN“ processing and the target RN“ cleavage sites [ ]. “ccording to their results, target mRN“ is cleaved in the centre of the region that is recognized by complimentary guide siRN“, which is – -nt away from the ′ terminus of siRN“ [ ]. The RN“i process is completed by the last step of siRN“ molecule amplification. It is well established that the next generation of siRN“s is derived from the priming on the target mRN“ by RN“-dependent RN“ polymerase RdRp enzyme by existing siRN“s. The second generation of siRN“s is effective in inducing a secondary RN“ interference that is defined as transitive RN“i. The transitive RN“i causes a systemic genetic interference in plants and C. elegans. Interestingly, transitive and systemic RN“i is absent in Drosophila and mammals owing to the lack of RdRp in both organisms [ ]. “n illustration of the function of RN“i is demonstrated in Figure . “ multitude of studies suggests a possible link between RN“i and chromatin remodeling [ ]. The dsRN“ works at TGS and PTGS in plants, where both pathways related and assist in gene silencing. Only TGS is heritable and drives methylation of endogenous sequences. Multiple proteins, including Polycomb in Drosophila and C. elegans [ ], and Piwi in Drosophila [ ], execute silencing at both TGS and PTGS levels. Volpe and his colleagues documented that RN“i complex proteins, including Dicer, “gronaute, and RdRp, assist in centromeric silencing in Schizosaccharomyces pombe [ ]. This suggests that RN“i contributes to the maintenance of genomic stability [ ].

. Enzymes involved in RNAi . . Dicer Dicer was first characterized and defined in Drosophila by ”ernstein et al. [ ]. Dicer belongs to the RNase III-class and assists in “TP-dependent siRN“ generation from long dsRN“s. Importantly, human Dicer does not require “TP for the cleavage of long dsRN“s [ ]. Structurally, Dicer is a large ~ -kDa multi-modular protein that acts as an antiparallel dimer. Dicer has multiple domains, including an N-terminal putative DExH/DE“H box RN“ helicase/“TPase domain, an evolutionarily conserved P“Z domain, two neighboring domains that resemble RNase III, and a dsRN“-binding domain. P“Z domain in dicer helps in recognizing the end of dsRN“, whereas RNase III domain helps in the cleavage of dsRN“. Function of other domains is not fully known. Dicer orthologs has been defined in many

RNA Interference Technology — Applications and Limitations http://dx.doi.org/10.5772/61760

Figure . Mechanism of RN“i-mediated silencing. The model demonstrates double-stranded RN“ dsRN“ can gener‐ ate either from exogenous natural sources, such as a viral infection, exogenous artificial sources such as transfection, or natural synthesis. The dsRN“ is then processed by a multimeric Dicer enzyme to generate siRN“ that can be further amplified by RN“-dependent RN“ polymerase RdRp . The siRN“ subsequently interacts with an array of proteins to form RN“-induced silencing complex RISC that is activated in an “TP-dependent manner. The activated RISC RISC* can then induce chromatin remodeling or TGS, or induce target RN“ cleavage, or cause miRN“-mediated translational inhibition.

organisms, including S. pombe, Arabidopsis thaliana C“RPEL F“CTORY [C“F] , Drosophila DCR- and DCR- , C. elegans DCR- , mouse, and humans. In addition to RN“i, Dicer also assists in the generation of microRN“s in multiple organisms [ ]. . . RNA-Induced Silencing Complex RISC RISC is a ribonucleoprotein complex that fragments mRN“s through the production of a sequence-specific nuclease. “t first, while working on Drosophila embryo extracts, Zamore and his colleagues identified ~ kDa precursor complex, which turns into an activated complex of kDa upon addition of “TP. However, Hannon and his colleagues found a -kDa complex from Drosophila S cells [ , ]. The siRN“ is an important part of RISC and was the first to be identified. It acts as a template and guides RISC to the target mRN“ molecule. To date, a number of RISC protein components are known in Drosophila and mammalian species. Interestingly, these components are not completely overlapping, which suggests the develop‐ mental stage-specific or evolutionarily non-conserved nature of the components of RISC complex [ ].

25

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RNA Interference

The first RISC protein component identified was “gronaute- , a C. elegans RDE- homologue [ ]. “rgonaute “GO proteins are part of an evolutionarily conserved protein family and they play a central role in RN“i, determination of stem cell developmental regulation, and tumorigenesis. “GOs are ~ kDa highly basic proteins that contain N-terminal P“Z and mid- and C-terminal PIWI domains [ ]. The P“Z domain is an RN“-binding module, which is involved in protein–protein interactions, whereas PIWI is essentially required for target cleavage. Some “GO proteins that are involved in RN“i are listed in the Table . Species

Argonaute homolog

Essentiality for RNAi

Arabidopsis

“GO

Essential for co-suppression and

Citations

PTGS ZWILE

Non-essential

Tetrahymena

TWI

Essential for DN“ elimination

Neurospora

QDE

Required component of RISC

C. elegans

RDE-

Forms complex with Dicer

“LG-

Nonessential

“LG-

Nonessential

Drosophila

PPW-

Essential for germline RN“i

“ubergine

Localizes with dsR”P Staufen and RN“ helicase Vasa. Essential for maturation-dependent RN“i generation.

d“gro

Essential in embryos, acts downstream of RN“i generation

d“go d“go

Required component of RISC Prediction based on DN“ sequence

PIWI Mammals human

EIF C /h“go

Essential for PTGS and TGS Part of RISC complex and catalyzes the miRN“ –directed cleavage

Table . “rgonaute homolog proteins in RN“i

Some RISC components are non-“GO proteins, including dFXR and VIG in Drosophila, the fragile X mental retardation FMR homolog in Drosophila, and germin / in mammals [ ]. . . RNA helicase RN“ helicases cause unwinding of dsRN“. However, Dicer contains its own helicase activity in the N-terminal helicase domain. Hence, the helicase proteins putatively function down‐

RNA Interference Technology — Applications and Limitations http://dx.doi.org/10.5772/61760

stream of the RISC complex. Two major RN“ helicase families are involved in RN“i [ ]. SDE from A. thaliana and its homologous proteins in mouse, human, and Drosophila constitute the first such helicase family. The second family contains Upf p from yeast and an Upf homo‐ logue SMG- in C. elegans. The Upf /SMG- is characterized by cysteine-rich motif conserved across species and multiple C-terminal Ser-Gln SQ doublets. MUT- , a DE“H-box helicase in C. elegans is also putatively involved in transposon suppression. “nother RN“ helicase Germin resides in complex with human “GO protein EIF C /h“go [ ]. . . RNA-dependent RNA polymerase RdRp RdRp catalyzes the amplification and triggering of RN“i, which is usually in small amounts. RdRp catalyzes the siRN“-primed amplification by polymerase chain reaction to convert mRN“ into dsRN“s, a long form that is cleaved to produce new siRN“s [ ]. Lipardi and his colleagues demonstrated RdRp-like activity in Drosophila embryo extracts, but the enzyme responsible for the RdRp activity in the Drosophila or human is not known. Some of the RdRps involved in RN“i have been summarized in Table . Species

RdRp homolog

Arabidopsis

SDE

Essentiality for RNAi

Citations

Essential for PTGS by transgenes but not by viruses

Neurospora

QDE

Essential for co-suppression

C. elegans

EGO

Essential for germline RN“i

RRF

Essential for RN“i in soma

RDE

Forms complexes with Dicer

Table . RdRps involved in RN“i

. Various small RNA isoforms related to RNAi . . Small interfering RNAs siRNAs Small interfering RN“s are – -nt-long double-stranded RN“ molecules with – -nt overhangs at the ′ termini. siRN“s are normally generated, as mentioned in the above sections, by the cleavage of long double-stranded RN“s by RNase III Dicer [ ]. siRN“s must be phosphorylated at the ′ termini by endogenous kinases to enter into the RISC complex [ ]. It is thought that the hydroxylated ′ termini are essential for the siRN“-primed amplification step catalyzed by RdRps. However, Zamore et al. showed that non-priming alterations in the ′ hydroxyl group did not adversely affect RN“i-mediated silencing [ ]. They went on to explain that siRN“s operate as guide RN“s for gene repression but not as primers in the human and Drosophila RN“i pathways [ ]. Conversely, Hamada et al. showed in mammalian cells that modifying the ′ end of the antisense strand of siRN“ abolished the RN“i effect,

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while modifying the ′ end of the sense strand did not affect the RN“i silencing [ ]. These findings support the model that each strand of siRN“ has different functions in the RN“i process, and the ′ hydroxylated end of the antisense strand may prime the amplification. “mbros et al. discovered endogenous siRN“ in more than genes in wild-type C. elegans [ ]. This suggests that siRN“ may be a globally conserved and common molecule among species. . . Micro RNAs miRNAs miRN“s are – -nt small RN“ species produced by Dicer-mediated cleavage of endogenous ~ -nt noncoding stem-loop precursors. The miRN“s, while allowing mismatches, can either repress the target mRN“ translation mostly in mammals or facilitate mRN“ destruction mostly in plants [ ]. miRN“s lin- and let- were the first ones to be identified in C. elegans [ ]. So far, about different miRN“s have been identified in plants, animals, and lower species. While some miRN“s are evolutionarily conserved, others are specific for some developmental stages or are species-specific. Different terminologies are referred to in literature. “ccording to one terminology, the miRN“s with well-characterized functions e.g., lin-4 and let-7 are referred to as small temporal RN“s stRN“s , while other similar small RN“s of unknown functions are called miRN“s [ ]. Multiple miRN“s have been character‐ ized for their physiological roles in cancer and other diseases [ , ]. Comparisons between siRN“ and miRN“ have been listed in the Table . Resemblences siRNA

miRNA

. The siRN“s require processing from long dsRN“s.

. The miRN“s require processing from stem-loop precursors that are ~

nt long.

. “n RNase III enzyme Dicer is required for processing. . Dicer is required. . The siRN“s are usually ~

nt long.

. The miRN“s are also ~

nt long.

Disparities siRNA . The siRN“s are double-stranded structures with -nt

miRNA . The miRN“s are single-stranded structures.

′ overhangs that are formed during cleavage by Dicer. . The siRN“ require high homology with the mRN“ to . The miRN“s can function even with a few mismatched bind and cleave. . The siRN“s mediate target mRN“ cleavage by RISC.

nucleotides. . The miRN“s can either block target mRN“ translation by binding to it or mediate target mRN“ cleavage by RISC.

. The siRN“s are usually triggered by transgene incorporation, viral infection, or active transposons.

. The miRN“s are constitutively expressed cellular RN“ moieties with potential roles in development, and cell proliferation and death.

Table . Comparative characteristics of siRN“ and miRN“

RNA Interference Technology — Applications and Limitations http://dx.doi.org/10.5772/61760

. . Tiny noncoding RNAs tncRNAs “mbros and his colleagues discovered the first tncRN“s in C. elegans. They identified and characterized new tncRN“s in C. elegans by performing cDN“ sequencing and comparative genetics [ ]. The tncRN“s are very similar to miRN“s with regard to their size, singlestranded structure, and lack of a precise complementarity to a given mRN“. However, they are distinct with regard to their lack of processing from a miRN“-like hairpin precursor , and phylogenetic nonconservation. Similarly to miRN“, tncRN“s are transcribed from noncoding sequences. However, their developmental role is not fully understood. “ccording to “mbros and his colleagues, it is plausible that some of the miRN“s might be processed from noncoding mRN“s in the course of RN“i [ ].

. Evolutionary relevance of RNAi in the immunological responses RN“i may provide a systemic way to immunize an organism against the invasive nucleic acids from viruses and transposons via inducing the RN“i responses. Virus-induced gene silencing VIGS in plants is accomplished by RN“i. Multiple genetic links between RN“i and virulence are known. Many plant viruses code for viral suppressors of gene silencing VSGS . VSGS acts as a virulence determinant, and hence, is required for developing antivirulence response in the host. In response to the virulence, the host can also modify its PTGS/RN“i mechanisms to prevent future infections. RN“i can even target DN“ virus amplification in plants [ ]. VIGS mechanisms exist not only in plants and nematodes but also in other species for example, flock house virus FHV , a virus that infects Drosophila, also codes for a potential silencing suppressor b [ ]. Nonetheless, the precise function of RN“i in mammalian antiviral defense is not clear. RN“i also plays a crucial role in the development process of multicellular organisms. When mutated, CARPEL FACTORY, a Dicer homologue in Arabidopsis, can cause developmentally defective leaves and induce overproliferation of floral meristems. Inactivation of Dicer by mutations causes developmental problems and sterility in C. elegans. Mutations in “GO protein influence normal development in Drosophila as well. Hence, components of RN“i pathway play a significant role in normal development, but such components and the affiliated gene products play crucial roles in related but distinct gene regulation pathways [ ]. “ potential role of RN“i and human disease pathogenesis has been proposed due to associa‐ tion of RN“ binding proteins with RISC complex, such as Vasa intronic gene VIG and the fragile X mental retardation protein FMRP Drosophila homologue [ ].

. RNAi as a functional genomics tool and its applications for therapy RN“i technology is applicable for gene silencing in many species. RN“i has been used extensively in C. elegans for functional genomics. High-throughput investigation of most of the

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~

,

genes has been accomplished. “hringer and his colleagues produced an RN“i library,

representing ~

% of the genes of C. elegans [

]. This strategy has been successfully attempted

in multiple other model organisms, including human [ ]. RN“i has also been utilized successfully in mammalian cells [ ]. Various methods have been employed for siRN“ knockdown of specific genes in mammalian cells. DN“-vector-mediated RN“i silences genes transiently in mammalian cells, while other expression systems are used for stable silencing. The promoters of RN“ polymerase pol II and III U and H , alone or together have been used for stable silencing. Furthermore, tRN“ promoter-based systems have been used for this purpose. However, pol III-based short hairpin RN“ shRN“ expres‐ sion systems e.g., H RN“ pol-based pSuper vector are suitable choices. Retroviral-vectorbased delivery of siRN“s has also been utilized for more efficient silencing. Two classes of retrovirus vectors have been employed:

HIV- -derived lentivirus vectors and

Oncore‐

trovirus-based vectors, such as Moloney murine leukemia virus MoMuLV and Murine stem cell virus MSCV . Transgenic mice have been established with germline transmission of a shRN“ expression cassette for silencing of genes not targeted by homologous recombinationbased approaches [ ]. Desirable applications of this technique include inducible and cell typespecific expression patterns. The use of RN“i is not limited to the determination of mammalian gene function, and also could be used for treating viral infections and cancer [

,

]. Viral and human genes that are

needed for viral replication can be attacked to generate viral-resistant host cells or to treat viral infections [ ]. Oncogenes, which accelerate cancer growth, can be targeted by RN“i [ ,

].

Targeting of molecules important for neovascularization could prevent tumor growth [ ]. This book presented several chapters with detailed discussions of therapeutic aspects of the RN“i in immune, blood, cancer, and brain diseases. We refer readers to those chapters by Hu et al. Gu and Cho and Kim rather to continue repeated information here.

. Conclusions Fast progress in RN“i technology has shown promise for use in reverse genetics and therapy. However, mechanistic complexities of this technology still need to be determined. RN“i has now been established as a revolutionary tool for functional genomics in organisms. Multiple studies have defined the role of RN“i in mammalian and plant defense systems. “ plethora of studies have utilized RN“i technology to modulate gene expression. RN“i-based full genomic screens have allowed identification of specific genes, controlling a given trait with high accuracy. Further studies will continue to unravel the unlimited potential of RN“i to serve humankind.

RNA Interference Technology — Applications and Limitations http://dx.doi.org/10.5772/61760

Author details Devi Singh *, Sarika Chaudhary , Rajendra Kumar , Preeti Sirohi , Kamiya Mehla , “nil Sirohi , Shashi Kumar , Pooran Chand and Pankaj Kumar Singh *“ddress all correspondence to: devisingh

@gmail.com

Molecular ”iology laboratory, Department of Genetics and Plant ”reeding, Sardar Vallabhbhai Patel University of “griculture and Technology, Meerut, India Institute of Genomics and Integrative ”iology, New Delhi, India Department of “gri-”iotechnology, SardarVallabhbhai Patel University of “gricultureand Technology, Meerut, India Eppley Institute for Research in Cancer and “llied Diseases, UNMC, Omaha, Nebraska, US“ International Centre for Genetic Engineering and ”iotechnology, New Delhi, India

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Chapter 3

The Role of Immune Modulatory MicroRNAs in Tumors Barbara Seliger, Anne Meinhardt and Doerte Falke Additional information is available at the end of the chapter http://dx.doi.org/10.5772/61805

Abstract Tumors could evade the control of CD + T and/or NK cell-mediated surveillance by dis‐ tinct immune escape strategies. These include the aberrant expression of HL“ class I anti‐ gens, coinhibitory or costimulatory molecules, and components of the interferon IFN signal transduction pathway. In addition, alterations of the tumor microenvironment could interfere with a proper antitumoral immune response by downregulating or inhib‐ iting the frequency and/or activity of immune effector cells and professional antigen pre‐ senting cells. ”ased on the identification as major mediators of the posttranscriptional silencing of gene expression, microRN“s miRN“s have been suggested to play a key role in many biological processes known to be involved in neoplastic transformation. In‐ deed, miRN“ expression is frequently deregulated in many cancer types and could have tumor-suppressive as well as oncogenic potential. This review focused on the characteri‐ zation of miRN“s, which are involved in the control of the immune surveillance or im‐ mune escape of tumors and their use as potential diagnostic and prognostic biomarkers as well as therapeutic targets. Moreover, miRN“s can have dual activities by affecting the neoplastic and immunogenic phenotype of tumors. Keywords: “PM, IFN, immune escape, microRN“, tumor microenvironment

. Introduction Tumors have developed different strategies to evade immune recognition by cytotoxic T lymphocytes CTLs as well as natural killer NK cells. This is caused by alterations of the tumor itself, changes of the tumor microenvironment TME , reduced frequency, and impaired function of diverse immune subpopulations. The processes leading to immune evasion of tumors are diverse and could be associated with structural alterations and/or deregulation of genes/proteins from tumor cells, but also from different immune cells important for recogni‐ tion and killing of tumor cells or in the induction of immune suppression. The identification of microRN“s miRN“s , involved in the RN“ interference RN“i -based control of these

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immune modulatory molecules, clarified the complexity of the mechanisms conditioning tumor immune escape. This review is focused on the identification and characterization of immune modulatory miRN“s im-miRN“s in tumors, thereby altering the antitumoral immune response by miRN“i-mediated RN“i.

. The MHC class I antigen processing and presentation machinery APM The major histocompatibility complex class I MHC molecules present an array of peptide epitopes for surveillance by CD + T cells. These peptides are classically derived from proteins synthesized in the cytosol. Upon proteasomal degradation of ubiquitinated proteins, the yielded peptides are then transported into the endoplasmic reticulum ER via the heterodi‐ meric peptide transporter associated with antigen processing T“P . The peptide transport into the ER is “TP dependent and sequence specific. The T“P heterodimer associates in ER with a number of other proteins to form the peptide loading complex PLC . These include the chaperone tapasin, which recruits MHC class I heavy chain HC /β -microglobulin β -m dimers and calreticulin. The peptides are either trimmed by ER-resident aminopeptidases or directly loaded onto MHC class I molecules [ ]. Upon peptide loading, the PLC dissociates from the trimer consisting of the MHC class I HC, β -m, and peptide is then transported via the trans Golgi to the cell surface and exposed to CD + CTLs [ ].

. Immune stimulatory and immune inhibitory molecules and immune response “n effective T-cell response requires two signals. The first is mediated by the interaction with MHC class I antigens on the antigen-presenting cells “PC , and the second is mediated by the interaction of ” family members on “PC with CD or CTL“ on T cells. The prototypes of ” family members are ” - CD and ” - CD . During the last years, the ” family was growing consisting of ” -H PDL- , ” -H , ” -H , ” -H , and ” -H molecules [ , ]. While ” -H and ” -H represent coinhibitory molecules, ” -H was identified as costimu‐ latory molecule, which is mainly expressed on ” cells, monocytes and dendritic cells DC : ” H binds to the receptor ICOS, which results in activation of T cells through phosphatidylinositol- -kinase-dependent signal transduction pathways and in the induction of Th cell-mediated immune response, proliferation, and cytokine production [ ]. The role of ” -H is currently controversially discussed and depends on the cell types analyzed, demon‐ strating either costimulatory or coinhibitory activity. Regarding ” -H , its expression is primarily restricted to activated T cells, ” cells, monocytes, and DCs [ , ]. ” -H is not detected in the majority of normal tissues and cells but is overexpressed in a variety of tumor tissues. ” -H has been identified as ligand for the NK cell receptor NKp and is detectable on surface or in the cytosol of tumor cells and as soluble factor in the peritoneal fluid [ ], while it is not expressed on healthy cells. The interaction of ” -H with NKp is involved in NK cell

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responses [ ]. It is noteworthy that many other coinhibitory molecules have also been identi‐ fied and their role on immune responses is currently under investigation [ , ].

. Features of the interferon-γ-mediated signal transduction Interferons IFN are a group of pleiotropic cytokines that play a key role in the intercellular communication during innate and adaptive immune responses, in particular in the host defense against viral and bacterial infections and neoplastic transformation [ ]. The IFN family could be classified into type I and type II IFNs, which differ in their activity regarding immune modulation [ ]. IFN- belongs to the type II IFN and is a central regulator of immune responses by controlling and modulating the expression of targets essential for cell-cell communication and cellular interactions. It is secreted by activated T cells, NK cells, and macrophages and induced by DC and monocytes stimulated with bacterial cell wall components [ ]. IFN- exerts its activity by binding to its heterodimeric receptor consisting of IFN- -R and IFN- -R subunits [ , ]. This results in the dimerization of the receptor subunits followed by activation trans‐ phosphorylation of the receptor-associated tyrosine kinases J“K and J“K belonging to the Janus kinase J“K family and phosphorylation and dimerization of the J“K-associated ST“T transcription factor. The activated ST“T is translocated into the nucleus and recruited to the IFN- -activated sequence G“S element of the promoters of the ST“T target genes leading to their transcriptional activation. IFN- -regulated genes can be classified into primary and secondary responsive genes. Primary responsive genes are induced early due to the binding of ST“T dimers to the G“S element in the promoter region of target genes, like IRF , CXCL , and CXCL [ ]. IRF binds to IFNstimulated response elements ISRE and modulates gene induction of the secondary respon‐ sive genes. IFN- induced the transcription of MHC class I and class II antigens and of many “PM components and at high concentrations could lead to a caspase-dependent apoptosis. In addition, IFN- is involved in amplifying toll-like receptor TLR signaling by increasing or inhibiting the transcription of TLRs, chemokines, and cytokines [ , ]. Furthermore, IFNpromotes the induction of SOCs proteins suppressor of cytokine signaling , which inhibit IFNsignaling by a negative feedback loop, resulting in the inactivation of J“K and J“K [ ]. Moreover, IFN- signaling is controlled by inhibiting J“K , J“K , and IFN- -R via dephos‐ phorylation mediated by SH -domain-containing protein tyrosine phosphatase [ ], by proteasomal degradation of J“K and J“K [ ] and by inhibition of ST“T , which is mediated by the protein inhibitor of activated ST“T [ ].

. Distinct levels of tumor immune escape Tumors have developed different strategies to escape immune surveillance, which could occur at the level of immune cells, tumor micromilieu, and the tumor itself Figure . The frequency,

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activity, and function of CD + and CD + T lymphocytes, DC, NK cells, and ” cells are often downregulated in peripheral blood of tumor patients, while the number of immune-suppres‐ sive myeloid-derived suppressor cells MDSC , NKT cells, and regulatory T cells Treg is upregulated [ - ].

Figure . Tumor immune escape mechanisms containing “ loss of tumor antigen expression ” variations in tumor antigen processing C defects in the peptide transporter T“P, chaperone tapasin, and protease ER“P D defects in expression of MHC class I heavy chain and β -microglobulin E release of anti-inflammatory cytokines F downre‐ gulation of IFN- -R , J“K , J“K , ST“T , and ST“T G altered phosphorylation states of ST“T H impaired up‐ regulation of IFN- regulated genes MHC class I, “PM I upregulated expression of HL“-G J altered methylation pattern of IRF K overexpression of SOCS and L protection from T-cell-mediated apoptosis.

The tumor microenvironment TME consists of various cellular and soluble factors and is of clinical relevance since its composition significantly correlates with the tumor patients’ outcome. These include different cellular components, such as fibroblasts, blood vessels, immune cells, stroma cells, extracellular matrix, and soluble factors such as immune-suppres‐ sive cytokines, like interleukin IL - , transforming growth factor TGF -β, metabolites, arginase and prostaglandin, hypoxia, and pH, which negatively interfere with the antitumoral immune responses.

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In tumors, an aggressive and deregulated growth of neoplastic transformed cells, which overexpress proangiogenic factors, such as the vascular endothelial growth factor VEGF , leads to the development of organized blood vessels. These blood vessels are fundamentally different from the normal vasculature. Tumor-associated fibroblasts T“F represent the major constituents of the tumor stroma and produce growth factors, including the VEGF and inhibitory cytokines that activate extracellular matrix thereby contributing to the tumor growth. Furthermore, cancer is often driven by inflammation mediated by monocytes and tumorassociated macrophages T“M , which belong to the innate immune cells. Macrophages could be classified in type and macrophages. While M macrophages express a series of proin‐ flammatory cytokines, chemokines and effector molecules, the M macrophages express a wide array of anti-inflammatory molecules, including IL- , IL- , TGF-β, and adenosine. T“Ms are mainly of the M phenotype and secrete different cytokines, chemokines and proteases, which promote tumor angiogenesis, growth, metastasis as well as immune sup‐ pression. In addition to T“M and T“F, MDSC represent a heterogeneous population derived from myeloid progenitors [ ]. They can promote tumor growth by enhancing angiogenesis or suppression of innate and adaptive immune responses. Regarding the innate immune cells, MDSCs suppress NK cell cytotoxicity, promote M macrophage differentiation, and modulate the priming activity of mature DC [ ]. Moreover, MDSCs suppress T-cell responses by induction of apoptosis, secretion of immune modulatory factors, modulation of amino acid metabolism, restriction of T-cell homing, and induction of Treg [ - ]. Tregs suppress the activity of immune cells and maintain immune tolerance to self-antigens. They express CD , CD and FoxP [ ]. The elevated numbers of Tregs in cancer is due to their efficient migration into the tumor sites [ ], local expansion in the tumor environment[ ], and de novo generation within the tumor [ ]. . . Alterations of the tumors Immune escape mechanisms include loss or downregulation of HL“ class I antigens and/or components of the antigen processing machinery “PM , upregulation of nonclassical HL“G and HL“-E antigens, and coinhibitory molecules, including PDL- , as well as alterations of signaling transduction cascades, including in particular the IFN signaling pathway [ ]. The frequency of these different mechanisms highly varied between tumor sub types and is often correlated with a worse prognosis and reduced survival of tumor patients. 5.1.1. MHC class I abnormalities The classical MHC class I pathway and the “PM components are involved in eradication of developing tumors [ ]. Since CD + CTLs recognize and eliminate cells presenting tumor antigens via HL“ class I molecules, loss of HL“ class I expression results in evasion of CTLmediated cell death [ ]. “bnormalities of HL“ class I antigens are often due to downregula‐ tion of various components of the MHC class I “PM, in particular of T“P, tapasin, β -m, and MHC class I HC. Structural alterations of these components are rare, while MHC class I defects

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are mainly due to deregulation of the different components, which could be controlled at the transcriptional, epigenetic methylation, acetylation , posttranscriptional e.g., microRN“s, protein degradation , or posttranslational phosphorylation level. HL“-G has been demonstrated as a nonclassical HL“ class I antigen, which is in general only expressed on immune privileged organs, but also on many tumors of distinct origin [ ]. The overexpression of HL“-G or secretion of soluble HL“-G are directly associated with tumor progression and reduced patients’ survival. It suppresses antitumoral immune responses by binding to receptors of various immune populations, thereby inhibiting the sensitivity to CTLand NK cell-mediated lysis in particular [ ]. In contrast, tumor cells with deficient expression of classical HL“ class I molecules are eradicated by NK cells. 5.1.2. Check points as important regulators of immune response During carcinogenesis, members of the ” -family play a key regulatory role of both stimulato‐ ry and inhibitory T-cell responses, which depends on the available ” ligand and receptor on the respective target and immune cells [ , ]. Interestingly, ” -H and ” -H were often overexpressed on tumors leading to impaired immune recognition. ”y interaction with these coinhibitory molecules, the intensity of the T-cell responses is reduced by raising the thresh‐ old of activation, halting proliferation, enhancing apoptosis, and inhibiting the differentia‐ tion of effector cells [ ]. 5.1.3. Role of IFN-γ in cancer immunogenicity “bnormalities of MHC class I expression on tumor cells due to the downregulation or loss of “PM component expression are common mechanisms, by which tumor cells can escape from anti-tumor-specific immunity [ , ]. In addition, tumor cells are often not susceptible to treatment with IFN- , which could be due to structural alterations or deregulation of constit‐ uents of the IFN signal pathway. Several studies confirmed that defects in the IFN- receptor signaling cascade could be occur at multiple steps of this pathway, including lack of the expression of the IFN- -R , abnormal forms of J“K , lack of expression of J“K [ ], altered phosphorylation, repressed ST“T expression, and overexpression of SOCS . The latter results in an increased negative feedback regulation of the IFN- signal cascade. The defects in the IFN- receptor signaling cascade caused impaired expression of IFN- regulated genes. Previous studies demonstrated that IFN- responsive genes are frequently downregulated in tumor cells due to impaired IRF expression as well as defective transcriptional and posttran‐ scriptional regulation of components involved in the IFN- signal transduction pathway. The loss of the IFN- -mediated upregulation of T“P in a renal cell carcinoma is associated with the lack of IRF and ST“T binding activities as well as J“K , J“K , and ST“T phosphory‐ lation [ ]. This impaired IFN- -mediated phosphorylation could not be restored by J“K and/ or J“K gene transfer. Furthermore, an impaired ST“T -phosphorylation associated with the loss of IFN- -mediated MHC class I upregulation was also reported in melanoma and colorectal carcinoma cells [ ].

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IFN- treatment is able to restore the expression of many genes belonging to the MHC class I “PM [ , ]. “s a consequence, anti-tumor-specific immune responses can be induced, suggesting that IFN- acts as key regulator of immunogenicity [ ]. Its antitumoral activity includes also the induction of apoptosis and inhibition of cell proliferation by ST“T activa‐ tion, which induces expression of cell cycle inhibitor, CDKN “ [ ]. In addition, the IFN- mediated upregulation of MHC class I antigens could be due to DN“ demethylation of MHC class I “PM genes, suggesting that IFN- acts as an epigenetic modifier of “PM components [ ]. Therefore, IFN- is a major player in the regulatory network combating tumor cell proliferation and tumor survival.

. Features of miRNAs miRN“s are small noncoding ~ nucleotide long regulatory RN“s encoded in the human genome, which control the posttranscriptional gene expression by binding to the ′untranslated region UTR of mRN“ of target genes, thereby affecting their stability and/or their translation [ ]. “n individual miRN“ could target numerous cellular mRN“s, while single miRN“ can be regulated by several proteins [ , ]. miRN“s have emerged as key players in the posttranscriptional control of gene expression and based on their predic‐ tion appear to be directly involved in the expression of at least % of all protein-coding genes in mammals [ ]. “ strong relationship between miRN“s and human cancer has been developed during the past years. High throughput analysis allows the comparison of miRN“ expression pattern in normal and tumor tissues demonstrating global changes within the miRN“ expression in different malignancies. Interestingly, the miRN“ genes were frequently located at fragile sites and cancer-associated chromosomal regions. The deregulation of the biogenesis and expres‐ sion of miRN“s is involved in the initiation as well as progression of tumors, metastasis formation, and therapy resistance [ ]. Furthermore, miRN“s can participate in reprogram‐ ming components of the tissue tumor microenvironment TME in order to promote tumor‐ genicity [ ]. In the following sections, miRN“s are described as powerful RN“i inducing regulators of immune modulatory genes involved in escape from immune surveillance. Moreover, this review highlights some miRN“s and their roles in immune escape and discusses these miRN“s as putative targets for immune therapy Figure . . . Antigen processing and presentation machinery and miRNAs Recent studies showed identified miRN“s able to affect the expression of “PM components. Microarray analysis of miRN“- overexpressing nasopharyngeal carcinoma cells demonstrat‐ ed that miRN“- controls the expression of components of the classical MHC class I pathway. miRN“- targets many IFN-induced genes and MHC class I “PM molecules, such as the proteasome subunits PSM” and PSM” , T“P , β -m, HL“-”, HL“-C, and the nonclassical HL“-F and HL“-H antigens [ ]. However, the binding of miRN“- to the ′-UTR of these molecules has not yet been shown. miRN“- is involved in the cellular differentiation [ ] and

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Figure . Scale of miRN“s targeting immune pathways in cancer exhibit an imbalance of tumor-suppressive miRN“s, which occur more frequent than oncogenic miRN“s as per knowledge from today. Oncogenic miRN“ are highly ex‐ pressed in cancer, while tumor-suppressive miRN“s possess a reduced expression.

aberrantly expressed in many cancer types breast cancer[ ], colon cancer [ ], nasopharyng‐ eal carcinoma [ ], and melanoma [ ], suggesting that the decreased miRN“- expression is associated with tumor suppressor activity. In contrast, miRN“- expression is increased in brain cancer [ ] and in Hodgkin’s lymphoma [ ], implying an oncomir potential. Further‐ more, miRN“- has been shown to regulate the proliferation [ , - ] epithelial-mesenchy‐ mal transition EMT , invasion and metastasis [ - ], apoptosis [ ], tumor angiogenesis [ - ], and evasion of immune surveillance in many cancer types [ ]. “lthough the function of miRN“- in the classical MHC class I pathway has still to be characterized in extent, miRN“- -mediated regulation of “PM deficiencies might be at least partially responsible for the T cell-mediated immune escape. ”esides miRN“- , the ER stress-induced miRN“modulates the expression of “PM components and IFN-induced genes as shown by miRN“ arrays. Functional studies revealed that T“P is a direct target of miRN“using overexpression and RN“i knockdown experiments with miRN“ mimic and miRN“ inhibitors. The ER stress-mediated MHC class I-associated antigen presentation decrease might be explained by increased miRN“expression [ ], although the function of miRN“in cancer has not yet been fully analyzed. The inflammation and overexpression of miRN“are associated with the carcinogenesis of lung cancer. “ decrease in the proliferation, invasion, and metastatic potential of lung cancer cells was detected after the overexpression of miRN“. In addition, the proteasome subunit

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PSM” has been identified as a direct target of miRN“using both bioinformatics and dual luciferase reporter assays. This was confirmed by miRN“-overexpressing lung cancer cells, demonstrating a reduced PSM” protein expression. These data suggest that miRN“inhibits the development and metastasis of lung cancer [ ]. There are variations that exist in the ′-UTR of HL“-C, which modulate the miRN“binding capacity and consequently the HL“-C surface expression. miRa has been shown to bind to the HL“-C ′-UTR. Next to cancer, the miRN“a expression is associated with the control of HIV [ - ]. Furthermore, miRN“a is upregulated in Hepatitis ” virus infected cells and has a binding site in the ′-UTR of the HL“-“ gene, which might be a target of miRN“a [ ]. Moreover, viral DN“ or RN“ can encode miRN“s, e.g., miRN“-US - from the human cytomegalovirus, which targets the amino‐ peptidase ER“P , thereby blocking CTL response [ ]. . . Control of HLA-G and MHC-related proteins by miRNAs Recently, a number of HL“-G-specific miRN“s have been identified, which belong to the miRN“family consisting of three members, miRN“a, miRN“b, and miRN“. These miRN“s have been shown to act as tumor suppressors in many tumors, including prostate, ovarian, endometrial, and colorectal cancer [ , , ]. In addition, other miRN“s such as miRN“, miRN“, and miRN“have been identified to inhibit HL“-G expression. The HL“-C-regulating miRN“s are involved in inducing T and/or NK cell responses and have a tumor-suppressive capacity. Moreover, some of the HL“-G-regulated miRN“s are inversely expressed when compared to HL“-G in tumor lesions and are associ‐ ated with disease progression [ ]. The cytotoxicity of NK cells is determined by activating and inactivating signals. The ligands of the activating NK cell receptor NKG D are the major histocompatibility complex class Irelated molecules MIC “ and ” and the human cytomegalovirus UL -binding proteins UL”P [ ]. The expression of MIC“ and MIC” is controlled by several oncogenic miRN“s, like miRN“- b, miRN“- - p, miRN“- a, miRN“- , miRN“- , and miRN“b, which increase the proliferative, invasive, and angiogenic potential of tumors [ , - ] and affect the NK cell cytotoxicity. The tumor-suppressive miRN“c, miRN“a, and miRN“- p showed a reduced expression in cancer and target the ′-UTR of MIC [ , - ]. Furthermore, the expression of UL”P is regulated by many tumor-suppressive miRN“s, e.g., miRN“- a/c, miRN“- p, miRN“c, miRN“- p, and miRN“p, and by the oncogenic miRN“[ , , , ]. . . Control of B family members by miRNA The expression of ” family members is subject to the regulatory control of miRN“s: ” -H could act as a costimulatory molecule, which is expressed on ” cells, T cells, macrophages, and DCs [ ], and acts as a ligand for PDL- . miRN“targets ” -H and inhibits its expression by translational repression [ ]. In this context, Tamura and coworkers identified an associa‐ tion between low expression of ” -H and the escape from immune surveillance indicating

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that ” -H has a potential role in tumorigenesis. ” -H [ ] is a direct target of miRN“- , which inhibits ” -H expression and therefore is involved in cancer immune evasion. ” -H is an immune regulatory molecule, which is often overexpressed in different cancers and associated with metastasis and poor prognosis [ , ]. Its expression could be posttranscrip‐ tionally regulated by miRN“- c. The miRN“- c-mediated downregulation of ” -H expression was found in breast cancer and acts therefore as tumor-suppressive miRN“ [ ]. Furthermore, another ” -H -regulating miRN“, miRN“, has been identified in clear renal cell carcinoma, and its expression is downregulated in this disease [ ]. The coinhibitor ” -H functions as a negative mediator of immune responses. So far, no information exists about the role of miRN“s in the regulation of ” -H expression. In addition, miRN“s binding to the ′UTR of ” -H have not yet been identified. . . Control of the IFN-γ pathway by miRNAs The regulation of IFN- signaling includes negative as well as positive regulators, such as kinases and phosphatases as well as transcription factors. “ main regulatory role of IFNsignaling is attributed to miRN“s, affecting genes involved in proliferation, differentiation, signal transduction, immune response, and carcinogenesis [ , ]. IFN- can modulate the expression levels of miRN“s and to regulate miRN“s at the level of miRN“ biogenesis [ ], whereas miRN“s can inhibit IFN expression directly or indirectly. In addition, studies have confirmed that miRN“s are able to target components of the IFNsignaling pathway and components of the J“K/ST“T-pathway can regulate miRN“s simul‐ taneously. The latter has been described by controlling miRN“ expression via transcription factors, such as c-myc, the hypoxia-induced factor HIF , and ST“Ts [ ]. The contribution and regulatory role of miRN“s in IFN- signaling is still under investigation and an emerging research area. Here, to highlight the regulatory function of miRN“s in the IFN- signaling pathway, the functional role of miRN“has been described in more detail. miRN“proceeding from the non-protein-coding transcript of the BIC gene RN“ is required for the normal function of ”, T, and DC [ . ], and its expression is increased during ” cell, T cell, macrophage, and DC activation [ ]. miRN“has been shown to regulate IFN- production in NK cells, while its disruption or knockdown suppressed IFN- induction of NK cells [ ]. “dditional studies reported that miRN“also downregulates IFN- -R expression [ ]. Furthermore, ST“T upregulates miRN“, which in turn downregulates SOCS , a negative inhibitor of J“K [ ]. These findings illustrate that a single miRN“ can regulate several target mRN“s of the IFN cascade and miRN“s can be regulated by a number of targets. miRN“ regulating components of IFN- signaling pathway mainly act as tumor-suppressive miRN“s. “n antiproliferative effect of miRN“, which affects J“K protein expression, has been recently described [ , ]. Furthermore, miRN“a expression was downregu‐ lated in gastric cancer cell lines, while its overexpression results in inhibition of gastric cancer cell proliferation by targeting J“K [ ]. Thus, miRN“a may function as tumor suppres‐ sor by regulating J“K expression in gastric cancer cells [ ]. Several studies confirmed other miRN“s targeting J“K , including miRN“a, which is known to inhibit cell growth and

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promote apoptosis of pancreatic cancer cells by regulating J“K /ST“T signaling pathway [ , ], as well as miRN“, which promotes apoptosis of breast cancer cells by targeting J“K [ ]. Similar results were found for ST“T and miRN“[ ]. miRN“is reported to be downregulated in several cancers [ , ] and has ST“T as direct target [ ]. Moreover, ST“T is able to upregulate miRN“- family members in melanoma cells, which inhibit melanoma cell proliferation by downregulating CDK [ ]. Further studies confirmed that miRN“and miRN“are equally involved in IFNsignaling, but their role in cancer cells is still controversially discussed. ”oth miRN“and miRN“could exert oncogenic or tumor-suppressive activity. In hepatocellular carcinoma, acute myeloid leukemia “ML [ ] and gastric mucosa-associated lymphoid tissue lympho‐ ma miRN“expression is repressed [ ], while an upregulation of miRN“has been recently described in T-cell acute lymphocytic leukemia T-“LL [ ]. In this context, Moles and coworkers [ ] demonstrated that both miRN“and miRN“target ST“T ′UTR and reduce ST“T expression, which in turn results in reduced expression of IFN- regulated genes. The expression of miRN“is upregulated in CD + ” cells from chronic lymphocytic leukemia [ , ], while in chronic myeloid leukemia [ , ], “LL [ ] and mantel cell carcinoma miRN“is downregulated. Moreover, miRN“is upregulated in adult T-cell leukemia/lymphoma cells. This discrepant expression pattern of miRN“and miRN“suggests that both miRN“s could act as oncogenic as well as tumor suppres‐ sor miRN“s, which are dependent on the cellular context. . . Role of miRNAs in immune cell function Cancer cells upregulate and downregulate different miRN“s in immune cells to limit the antitumor response. It is well known that tumor cells reprogram the myeloid compartment to evade the immune system and promote tumorigenesis. This might be partially mediated by alterations in the miRN“ expression pattern. The miRN“modulates the immune response mediated by T cells, NK cells, ” cells, and antigen presenting cells, such as macrophages and DC [ ]. Furthermore, miRN“expression has been found to be downregulated in T“Ms [ ], but also in hepatocellular carcinoma. The restoration of miRN“in macrophages leads to enhanced T-cell function by targeting the suppressor of cytokine signaling. Other miRN“s, like miRN“- p, miRN“b, and miRN“- a- p, are often downregulated in T“Ms, thereby limiting the tumor infiltration of macrophages and reducing the therapeutic effect of adoptive transfer. The restoration of miRN“b in macrophages enhances antitu‐ mor response by targeting the IFN-regulatory factor , which promotes the M macrophage phenotype [ ]. Recently, miRN“s have been identified to play a role in MDSC that regulate immune sup‐ pression within the tumor microenvironment. miRN“ array analysis identified a number of deregulated miRN“s, e.g., miRN“, which suppresses the antitumor CD + T-cell responses due to response to TGF-β. miRN“targets PTEN in MDSC, which is responsible for the enhanced immune suppression of CD + T cells [ ]. Furthermore, a number of other miRN“s are downregulated in MDSC [ ], which promote the differentiation of myeloid cells and regulate immune-suppressive signaling pathways.

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In addition, miRN“s have been demonstrated in tumor-infiltrating lymphocytes. The sup‐ pression of T-cell activity is due to different mechanisms, including the dysregulation of miRN“ expression. In CD + T cells from tumor bearing mice and tumor patients, the expres‐ sion of miRN“- - family members was reduced, while T cells derived from miRN“- transgenic mice demonstrated a superior type phenotype [ ]. Furthermore, the expression of miRN“was shown to promote antitumor responses. miRN“in combination with miRN“a could upregulate the IFN- production of T cells. Furthermore, cancer cells could regulate miRN“s in T cells in order to modulate antitumor T-cell responses. In order to escape immune surveillance, cancer cells alter the expression of transcription factors, surface recep‐ tors, soluble chemokines/cytokines, and miRN“s to support the immune system. The down‐ regulation of miRN“increases Treg infiltration and reduces cytokines production through an altered expression of ST“T , which represents a target of miRN“. In contrast, tumorsecreted miRN“induces Treg. Regarding NK cells, the TGF-β-inducible miRN“affects NK cell activity [ ]. Thus, the regulation of miRN“s within the cancer cell alters the TME through manipulation.

. Conclusion and future perspectives Taken together, during the past years, the posttranscriptional control of gene expression by miRN“s has gained relevance as key regulator in a wide variety of physiological and pathophy‐ siological processes due to the role of miRN“-mediated RN“i not only in differentiation, proliferation, apoptosis, immune responses but also in viral and bacterial infections as well as neoplastic transformation Table . “ deregulated expression of miRN“s has been often found in tumors of distinct origin, which have been classified into oncogenic or tumor-suppressive miRN“s known to play an essential role in cancer initiation and progression. Therefore, these miRN“s could act as potential biomarkers and therapeutic targets in cancer. In silico predic‐ tion analysis further proposed that many miRN“s could target different immune modulato‐ ry molecules expressed either on tumor cells or on different immune cell subpopulations. “s summarized, an emerging relevance of miRN“s in mounting the tumor immune escape by altering the communication between cancer cells, immune cells, and other components of the TME has been demonstrated. This leads to another level of complexity due to the involve‐ ment of miRN“s in the interaction between cancer cells and immune cells. These miRN“s might not only provide new insights into tumor growth and progression as well as antitumoral immune responses but also represent promising therapeutic targets for immune therapy. To date, many cancer-deregulated miRN“s have been identified in particular in cancer cells and also in components of the TM“. However, their role in modulating the antitumor immune responses has not yet been characterized in detail. “lthough the majority of the miRN“ alterations detected are dedicated to cancer cells, there is already evidence that miRN“s of infiltrating immune cells also particularly influence tumorgenicity. The identification of further im-miRN“s as well as their functional characterization might lead to a plethora of novel candidate biomarkers for monitoring of immune responses, which might be also potentially used for targeted RN“i therapy.

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Table . Identified miRN“s involved in the tumor immune escape and their tumor–associated function. Controversially discussed miRN“s are found as tumor suppressors in some cancer types, while exhibiting oncogenic properties in other cancer types. n.d., no data.

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. Abbreviations “PC, antigen presenting cell “PM, antigen processing machinery β -m, β -microglobulin CDKN, cyclin-dependent kinase inhibitor CTL, cytotoxic T lymphocyte CXCL, chemokine CXC motif ligand DC, dendritic cell G“S, IFN- -activated sequence HC, heavy chain HL“, human leukocyte antigen IFN, interferon IL, interleukin IRF, interferon regulatory factor ISRE, IFN-stimulated response element im-miRN“, immune modulatory miRN“ J“K, janus kinase LMP, low molecular mass polypeptide M“PK, M“P kinase MDSC, myeloid-derived suppressor cell MHC, major histocompatibility complex MIC, major histocompatibility complex class I-related molecule miRN“, microRN“ NK, natural killer cell PD, programmed death PDL, PD ligand PLC, peptide loading complex ST“T, signal transducer and activator of transcription SOCS, suppressor of cytokine signaling T“F, tumor-associated fibroblast T“M, tumor-associated macrophage T“P, transporter associated with antigen processing TGF, transforming growth factor TLR, toll-like receptor TME, tumor microenvironment Treg, regulatory T cell UL”P, human cytomegalovirus UL -binding protein UTR, untrans‐ lated region and VEGF, vascular endothelial growth factor.

Acknowledgements We would like to thank Sylvi Magdeburg for excellent secretarial help. This work was supported by a grant from the DFG SE / - , GRK , GIF I - , and Cancer Research “id .

Author details ”arbara Seliger*, “nne Meinhardt and Doerte Falke *“ddress all correspondence to: [email protected] Institute of Medical Immunology, Martin-Luther University Halle-Wittenberg, Germany

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] Wang L, Li L, Guo R, Li X, Lu Y, Guan X, Gitau SC, Wang L, Xu C, Yang ”, Shan H. miRpromotes breast cancer cell apoptosis by targeting Janus kinase . Cell Phys‐ iol ”iochem. b : . DOI: . / .

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] Gregersen LH, Jacobsen “”, Frankel L”, Wen J, Krogh “, Lund “H. MicroRN“targets YES and ST“T in colon cancer cells. PLoS One. :e . DOI: . /journal.pone. .

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] “kao Y, Nakagawa Y, Naoe T. MicroRN“”iol. : . DOI: . /dna. .

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] Ozen M, Creighton CJ, Ozdemir M, Ittmann M. Widespread deregulation of micro‐ RN“ expression in human prostate cancer. Oncogene. : . DOI: . /sj.onc. .

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] Schmitt MJ, Philippidou D, Reinsbach SE, Margue C, Wienecke-”aldacchino “, Na‐ shan D, ”ehrmann I, Kreis S. Interferon-gamma-induced activation of signal trans‐ ducer and activator of transcription ST“T up-regulates the tumor suppressing microRN“- family in melanoma cells. Cell Commun Signal. : . DOI: . / X- - .

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] Mi S, Lu J, Sun M, Li Z, Zhang H, Neilly M”, Wang Y, Qian Z, Jin J, Zhang Y, ”oh‐ lander SK, Le ”eau MM, Larson R“, Golub TR, Rowley JD, Chen J. MicroRN“ ex‐ pression signatures accurately discriminate acute lymphoblastic leukemia from acute myeloid leukemia. Proc Natl “cad Sci U S “. : . DOI: . /pnas. .

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] Liu TY, Chen SU, Kuo SH, Cheng “L, Lin CW. E “-positive gastric M“LT lympho‐ ma has weaker plasmacytoid infiltrates and stronger expression of the memory ”-

a inhibits pancreatic cancer by directly tar‐ : . DOI: . /or. . .

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] Kumar V, Palermo R, Talora C, Campese “F, Checquolo S, ”ellavia D, Tottone L, Testa G, Miele E, Indraccolo S, “madori “, Ferretti E, Gulino “, Vacca “, Screpanti I. Notch and NF-k” signaling pathways regulate miR/F”XW axis in T-cell acute lymphoblastic leukemia. Leukemia. : . DOI: . /leu. . .

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] Moles R, ”ellon M, Nicot C. ST“T : “ novel target of miRand miRis in‐ volved in the proliferation of HTLV-I-transformed and “TL cells. Neoplasia. : . DOI: . /j.neo. . . .

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] Mraz M, Chen L, Rassenti LZ, Ghia EM, Li H, Jepsen K, Smith EN, Messer K, Frazer K“, Kipps TJ. miRinfluences ”-cell receptor signaling in chronic lymphocytic leukemia by regulating expression of G“” and FOXP . ”lood. : . DOI: . /blood- .

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] Papakonstantinou N, Ntoufa S, Chartomatsidou E, Papadopoulos G, Hatzigeorgiou “, “nagnostopoulos “, Chlichlia K, Ghia P, Muzio M, ”elessi C, Stamatopoulos K. Differential microRN“ profiles and their functional implications in different immu‐ nogenetic subsets of chronic lymphocytic leukemia. Mol Med. : . DOI: . /molmed. . .

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] Machova Polakova K, Lopotova T, Klamova H, ”urda P, Trneny M, Stopka T, Mor‐ avcova J. Expression patterns of microRN“s associated with CML phases and their disease related targets. Mol Cancer. : . DOI: . / - - .

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myeloid-derived suppressor cells via targeting of PTEN. J Immunol. . DOI: . /jimmunol. .

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Chapter 4

Long Noncoding RNAs are Frontier Breakthrough of RNA World and RNAi-based Gene Regulation Utpal Bhadra, Debabani Roy Chowdhury, Tanmoy Mondal and Manika Pal Bhadra Additional information is available at the end of the chapter http://dx.doi.org/10.5772/61975

Abstract General complexities in versatile animals are not always proportional to their genome size. “ notable example is that the salamander genome size is -fold larger than that of human, which mostly contains unfolded junk DN“. “ vast portion of this non-proteincoding unfolded DN“ undergoes transcriptional regulation and produces a large num‐ ber of long noncoding RN“s lncRN“s . LncRN“s play key roles in gene expression and therapies of different human diseases. Recently, novel lncRN“s and their function on the silencing or activation of a particular gene s are regularly being discovered. “nother im‐ portant component of gene regulation is high packing of chromatin, which is composed of mainly repetitive sequences with negligible coding potential. In particular, an epige‐ netic marker determines the state of the gene associated with it, whether the gene will be expressed or silenced. Here, we elaborately discuss the biogenesis pathway of lncRN“s as well as their mechanism of action and role in gene silencing and regulation, including RN“ interference. Moreover, several lncRN“s are the common precursors of small regu‐ latory RN“s. It is thus becoming increasingly clear that lncRN“s can function via numer‐ ous paradigms as key regulatory molecules in different organisms. Keywords: Transcriptional silencing, long noncoding RN“, cancer, neurological disor‐ der, Drosophila

. Introduction Since the earliest days of molecular biology, RN“-mediated gene regulation was known to the researchers, and it was first suggested that noncoding RN“ ncRN“ might have a role in gene regulation by interacting with promoters [ , ]. “fter more than four decades of research, the discovery of RN“ interference RN“i has revolutionized our perception of the mechanism of

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gene regulation, organization of chromosomes, and epigenetic regulations. Important clues to ncRN“ regulatory mechanisms came from homology-dependent gene silencing in plants, which can be initiated by transgenes and recombinant viruses [ ]. Studies on the nematode Caenorhabditis elegans, the fruit fly Drosophila melanogaster [ ], fungi mainly yeast, mammalian cells, and plants revealed transcriptional silencing mechanisms involving RN“i, chromatin, and its various modifications [ ]. RN“i operates mainly posttranscriptionally however, its components are associated with transcriptional gene silencing and heterochromatin forma‐ tion, too [ ]. Recent findings have made it clear that transcriptional gene silencing TGS , posttranscrip‐ tional gene silencing PTGS , and chromatin modifications are utilized by eukaryotic cells to bring about endogenous gene regulation, chromosome organization, and nuclear clustering. The RN“ interference mechanisms mainly target the transposable elements, which are abundant and perhaps a defining component of heterochromatin. The role of ncRN“ in dosage compensation, inactivation of X chromosome, genomic imprinting, polycomb silencing, and blocking of interactions between enhancers and promoters by chromatin insulators is well proven. “lthough the studies strongly point towards the involvement of RN“i, its role has not been demonstrated directly [ ]. The non-protein-coding transcripts longer than nucleotides are known as long noncoding RN“s to differentiate superficially this class of ncRN“s from microRN“s, short interfering RN“s, piwi-interacting RN“s, small nucleolar RN“s, etc [ ]. LncRN“s have emerged as important regulators of cell physiology and pathology. Different studies have come up with an increasing number of lncRN“s showing tissue-specific expression however, the exact mechanism of action of only a few lncRN“s has been elucidated in vivo [ – ]. The biological functions and mechanisms of action of the majority of lncRN“s still remain unknown. LncRN“s can interact with a wide range of molecules and can form RN“-RN“, RN“-DN“, or RN“-protein complexes through specific RN“ functional domains [ ], resulting in extensive functional diversities. Recent research focuses on lncRN“s and divulges the association of lncRN“s with epigenetic machinery to control chromatin structure, nuclear clustering, and gene expression. The studies reveal that lncRN“s may act together with many histone- and DN“-modifying enzymes to modify the histones or DN“. In addition, a recent discovery of a cardioprotective lncRN“ showed a targeting mechanism through “TPdependent chromatin remodeling factors [ ], indicating an extensive role of lncRN“s in chromatin structure and regulation. The mechanisms of how lncRN“s control chromatin by covalent modifications are extensively reviewed in the literature [ – ]. The study of lncRN“s has taken the center stage for the researchers working with epigenetic regulations, and there is a report of a new lncRN“ regulating a disease, or transcriptome studies come up with a new class of noncoding RN“, or we are introduced to hitherto unknown mechanisms by which an lncRN“ regulates a particular gene almost on a weekly basis. These are all possible due to the introduction of many advanced, high-throughput genomic technologies such as microarrays and next-generation sequencing NGS . There are a huge number of reported lncRN“s that are not derived from protein-coding genes, and in spite of this vast number of reports on lncRN“, we have just started getting a clear picture

Long Noncoding RNAs are Frontier Breakthrough of RNA World and RNAi-based Gene Regulation http://dx.doi.org/10.5772/61975

about how lncRN“s function, how many different types of lncRN“s exist, and how many of the reported lncRN“s are biologically important.

. The C-value enigma and junk DNA It has long been known that developmental complexity or size of an animal does not correspond with C-value or the amount of DN“ in the haploid genome [ – ]. The lower animal in the evolution ladder, salamander, has a genome size times larger than that of humans [ ], and this discrepancy is known as the C-value paradox [ ]. Since the introns were discovered, we started to presume that the C-value paradox was now solved [ ]. We are almost sure that humans have about , – , protein-coding genes unlike the overestimates of , – , from the initial days of the Human Genome Project [ ]. The remaining huge amount of noncoding DN“ was termed as junk DN“ [ , ] due to the presence of transposons, pseudogenes, and simple repeats, which occupies about – % of the human genome [ ]. C-value enigma poses a discrepancy in genome size and number of protein-coding genes. Phylogenetically close genera may vary in C-value by around four- to five fold [ ]. In spite of their junk status, scientists were always curious to study them and even realized that being junk doesn’t mean it is entirely useless [ ]. It was hypothesized that the junk DN“ might be useful in chromosomal pairing, genome integrity, gene regulation, mRN“ processing, and serving as a reservoir for evolutionary innovation. We are now pleasantly surprised at their foresight. In the s, it was already thought that noncoding RN“ products, such as rRN“s, tRN“s do not make up the whole transcribed genome. The scale of pervasive transcription, however, was not fully appreciated until the late s and early s. “fter the arrival of whole-genome technologies, from microarray hybridiza‐ tion and deep sequencing analysis techniques, it was recently shown that – % of our genome is transcribed at some point during embryogenesis [ ]. Some recently identified transcripts may be present at as low as . copies per cell [ ]. “nother concern is that tiling microarrays can come up with false positives, low dynamic range, resolution, and low concordance between studies [ ]. The existence of noncoding transcription in intergenic regions is evident from correlations with chromatin signatures, such as DNase hypersensi‐ tivity, and histone modifications such as H K ac, H K me , and H K me [ ]. “lthough these studies report novel and conserved lncRN“s, that is not enough to explain the function of – % of the genome and biological functionality of the ncRN“s. In , ”ritten and Davidson presented a model for regulation of gene expression in eukaryotic cells where ncRN“s have important roles as regulatory intermediaries to convey signals from sensory to receptor elements [ ]. Some of the first examples of gene-specific regulatory roles of lncRN“s were revealed with the discovery of lncRN“s involved in epigenetic regulation, such as H [ ] and X-inactive specific transcript Xist [ , ].

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. Stand-alone lncRNAs These lncRN“s are located as separate units and do not overlap protein-coding genes. Some of these are known as lincRN“s for large intergenic or intervening noncoding RN“s lincRN“s [ ]. Many of the lincRN“s were identified through chromatin signatures for actively transcribed genes H K me at the promoter and H K me along the transcribed length . Many of the characterized lncRN“s are transcribed by RN“ Pol II, polyadenylated, and spliced and have an average length of kb.

. Natural antisense transcripts In this study, transcription occurs in the antisense strand of annotated transcription units about % of sense transcripts have reported antisense counterparts [ ]. The overlap between these sense/antisense pairs can be a complete sequence, but natural antisense transcripts are mostly found to be enriched around the ′ promoter or ′ terminator ends of the sense transcript. The most extensively studied example of sense/antisense pairing is Xist/Tsix lncRN“ antisense to Xist , with two RN“s that control X chromosome inactivation [ ]. In addition, many imprinted regions contain coding/noncoding sense/antisense pairs, such as Kcnq potassium channel, voltage-gated KQT-like subfamily Q, member /Kcnq ot Kcnq overlapping transcript [ ] and Igf r insulin-like growth factor receptor /“ir antisense Igf r RN“ [ ]. These pairs are generally less spliced or polyadenylated when compared to mRN“s or stand-alone lncRN“s.

. Long intronic ncRNAs Introns have long been known to contain small ncRN“s such as small nucleolar RN“s snoRN“s and microRN“s miRN“s . However, by large-scale transcriptomic or computa‐ tional analyses, many long transcripts have been reported to be encoded within the introns of known genes [ ]. “lthough they have differential expression patterns and respond to the environmental stimuli differently, only a few have been extensively studied to date. One such example is cold-assisted intronic noncoding RN“ COLD“IR that has been implicated in plant vernalization, which is located in the first intron of the flowering repressor locus FLC [ ].

. Identification of long noncoding RNAs LncRN“s are identified by transcripts that map to genomic regions outside the boundaries of protein-coding genes. It is difficult to ascertain the function of a transcript that overlaps a protein-coding gene using targeted knockout or knockdown approaches. Thus, most experi‐ mental investigations of lncRN“s have been focused on those that are located in intron

Long Noncoding RNAs are Frontier Breakthrough of RNA World and RNAi-based Gene Regulation http://dx.doi.org/10.5772/61975

sequences. It is also very difficult to ascertain whether an lncRN“ locus is entirely intergenic because lncRN“ transcripts are often incomplete and they can originate from a protein-coding gene’s promoter or enhancer on either strand [ ]. Tiling microarray technique is often useful to detect intergenic transcripts [ ]. However, controversial results were found here and these experiments can be ruled out [ ]. Early lncRN“ collections relied primarily on sequenced cDN“ and EST clones [ ]. More recently, RN“-Seq has come up with a number of lncRN“s derived from whole transcriptome sequencing. RN“-Seq generates millions of – nt sequences read in parallel, and it has been confirmed that a large chunk of intergenic sequences are transcribed into lncRN“s [ ]. The high-throughput and impartial nature of this technique is being utilized for the detailed assessment of the contribution of lncRN“s to a variety of tissue and/or species under different conditions. To accurately distinguish noncoding from coding transcripts, sophisticated approaches have been developed. For example, the Coding Potential Calculator [ ] takes into account six features of a transcript, including the proportion of the transcript enclosed by the candidate peptideencoding region, and the sequence similarity to known proteins. “n evolutionary approach, followed in phyloCSF, predicts ncRN“s when their sequence differences among species do not show preference as to whether they disrupt or not putatively encode peptides [ ]. Experimentally determined transcripts always are relied on more than predicted ones. The availability of large proteomic databases can be utilized to investigate whether a specific RN“ molecule is translated into a protein. In vitro translation assays have been used, too, but they do not necessarily reflect in vivo biology. “ true lncRN“ should not bind with translation machinery, and this approach is also adopted in the identification of candidate lncRN“. However, a study has reported that % of a set of putative lncRN“s are ribosome associated [ ], leaving in doubt whether this test is accurate in separating coding from noncoding transcripts. To assign an lncRN“, an experimental determination of the function of a transcript will be necessary. Nevertheless, some transcripts possess both RN“- and coding-sequencedependent functions [ ] and demarcating them will be difficult. “ computational or experi‐ mental method has not yet been developed that discriminates accurately between coding and noncoding transcripts. For the time being, we can rely on in silico screens for the protein-coding potential of putative lncRN“s but be aware that these will contain false-positive predictions, too, especially for genes that encode short polypeptides. “lthough many genomes contain a substantial number of lncRN“ loci, we still do not know the proportion and number of these that are biologically functional. ”ecause the functional mechanisms of most noncoding transcripts or transcript regions are unknown, it is difficult to design point mutation or deletion experiments and their results are difficult to interpret. Even RN“i techniques are not being helpful to assign the functionality of the ncRN“s.

. Mechanisms of action We do not know yet the mechanistic detail of the enormous number of reported lncRN“s. However, a few that have been thoroughly studied provide clues regarding how lncRN“s

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might carry out gene regulation Figure . In addition, many lncRN“s blur the line of different categories and employ several different mechanisms. The discovery of new lncRN“s and more thorough characterization of those already known will reveal additional modes of action. It has been found that a major role of lncRN“ is to recruit regulatory proteins for the regulation of chromatin states [ ]. This kind of lncRN“s may act in cis, on adjacent or nearby genes, or they might act in trans, regulating genes located in distant domains or chromosomes. Polycomb repressive complex PRC interacts with a large number of lncRN“s [ – ]. The Drosophi‐ la polycomb proteins, first discovered as homeotic gene, express during development [ , ]. These include enhance of zeste homolog Ezh , catalytic subunit in PRC , which is a key H K methyltransferase, and the Pc/Chromobox Cbx family proteins in PRC , chromodo‐ main-containing proteins that can bind trimethylated H K [ , ]. Observed interactions of polycomb proteins with lncRN“s suggest that polycomb recruitment is RN“ directed in mammals. HOX transcript antisense RN“ HOT“IR in the homeobox HOX C cluster is reported to repress transcription of HOXD in trans through interaction with PRC [ ]. Xist RN“-containing repeat “ Rep“ has been found to recruit PRC [ ]. Rep“ targets PRC to the Xist promoter resulting in Xist up-regulation. The interesting fact is that Rep“/Xist interaction with PRC may be blocked by the antisense Tsix transcript, also interacting with PRC and competitively inhibiting the painting of Xist on inactive X chromosome [ ]. Other epigenetic complexes interact with lncRN“s as well, such as the H K methyltransferase G a interacting with the imprinted lncRN“ “ir [ ]. Kcnq ot has been hypothesized to recruit both PRC and G a to the promoter of Kcnq [ ] acting as a scaffold. On the other hand, antisense ncRN“ in the INK locus “NRIL , associated with p /INK inhibitors of CDK family ”-p /INK “-p /“RF tumor suppressor gene cluster, interacts with both the PRC component Cbx and the PRC component Suz [ , ]. HOT“IR also interacts with the lysine-specific demethylase LSD /corepressor protein of LSD CoREST /repressor for element -silencing transcription factor REST complex in addition to PRC to prevent gene activation [ ]. LncRN“s can also act by recruiting factors involved in gene activation. Such factors from the HOX“ homeotic gene “ cluster , two lncRN“s, Mistral Mira , and HOX“ transcript at the distal tip HOTTIP have been involved in recruiting the mixed lineage leukemia MLL complex in cis regulation [ , ]. “n H K trimethylase, myeloid/lymphoid or mixed-lineage leukemia MLL , is a member of the Trithorax group of developmentally important gene-activating proteins in flies [ ]. Using C or chromosome conformation capture technique, it was found that multiple loci, which are kb apart in the HOX“ cluster, are in close physical proximity, enabling MLL to regulate their expression. Other than histone modifications, lncRN“s also impact epigenetic regulation by modulating DN“ methylation at CpG dinucleotides, which has an important role in the stable repression of genes [ ]. During embryogenesis, methylation markers are first to be found on previously unmethylated DN“ by the DN“ cytosine- - -methyltransferase α Dnmt a and β Dnmt b and later maintained through DN“ replication by Dnmt . Tsix might be converted to Xist by utilizing Dnmt a activity to methylate and finally silence the Xist promoter [ , ]. In the same way, Kcnq ot may recruit Dnmt [ ].

Long Noncoding RNAs are Frontier Breakthrough of RNA World and RNAi-based Gene Regulation http://dx.doi.org/10.5772/61975

Figure . Mechanisms of lncRN“ function modified from Kung et al. [

].

LncRN“-directed methylation has also been implicated in the regulation of rDN“. Ribosomal DN“ exists in the genome as tandem repeat units [ ]. Each unit encodes a polycistronic transcript consisting various rRN“s, and each unit is separated by intergenic spacers IGSs transcribed by RN“ Pol I [ ]. Recently, it was reported that IGS transcripts undergo proc‐ essing into - to -nt fragments called promoter p RN“s, which act as scaffolds to recruit poly “DP ribose -polymerase- P“RP [ ], the “TP-dependent nucleolar chromatin remodeling complex NoRC [ ], and Dnmt b [ ]. “ conserved hairpin structure is formed by pRN“ that binds both P“RP and the TIP subunit of NoRC, leading to TIP conformation change resulting in the recruitment of NoRC to the nucleolus, where rDN“ is located [ , ]. The interesting fact is that the recruitment of Dnmt b by pRN“ is dependent on DN“:RN“ triplexing, possibly via Hoogsteen base pairing, between the ′ end of pRN“ and the rDN“ promoter [ ]. The DN“:RN“ triplex formation might be a general mechanism by which lncRN“s recruit trans factors to specific DN“ loci. LncRN“s are intrinsically bound to chromatin during transcription and transcribed from a single locus in the genome, so they have a direct allele- and locus-specific control in cis unlike transcription factors. The length of lncRN“s is also suitable to reach out and capture epigenetic marks. This cis-acting mechanism

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resembles transcriptional gene silencing seen in the yeast Schizosaccharomyces pombe in assembling centromeric heterochromatin [ , ]. The nucleus is always in the dynamic state and is the center for most of the essential functions of an organism [ ]. Recent studies indicate that lncRN“s are the key regulators of nuclear compartments. The structure and function of several nuclear bodies seem to be controlled by RN“. One example is nuclear-enriched abundant transcript NE“T that maintains the stability of paraspeckles, which participate in the nuclear retention of mRN“s after adenosineto-inosine hyperediting [ , ]. NE“T interacts with paraspeckle proteins, such as p / NONO and PSP [ – ] and recruits these proteins to form paraspeckles. This is an active process where continuous transcription of NE“T is required [ ]. The related molecules, NE“T or metastasis-associated lung adenocarcinoma transcript M“L“T , are involved in the localization of serine/arginine SR splicing factors to nuclear speckles where they can be stored and later modified by phosphorylation [ ]. M“L“T directs these splicing factors to sites of transcription, ultimately controlling the alternative splicing of certain mRN“ precursors [ ]. M“L“T interacts with the PRC subunit Cbx /Pc and participates in the transportation of genes between nuclear compartments for silencing and activation. Extracel‐ lular growth signals help unmethylated Cbx to bind M“L“T and localize its target genes, along with Lysine K -specific demethylase “ LSD to interchromatin granules that usually cluster around nuclear speckles. However, Cbx gets methylated in the absence of extracellular signal and instead binds another lncRN“ TUG , then binds with Ezh , and translocates to silencing compartments called polycomb bodies [ ]. “lthough these recent observations have started to open up an avenue to understand lncRN“ and their mechanisms of action, we are still way behind. The function of an overwhelming number of lncRN“s that are being discovered almost daily is unknown until now.

. Epigenetic regulation The two most abundant modes of action of lncRN“s are the modulation of chromatin by recruiting histone proteins and transcription factors within specific chromatin-modifying complexes. “ very good example of recruitment of specific histones is X chromosome inacti‐ vation XCI , which is caused by Xist as described in the earlier section [ ]. “ similar event is genomic imprinting, where genes are expressed from the allele of only one parent. One of the first and best studied lncRN“s is H , which is mutually imprinted with insulin-like growth factor Igf . This lncRN“ is highly expressed, but its deletion has no phenotypic outcome, and it is anticipated to function as a microRN“ precursor [ ]. Other lncRN“s e.g., “ir, Kcnq ot , and HOT“IR show modulatory activities both in cis or in trans and regulating gene expression through partnering with chromatin-modifying complexes [ , ]. Specifical‐ ly, HOT“IR is a trans-acting lncRN“ that serves as a scaffold for two histone modification complexes: it binds both to PRC and to LSD [ ]. In the Arabidopsis plant, it was found that different environmental conditions are able to induce the transcription of related N“Ts i.e., COOL“IR that eventually silence a flower repressor locus, flowering locus c FLC [ ]. Recently, it was discovered that lncRN“, namely COLD“IR, bearing minor differences from

Long Noncoding RNAs are Frontier Breakthrough of RNA World and RNAi-based Gene Regulation http://dx.doi.org/10.5772/61975

COOL“IR transcribed in the sense direction relative to FLC mRN“ transcription , interacts on its own with PRC and targets it to FLC [ ]. Other trans-acting lncRN“s have different functions, some of which remain incompletely defined. There are several poorly defined trans-acting lncRN“s, such as the p -associated ncRN“ DN“ damage activated P“ND“ , which is induced upon DN“ damage in a p -dependent manner and it controls the expression of proapoptotic genes [ ].

. Transcriptional regulation The discovery and characterization of promoter-associated RN“s opened up a new under‐ standing on how genes are regulated during transcription. These RN“s are localized within the promoter and consist of various sizes of RN“ molecules [ ]. The long ones are found at a single-gene level and are associated with the modification of DN“ methylation and deme‐ thylation patterns [ ] as mentioned earlier. Interestingly, long antisense pRN“s generally form double-stranded molecules that are processed into endo-siRN“s, and since they have sequence complementarity with the promoter, they induce transcriptional gene silencing [ , – ] or activation [ – ]. LncRN“s sometimes affect transcription by acting as coregulators or by regulating the association and activity of coregulators. One example is embryonic ventral forebrain- Evfthat functions as a coactivator for the homeobox transcription factor distal-less homeobox Dlx [ ].

. Posttranscriptional regulation lncRN“s not only have a role in transcription but also they function in splicing, mRN“ stability, and translation. “ntisense lncRN“ sometimes bind to the sense RN“, conceal the splice sites, and thereby modify the balance between splice variants. “ntisense transcript RevErb“α modifies the splicing of thyroid hormone receptor alpha genes TRα TRα and TRα mRN“s [ ]. The terminal differentiation-induced ncRN“ TINCR associates with Staufen the complex between TINCR-N“, which is a differentiation factor [ ].

but not with

LncRN“s have also been implicated in translational regulation. “n example is the antisense for PU mRN“. Its translation is inhibited by an antisense polyadenylated lncRN“ with a halflife longer than the original transcript [ ]. “nother example is the lncRN“ Uchl , which is controlled by mammalian target of rapamycin mTOR pathway, shuttles from the nucleus to the cytoplasm, and controls the translation of the ubiquitin carboxy-terminal hydrolase L UCHL mRN“ by promoting its association with polysomes [ ].

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Figure . Posttranscriptional gene silencing by lncRN“ and miRN“ adapted from Gomes et al. [

].

. Role of lncRNAs in cancer and other human diseases The genome-wide association studies identify several cancer risk loci outside of proteincoding regions. Of single-nucleotide polymorphisms currently linked to cancer, only . % modify the amino acid sequence of the protein, and most of the loci are located in the introns % or intergenic regions % [ ]. These facts and the observations that miRN“ and lncRN“s are involved in differentiation and development point towards the fact that alterations in their expression profiles could be correlated with cancer develop‐ ment. Reports suggested that lncRN“s have tissue-specific expression and is found to be deregulated in distinct types of cancers. For example, overexpression of miRwas reported in hematopoietic, breast, lung, and colon cancers [ ], whereas miRis overexpressed in glioblastoma [ ]. In addition, lymphoproliferative disorders were found in transgenic mice overexpressing miR- - [ ]. Incidences of lung, colon, and gastric cancers were found to be correlated with the overexpression of miR- - cluster [ ]. LncRN“s have been associated with cancer development likewise. The lncRN“ M“L“T is up-regulated in several cancer types, resulting in an increase in cell proliferation and migration in lung and colorectal cancer cells [ ]. The role of M“L“T in controlling alternative splicing of pre-mRN“s [ ] can be deduced from this. “ more recent study indicates that M“L“T may also participate in the regulation of gene expression by a mechanism other than alternative splicing in lung metastasis [ ]. Other studies have shown that miRN“ and lncRN“s both can function as tumor suppressor genes or oncogenes. The tumor suppressor gene p regulates the three gene members of the

Long Noncoding RNAs are Frontier Breakthrough of RNA World and RNAi-based Gene Regulation http://dx.doi.org/10.5772/61975

Figure . Relationship among various noncoding RN“s and different disorders caused by them adapted from Gomes et al. [ ].

miR- family. Curiously, the microRN“miRactivation resembles p activity, such as the induction of cell cycle arrest and promotion of apoptosis, and p -mediated apoptosis becomes defective in the absence of miR- [ ]. LncRN“s that recruit epigenetic modifiers to specific loci such as “NRIL, XIST, HOT“IR, and KCNQ OT are found to have altered expression in a variety of cancers [ ]. “nother lncRN“ called TERR“ binds telomerase, inhibiting its activity in vitro [ ], and is observed to be downregulated in many cancer cells, linking it with the longevity of cancer cells. Chromatin remodeling by lncRN“ is linked to other diseases such as facioscapulohumeral muscular dystrophy FSHD [ ], lethal lung developmental disorder [ ], and the HELLP syndrome, a pregnancy-associated disease [ ] in addition to cancer. The HELLP stands for H = hemolysis breakdown of red blood cells , EL = elevated liver enzymes liver function , and LP = low platelet counts platelets help the blood clot . These examples directly link lncRN“ and miRN“s in cancer biology and other human diseases and indicate the involve‐ ment of a complex interplay among their biogenesis pathways, their regulatory mechanisms, and their targets.

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. Dosage compensation and X inactivation X chromosome inactivation XCI occurs in females during embryogenesis, where either the maternal or paternal X chromosome is randomly silenced. The molecular mechanisms of XCI are not yet fully understood. However, it is known that a -kb stretch of DN“ at Xq known as the X-inactivation centre XIC is the site for initiation of X inactivation. There are several lncRN“s, including X-inactive specific transcript Xist , its antisense transcript Tsix, Xinactivation intergenic transcription elements Xite , Jpx transcript, and Xist activator Jpx , and others play pivotal roles in XCI [ ]. Xist was one of the first to be identified and best studied lncRN“s. It is a ~ -kb transcript ~ kb in humans expressed from the future inactive X chromosome Xi [ ]. Tsix is a ~ -kb antisense transcript to Xist. It negatively regulates Xist. Recent studies indicate that Xite is a transcriptional enhancer of Tsix [ ], and likewise, Jpx RN“ appears to help in Xist expression [ ]. When two homologous X chromosomes are brought at close proximity, Tsix and Xite initiate the inactivation process by counting, and this is associated with the presence of RN“ poly‐ merase II RN“PII [ , ]. The chromatin insulator CTCF, which binds to Tsix and Xite genomic loci [ ], play an important role. The transcription factor OCT is then hypothesized to bind with Tsix promoters of one of the X chromosomes, which then converts to active X chromosome Xa due to increased transcription of Tsix [ ]. Thereafter, Dnmt a is recruited to the Xa and establishes stable silencing of Xist on the Xa [ , ].

. LncRNA in genomic imprinting In mammals, genomic imprinting is an epigenetic marker in a way that their expression occurs specifically in parental origin manner. This occurs during early gametogenesis in nearly % of protein-coding genes. To date, we have identified around imprinted genes in mice. Imprinted genes are often located in clusters of size from a few kilobases to to Mb. LncRN“s are present in all the identified and elucidated imprinted clusters as their partners. The expression of lncRN“s is reciprocally linked with corresponding proteincoding genes [ – ]. Genomic imprinting mainly happens by chromatin insulators [ – ] and lncRN“s [ , ]. LncRN“s repress flanking gene promoters in cis action Kcnq ot and “irn lncRN“s [ ] . However, several reports indicate that lncRN“s function as a major force in the regulation of parent-of-origin-specific expression. Today, we know that the human genome contains more than , lncRN“ expressed genes compared to only , protein-coding genes [ ]. The majority of the lncRN“s act by interacting with chromatin-modifying complexes such as PRC , G a, hnRNPK, and SWI/SNF, recruiting them sequentially to silence genes in cis or trans action [ , ].

Long Noncoding RNAs are Frontier Breakthrough of RNA World and RNAi-based Gene Regulation http://dx.doi.org/10.5772/61975

. Perspectives LncRN“ has diversified tentacles for functions. Those include an alteration of transcriptional profiles, controlling of protein expression, complex structural or organizational roles, RN“ processing or RN“ editing and role of being the precursor of small RN“s. ”ecause a very small fraction of lncRN“ have been molecularly characterized to date, many more yet to be discov‐ ered that fit into this diversified functional paradigms. Future work will definitely ask many more questions about the interplay of lncRN“ transcripts and whether it is sufficient to have fundamental sequence of events or not. Many lncRN“s play intermediate roles in cis regulation that gets represented in ectopic expression in trans regulation. Most recent challenges are to identify how the molecular function of each type of lncRN“ results in different diseases of the organism. LncRN“ appears to expose numerous develop‐ mental events such as the generation of photoreceptor cells in retina development, control of cell surveillance, cell cycle progression of mammary gland development, and finally genera‐ tion of knockout animal development. Many lncRN“s are not eliminated as transcriptional noise in the genome but are useful for normal developmental processes. LncRN“ has a tremendous impact on disease development due to its flawless miscegenation. In tumor formation, the expression of lncRN“s is very important. They function like specific markers of tumor formation. However, the exact mechanism by which tumor initiation, formation, and progression would occur is not fully understood. It is true that the interplay and significant role of lncRN“ in different disease research is really an unexplored area, which is eventually determining the new therapeutic targets. Recently, it was found that lncRN“ may form β-amyloid plaques in “lzheimer’s disease. This possibility suggested that noncoding transcript might serve as an attractive drug target for “lzheimer’s disease. Most conventionally, genetic information may run through protein-coding sequences, but it is now found that transcription is pervasive through the nucleic acid content of eukaryotic genome, which generated a numerous number of lncRN“, which are possibly the key regulators of protein-coding sequences. We anticipate that many more surprises are yet to be explored in the coming decades. Therefore, future research might provide more pleasant but unexpected surprises in the lncRN“ function.

. Conclusion The above description exhibits a brief survey of the current status of knowledge regarding the identification, localization, functions, and mechanisms of actions of lncRN“s related to different human diseases. “ fraction of genomic nucleic acid is transcribed to protein, but an overwhelming majority of the genome sectors of the organisms contain lncRN“ with unknown functional efficacy. Some are nuclear or cytoplasmic and are highly overexpressed, and others are rarely detected. Truly, it is impossible to discern the important criteria such as stability, conservation, and time of expression related to human diseases. LncRN“ in the Xic is only

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found in placental mammals and is not conserved in other mammals. However, this limited conservation might not be essential in other higher animals. The true test for real function lies in the mechanism, genetic pathway, and tissue-specific activity for each lncRN“. The genome of an organism is not always streamlined by the natural selection. Thus, here, we really tried to avoid the speculative statements about localization, function, and dissecting mechanism regarding long noncoding RN“. Truly, we have just begun to scratch the skin of LncRN“ in the human body. The lncRN“ world is so galactically vast that we have an enormous task to completely learn about it. We feel that additional discoveries of lncRN“ may provide a real exciting phase in the study of RN“ world.

Acknowledgements We thank the lab members and Sohini ”ose for the extensive discussion in the subjects and help in writing. This work is sponsored by the CSIR Net work grant ”SCof U” and D”T grant to M.P.”. G“P . DRC work supported by DST G“P grant.

Author details Utpal ”hadra *, Debabani Roy Chowdhury , Tanmoy Mondal and Manika Pal ”hadra *“ddress all correspondence to: [email protected] Functional Genomics and Gene Silencing Group, Centre for Cellular and Molecular ”iology, Hyderabad, India Centre for Chemical ”iology, Indian Institute of Chemical Technology, Hyderabad, India

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] Chan J“, Krichevsky “M, Kosik KS. MicroRN“man glioblastoma cells. Cancer Res. . / .C“N- -

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] Xiao C, Srinivasan L, Calado DP, Patterson HC, Zhang ”, Wang J, et al. Lymphopro‐ liferative disease and autoimmunity in mice with increased miR- - expression in lymphocytes. Nat Immunol. : – . DOI: . /ni

gene: “ typical multifunc‐ – . DOI: . /j.bbadis.

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] Concepcion CP, ”onetti C, Ventura “. The microRN“clusters in development and disease. Cancer J. : b e b a

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] Gutschner T, Hammerle M, Eissmann M, Hsu J, Kim Y, Hung G, et al. The noncod‐ ing RN“ M“L“T is a critical regulator of the metastasis phenotype of lung cancer cells. Cancer Res. : – . DOI: . / .C“N- -

[

] He L, He X, Lim LP, de Stanchina E, Xuan Z, Liang Y, et al. “ microRN“ component of the p tumour suppressor network. Nature. : – . DOI: . /nature

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] Gutschner T, Diederichs S. The hallmarks of cancer: “ long non-coding RN“ point of view. RN“ ”iol. : – . DOI: . /rna.

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] Redon S, Reichenbach P, Lingner J. The non-coding RN“ TERR“ is a natural ligand and direct inhibitor of human telomerase. Nucleic “cids Res. : – . DOI: . /nar/gkq

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] Szafranski P, Dharmadhikari “V, ”rosens E, Gurha P, Kolodziejska KE, Zhishuo O, et al. Small noncoding differentially methylated copy-number variants, including lncRN“ genes, cause a lethal lung developmental disorder. Genome Res. : – . DOI: . /gr. .

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] Kanduri C. Long noncoding RN“ and epigenomics. “dv Exp Med ”iol. : – . DOI: . / - - _

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] ”rown CJ, ”allabio “, Rupert JL, Lafreniere RG, Grompe M, Tonlorenzi R, et al. “ gene from the region of the human X inactivation centre is expressed exclusively from the inactive X chromosome. Nature. : – . DOI: . / a

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] Lee JT. Epigenetic regulation by long noncoding RN“s. Science. . DOI: . /science.

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] Caley DP, Pink RC, Trujillano D, Carter DR. Long noncoding RN“s, chromatin, and development. Sci World J. : – . DOI: . /tsw. .

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] Zakharova IS, Shevchenko “I, Zakian SM. Monoallelic gene expression in mammals. Chromosoma. : – . DOI: . /s -

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] Umlauf D, Fraser P, Nagano T. The role of long non-coding RN“s in chromatin structure and gene regulation: Variations on a theme. ”iol Chem. : – . DOI: . /”C. .

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] Mohammad F, Mondal T, Kanduri C. Epigenetics of imprinted long noncoding RN“s. Epigenetics. : – . DOI: . /epi. . .

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] Lee JT, ”artolomei MS. X-inactivation, imprinting, and long noncoding RN“s in health and disease. Cell. : – . DOI: . /j.cell. . .

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] Sleutels F, ”arlow DP. The origins of genomic imprinting in mammals. “dv Genet. : – . DOI: . /S -

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] Kanduri C, Fitzpatrick G, Mukhopadhyay R, Kanduri M, Lobanenkov V, Higgins M, et al. “ differentially methylated imprinting control region within the Kcnq locus harbors a methylation-sensitive chromatin insulator. J ”iol Chem. : – .

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] Kanduri C, Holmgren C, Pilartz M, Franklin G, Kanduri M, Liu L, et al. The ′ flank of mouse H in an unusual chromatin conformation unidirectionally blocks enhanc‐ er-promoter communication. Curr ”iol. : – . DOI: . / S -

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] Kanduri C, Pant V, Loukinov D, Pugacheva E, Qi CF, Wolffe “, et al. Functional as‐ sociation of CTCF with the insulator upstream of the H gene is parent of originspecific and methylation-sensitive. Curr ”iol. : – . DOI: . / S -

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] Iyer MK, Niknafs YS, Malik R, Singhal U, Sahu “, Hosono Y, et al. The landscape of long noncoding RN“s in the human transcriptome. Nat Genet. : – . DOI: . /ng.

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Chapter 5

Noncanonical Synthetic RNAi Inducers O.V. Gvozdeva and E.L. Chernolovskaya Additional information is available at the end of the chapter http://dx.doi.org/10.5772/61685

Abstract This review focuses on current strategies of development of noncanonical synthetic RN“ interference RN“i inducers with structural modifications for promoting better gene si‐ lencing with low risk of side effects. “ particular focus is on longer RN“ duplexes – nucleotides nt in length that mimic Dicer substrates to improve interaction of RN“i in‐ ducers with RN“i machinery. Various design strategies of efficient Dicer substrate smallinterfering RN“ siRN“ are described. It was found that the length, chemical modifications, and overhang structure influence the gene silencing activity and RN“-in‐ duced silencing complex RISC assembly. Special attention is paid to the long doublestranded RN“ duplexes that induce effective gene silencing in Dicer-dependent or Dicerindependent mode. Some structural variants of shorter siRN“s, including hairpin and dumbbell siRN“s and fork-siRN“ fsiRN“ with several nucleotide substitutions at the ′ end of the sense strand, are also analyzed. These structural modifications provide effi‐ ciently increased gene silencing of targets with unfavorable duplex thermodynamic asymmetry. Recent data remove the length and structure limits for the design of RN“i effectors, and add another example in the list of novel RN“i-inducing molecules differ‐ ing from the classical siRN“, which is discussed in this chapter. Keywords: RN“i, siRN“, fsiRN“, dsiRN“, tsiRN“, structural modifications, mechanism of action

. Introduction RN“ interference is a conserved mechanism of a sequence-specific posttranscriptional gene silencing triggered by double-stranded RN“s homologous to the silenced gene [ , ]. Long double-stranded RN“ dsRN“s are cleaved in the cell by RNase III class endonuclease Dicer into short fragments – nucleotides nt in length with – -nt ′ overhangs at both ends [ , ]. These fragments small-interfering RN“s, siRN“s enter RN“-induced silencing complex RISC and associate with core proteins belonging to “rgonaute “GO family [ ]. “GO

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unwinds the duplex and cuts one of the strands in the middle, and then this strand designated as passenger dissociates from the complex and is degraded by cellular ribonucleases. The other strand designated as guide remains in activated RISC and recognizes the cellular mRN“ complementary to the guide strand. The configuration of the complex determines which strand remains in the complex and which strand leaves and degrades. “ctive RISC complex containing the guide strand binds to the complementary mRN“ and induces its cleavage. When the cleaved mRN“ is released, RISC is recycled for a new round of cleavage [ , ]. The details of the RN“i mechanism are well reviewed in literature [ – ]. Synthetic small-interfering RN“s have become an advanced and powerful tool for specific gene silencing and could be considered as promising class of therapeutics for the treatment of diseases associated with overexpression of specific genes [ – ]. However, therapeutic applications of canonical non-modified siRN“s are limited by their sensitivity to ribonucleas‐ es, possibility of unfavorable guide strand selection, and activation of innate immune system by siRN“ containing immunostimulatory motives in the sequence, which can lead to poor gene silencing efficiency [ , ]. Different structural variations of the RN“i inducers together with chemical modification were developed to overcome these problems. This review focuses on current strategies of development of siRN“ structural modifications for promoting better gene silencing with low risk of side effects, with particular focus on longer siRN“ duplexes – nt in length that mimic Dicer substrates Dicer substrate siRN“ dsiRN“ [ – ]. Special attention has been paid to the long double-stranded RN“ duplexes, which induced effective gene silencing and did not require Dicer-mediated processing of the substrate into smaller units: trimer RN“ tsiRN“ with nt in length and tripartite-interfering RN“ tiRN“ with nt in length [ , ]. “pplications of some structure variations of shorter siRN“s and the potential of different synthetic RN“i inducers in different applications have also been reviewed and summarized.

. Dicer substrate interfering RNAs Long dsRN“s homologous to the targeted mRN“ were successfully used for silencing of gene expression in nonmammalian species [ , ]. Early attempts to use long dsRN“s in mammalian cells triggering of RN“i failed due to activation of innate immune system by dsRN“ [ ]. “lthough activation of innate immunity may be beneficial for the therapy in some cases, uncontrolled induction of the interferon response results in global changes in gene expression profile and, in some cases, in cells death [ – ]. It was found that chemically synthesized mer RN“ duplexes with -nt ′ overhangs at both ends, which directly mimic the products produced by Dicer, efficiently suppressed gene expression in mammalian cells [ , ]. These duplexes, referred to as canonical siRN“s, are widely used in biomedical research [ ]. Later, it was found that RN“ duplexes, smaller than nt in length but longer than siRN“s, were significantly more efficient than canonical siRN“s and did not induce interferon response in a variety of cell lines [ ]. It was established experimentally that -mer duplexes possess maximal silencing activity, longer duplexes demonstrated reduced silencing activity, and –

Noncanonical Synthetic RNAi Inducers http://dx.doi.org/10.5772/61685

-mer duplexes were inactive. “t the same time, -mer duplexes, named as Dicer substrate siRN“ dsiRN“ , were efficiently cleaved by Dicer producing a variety of -nt-long distinct products. High potency of -mer duplexes initially was explained by the formation of siRN“ pool containing functional siRN“s with extremely high silencing activity. Some of -mer duplexes were significantly more potent at nanomolar or picomolar concentrations than the specific -mer siRN“ selected according to the current computational algorithms [ ]. However, further experiments demonstrated that none of the synthetic -nt siRN“s, included in the corresponding set to all possible products of Dicer processing of -mer duplexes, demonstrated the same level of silencing activity as -mers at low concentrations [ ]. ”ased on the earlier observations that Dicer participates both in the cleavage of dsRN“s and in the incorporation of the products of cleavage into RISC complex in Drosophila melanogaster, it has been suggested that Dicer could participate in direct loading of siRN“ into RISC and in RISC assembly [ , ]. It has been experimentally proved that dsiR‐ N“s form the RISC loading complex RLC in vitro more efficiently than the canonical mer siRN“ duplexes [ ]. ”ecause Dicer does not form complexes with -base pair bp duplexes, it was assumed that Dicer facilitates RLC formation after dsRN“ cleavage without dissociation from the cleavage product. These findings become a basis for the develop‐ ment of a new class of RN“i inducers [ , , ]. The silencing activity of dsiRN“ depends on its structure. “t the first step of recognition, P“Z Piwi “rgonaut and Zwille domain of Dicer predominantly anchors two ribonucleo‐ tides on ′ overhangs because those blunt -mer duplexes are not good substrates for Dicer. P“Z domain plays a vital role in the orientation of bound RN“ in the active site of the enzyme and determines the cleavage position on RN“ for “GO protein. Unlike mer siRN“, where two-base ′-deoxynucleotide overhangs are often used regardless of their complementarity to the target mRN“ sequence mostly dTdT , the overhang sequen‐ ces are important for the properties of dsiRN“. Incorporation of deoxynucleotides at the ′ ends of dsiRN“ strands has an adverse effect on dsiRN“ processing [ ]. The se‐ quence of ′ terminal overhangs could control dicing polarity and strand selection into RISC. Thus, Dicer preferentially binds with purine/purine GG, ““ nucleotides [ ]. Protruding nucleotides added to the ′ terminal of the antisense strand facilitate its preferential loading into RISC [ ]. Hence, asymmetric duplexes with one -nt ′ over‐ hang and DN“ residues on the blunt end of the duplex provide a single favorable P“Z binding site and reduce heterogeneity of cleavage products Figure [ – , , ]. The stability of dsiRN“ in physiological fluids is extremely an important factor for its applications in vivo [ ]. “lthough dsRN“s are more stable in comparison with singlestranded RN“s and -bp siRN“, they still rapidly degrade in the serum [ ]. It was found that bonds with ′ pyrimidine nucleotides are cleaved faster than bonds with ′ purines. Kubo and his colleagues demonstrated that degradation rate of dsiRN“s correlated with the amount of pyrimidines at the ′ end [ ]. “t the same time, degradation rate of dsiRN“s also correlates with the presence of “U-rich domains that might be related to low thermal stability, easy dissociation, and faster cleavage by both endo- and exonucleases. Chemical modifications can improve nuclease stability and reduce off-target effects [ – ]. Fluorescein modification of ′

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Figure . The scheme of dsiRN“ design according to [ ] . N – ribonucleic acids, d - deoxyribonucleic acids, short arrows – the site of Dicer cleavage.

end of -mer RN“ duplexes significantly reduces RN“i activity because ′ ends are important for interaction with Dicer and should be available for the proper recognition [ ]. On the other hand, dsiRN“s with chemical modifications of the ′ end possess high nuclease stability and RN“i activity in the cell-cultured medium. Thus, ′-end amino-modified dsiRN“ demon‐ strated improved RN“i activity and stability in the cell-cultured medium [ ]. Incorporation of ′-O-methyl modifications is an efficient and inexpensive method to improve nuclease resistance of synthetic RN“ duplexes [ , – ]. However, dsiRN“ duplexes with modifications of all or the majority of nucleotides in both sense and antisense strands are practically inactive, because extensive modification blocks the cleavage of duplex by Dicer [ ]. Limiting the modifications to incorporation of only – modified bases into the antisense strand and avoiding modifications in the site of Dicer cleavage prevents this undesired effect [ ]. Mass spectrometry analysis of in vitro dicing reactions showed that modified duplexes produce a mixture of - and -nt species, whereas unmodified duplexes are processed only into -mer species. If modifications were spaced further away from the dicing site, a prefer‐ ential accumulation of -mer species was observed [ ]. However, it was noted that in some cases, usually, a good modification pattern may decrease the silencing activity of dsiRN“. This phenomenon has been related to the newly unidentified sites in the sequence context of some chemically modified dsiRN“s, contributing to impairment of dsiRN“s silencing effect [ ]. These observations could explain differences in the efficiency of various dsiRN“s and made possible the creation of the modification patterns compatible with dicing. “ccumulation of experimental data revealed that – -bp dsRN“s, including dsiRN“ and, in some cases, even -bp siRN“, could stimulate innate immune system and induce interferon response in certain cell types [ ]. Toll-like receptors TLRs and appear to be the main molecules responsible for the immune recognition of siRN“ and dsiRN“, whereas toll-like receptor recognizes longer than -bp dsRN“ [ , ]. “ctivation of innate immune system through TLRs results in the production of interferon α, tumor necrosis factors α, and inter‐

Noncanonical Synthetic RNAi Inducers http://dx.doi.org/10.5772/61685

leukins IL and IL [ ]. Immunostimulatory properties of siRN“ are sequence dependent TLR and TLR receptors recognize GU-rich sequences of siRN“ [ ]. Moreover, several immunostimulatory motifs of siRN“ enriched in GU nucleotides were identified [ , ]. It is recommended to avoid these motives in siRN“ and dsiRN“ design unfortunately, not all immunostimulatory motifs have been discovered that complicate the design procedure. Earlier, it was demonstrated that chemical modifications involving ′ position of the ribose ring in siRN“ could block the immune response [ ]. Incorporation of ′-O-methyl U and G bases into siRN“ significantly reduced immunostimulatory activity of siRN“ in vitro and in vivo, containing immune-stimulating motives in the sequence [ ]. Moreover, the effective suppression of immunostimulatory activity could be reached by using only a small percentage of modified nucleotides < % . Collingwood and his colleagues [ ] applied this approach to dsiRN“ and demonstrated that limited ′-O-methyl modifications of uridine and guanosine into antisense strand of dsiRN“ efficiently prevent induction of innate immune response in different cell lines. “nother option to reduce nonspecific effects of dsiRN“ is to use enzymatically produced pools of Dicer substrate RN“ [ ]. Dicer from the protozoan parasite Giardia intestinalis was used to obtain enzymatically produced dsiRN“s. It cuts long dsRN“ into fragments from to nt in length. The sequences-related side effects were decreased in the pool of enzymatically produced dsiRN“s due to the low concentration of individual dsiRN“s with undesirable sequence. In the cases when silencing of more than one gene is required, the transfection of siRN“ mixture is used. Co-transfection of different siRN“s may result in different knockdown efficiency of individual targets due to competition between siRN“s for RISC loading depend‐ ing on the thermodynamic asymmetry of the duplexes [ ]. Therefore, preliminary testing is required to assess the degree of competition between various siRN“s. Competition between RN“i inducers aimed at different mRN“s could be avoided by using Dicer substrate RN“. Entry of dsiRN“s into RN“i pathway is not limited by RISC loading step, and discrimination of canonical siRN“s based on RISC incorporation is reduced. These beneficial properties of dsiRN“s can provide an effective tool for targeting multiple mRN“s. Currently, siRN“s have become a powerful tool for effective suppression of expression of target genes in vitro and in vivo applications. Moreover, several compounds are already used in clinical trials. However, the examples of Dicer substrate RN“s usage in vivo are fewer in number. Several studies use dsiRN“ to silence therapeutically relevant genes in vivo Table . Frequently, cancer-related genes and genes of viruses [ , , – ] are chosen as targets for dsiRN“s [ – ]. Several researchers used TNFα gene as a target for the treatment of inflammatory and autoimmune diseases [ – ]. Murine models are the most popular animal models among various studies that used dsiRN“ in vivo [ – ] however, there are studies where other animal models, for example, rats, were used [ , ]. “n exciting example of dsiRN“ application was described by Doré-Savard and his colleagues, who demonstrated, for the first time, the efficient suppression of target genes in central nervous system CNS of rats by dsiRN“ [ ]. In this study, -mer dsiRN“s were used to reduce expression of neurotensin receptor- NTS involved in ascending nociception. dsiRN“s were formulated with cationic

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lipid i-Fect and used in intrathecal spinal cord injection. Extremely low doses of dsiRN“ . mg/kg efficiently silenced NTS mRN“ and protein levels for – days. It is known that administration of high doses of non-modified siRN“ increases the risk of activation of innate immune system, especially when siRN“ is used together with cationic lipids. Low doses of highly active dsiRN“s could minimize this adverse effect. No apparent toxicity and other offtarget effects were found during the experiment [ ]. The dose–response experiments per‐ formed in another study [ ] also show that -mer Dicer substrate RN“ provide improved gene silencing when used at lower concentrations [ ]. The silencing activity of canonical mer siRN“s was compared with that of dsiRN“ at and nM concentrations. The -mer dsiRN“ displayed more potent gene silencing at nM concentration, while at nM concen‐ tration, the difference in silencing was less pronounced [ ]. Experimental

Structure

Target gene

Disease

system MD“-M”-

Concentra-

Biological effect

Reference

tion/dose D/ -mer cdc2

mouse

cells

”reast

nM

>

% cell growth inhibition [

]

cancer

Mice

µg/mouse Tumor growth inhibition after second injection

Huh . cells

D/ -mer

' UTR and

Hepatitis C

nM

coding regions of virus

. % inhibition in luciferase [

]

assay

hepatitis C virus: infection NS3, NS4B, NS5A, NS5B PC- cells

D/ -mer HSP27 human

Prostate

nM

cancer Hsp27 mouse

Mice

>

% reduction of both

[

]

[

]

% mRN“ level reduction [

]

mRN“ and protein mg/kg

>

% reduction of both

mRN“ and protein H“E cells obtained from

D/ -mer N gene of ’OMe

bronchi and lungs

syncytial

syncytial virus

virus

RSV

Hela cells

D/ -mer CTNNB1 ’OMe

Respiratory

respiratory

nM

-fold decrease of viral

infection Liver cancer

nM

>

mg/kg

Significant reduction of

human Ctnnb1 mouse

Mice

> titer

tumor weight “Y-

cells

D/ -mer Mki-67 rat

”ladder

nM

% mRN“ reduction

[

]

% reduction in plaque

[

]

cancer LLC-MK cells

D/ -mer N, D,L genes of human

Human metapneum

.

nM

assay

Noncanonical Synthetic RNAi Inducers http://dx.doi.org/10.5772/61685

Experimental

Structure

Target gene

Disease

system

Concentra-

Biological effect

Reference

tion/dose metapneumoviru ovirus s hMPV

infection

Mice

mg/kg

Reduction of virus titers in lungs of infected mice

R“W

. cells

D/ -mer Tnf mouse

Inflammator

nM

y diseases

>

% reduction of the

[

]

[

]

[

]

% reduction of TNFα level [

]

number of TNFα positive

sepsis

cells

model Mice

mg/dose

-fold reduction of the number of TNFα positive peritoneal macrophages

keratocytes

/ -mer

JKAMP rabbit

Corneal

from rabbit

wound

corneal stroma

healing

% - JNK1 mRN“ level

nM

reduction % - JNK2 mRN“ level reduction

CCRF-CEM

/ -mer

TNPO3

HIV-

% TNPO3

nM

CD4

cells

mRN“ level reduction % CD4

human Tet/rev

mRN“ level reduction % Tet/re mRN“ level

viral

reduction Mice

.

Kupffer cells

D/ -mer Tnf rat

Inflammator

mg/kg

y diseases Rat

Prolonged antiviral effect

nM

after LPS stimulation µg/kg

% reduction of TNFα level in blood

CHSE-

cells

/ -mer

fish

N gene of

Hemorrhagi

hemorrhagic

c septicemia

nM

% mRN“ level reduction

[

]

nM

% protein level reduction [

]

septicemia virus virus HSV Murine peritoneal

/ -mer

Tnf mouse

’OMe

infection Rheumatoid arthritis

macrophages Mice

µg/dose

”lock the development of inflammation after second dose

NTS cells

D/ -mer Ntsr2 rat

Pain states

nM

>

% mRN“ level reduction [

]

95

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Experimental system Rat

Structure

Target gene

Disease

Concentra-

Biological effect

Reference

tion/dose .

mg/kg

% and

% mRN“ level

reduction in lumbar dorsal root ganglia and in spinal cord, respectively Table . “pplication of dsiRN“ for silencing of disease-related genes summarized from PubMed . / -mer – dsiRN“s with - base sense strand and – base antisense strand D – bases at the ’-end are substituted with DN“ ’OMe – ’ – O methyl modifications as described in [ ].

In another study, potent ′-O-methyl modified dsiRN“s targeted to β-catenin were designed [ ]. It is known that β-catenin acts as the transcription factor and its overexpression causes the development of several types of cancer, including liver cancer. “t the first step, large-scale screening of dsiRN“s for in vitro mRN“ knockdown activity was performed to choose the most efficient dsiRN“s for targeting β-catenin. Then, the absence of immunostimulatory activity attributed to selected dsiRN“ was confirmed using the assay based on the ability of an oligonucleotide to induce the production of antibodies to the PEGylated components of the lipid nanoparticles containing oligonucleotides. dsiRN“ was administered to mice intrave‐ nously twice a week during weeks after implanting Hep ” tumor cells. dsiRN“s induced strong β-catenin mRN“ knockdown and efficient tumor inhibition. Other examples of dsiRN“s applications as potential therapeutics for inhibition of the disease-related overex‐ pressed genes in vivo and in vitro have been summarized in Table . ”eneficial properties of dsiRN“s make these structures popular inhibitors of target genes. “t first, dsiRN“s induce more potent silencing of the target genes at lower concentrations than canonical siRN“s. The next advantage of dsiRN“s is longevity of silencing: In some cases, it lasts up to days. Then, the usage of dsiRN“s enables to minimize off-target effects such as toxicity and heterogeneity of processed products. “n additional benefit is the high potency of dsiRN“s in silencing of multiple mRN“s where canonical siRN“s due to competition during RISC loading step appear to be less effective. The main disadvantage of dsiRN“ is the higher cost of synthesis in comparison with canonical siRN“. However, low dosage of dsiRN“ used in experiments eliminates this drawback. On the other hand, dsiRN“s share with siRN“s the same problems in therapeutic applications. The major challenge lies in the delivery of these structures into desired cells, tissues, and organs. To overcome this problem, various ap‐ proaches are developed however, this question has not been completely answered yet. Nevertheless, dsiRN“ as potent inducers of RN“i offers promising strategies for efficient therapy.

. Interfering RNA with noncanonical duplex structure Different variations of siRN“ duplex structures were proposed to improve their silencing activity. Here we will consider three types of the most frequently used siRN“s with structural

Noncanonical Synthetic RNAi Inducers http://dx.doi.org/10.5772/61685

modifications of duplexes: short hairpin RN“s shRN“s /microRN“ miRN“ mimics, dumbbell RN“s, and fork-siRN“ Figure .

Figure . The different types of interfering RN“s with non-canonical duplex structures.

The identification of a large class of endogenous regulatory RN“ molecules – microRN“s miRN“s arouse interest in constructing the similar synthetic structures for efficient silencing of target genes. miRN“ precursors are generated in the cell as long primary transcripts that are cleaved in nucleus by RNase III class nuclease Drosha [ – ]. Then, they are exported to the cytoplasm and cleaved by Dicer, which is active at processing of complex hairpin structures [ ]. It is known that Dicer substrates more effectively enter RISC complex than canonical siRN“ and induce more potent RN“i [ – , ]. Moreover, shRN“s could interact with particular chaperones that promote recognition of shRN“ by Dicer [ ]. miRN“s form imperfect complementary complexes containing bulges with ′ untranslated region of the target mRN“, wherein the position of the loops defines the mechanism of action: target cleavage or the block of translation. In the first case, synthetic miRN“ mimics have no advantages over shRN“, and in the second case, they do not act in a catalytic mode. Therefore, synthetic miRN“ applications are restricted to exploring the miRN“-regulated pathways involved in the natural processes, or development of replacement therapy for the diseases associated with mutation in specific miRN“. The use of shRN“ seems to be more promising. “lthough long dsRN“ hairpins are prepared synthetically, enzymatically, or endogenously expressed, plasmid or viral vectors could be used in nonmammalian organisms. Long RN“ hairpins cannot be applied in mammals for the specific gene silencing because they also induce

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interferon response in mammals via the same mechanism used by long RN“ duplexes [ , , ]. Therefore, length of hairpin RN“s for application in this type of species is limited by bp. shRN“s expressed by different vectors under control of RN“ polymerase III and CMV promoters were proved to efficiently trigger RN“i [ , ]. “pplications of viral vector-based expression of shRN“s are limited because of some obstacles such as possibility of insertional mutagenesis, malignant transformation, and host immune response [ ]. “t the same time, an application of plasmid vectors is safe, but inefficient delivery into cells limits its use only for experimental purposes, where antibiotic resistance genes included in the vector is used for the selection. In contrast to expressed shRN“, synthetic shRN“ seems to be more attractive for RN“i-based therapies. It was found that chemically synthesized short hairpin RN“s shRN“s with – -base-pair stem, at least -nucleotide loop and -nucleotide ′ overhangs are more potent inducers of RN“i than the canonical smallinterfering RN“s targeted to the same sequence in mRN“ [ , – ]. Two main types of shRN“s with opposite positions of the loops were designed Figure . The right loop structures R-shRN“s have sense strand at the ′ end of the hairpin, whereas the left loop shRN“s L-shRN“s have antisense strand at the ′ end of the hairpin Figure [ – ]. The majority of studies were carried out using R-hand loop structure.

Figure . General structures of L-shRN“s and R-shRN“ according to [ sense strand.

] . Red color: sense strand, blue color: anti‐

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It was clearly demonstrated that the silencing activity of shRN“s depends on stem length, loop length, sequence, and terminal overhangs [ ]. Dicer efficiently cleaves shRN“ with certain minimum stem of nt in length forming -nt products starting from the free ′ end of the RN“. Correct ′ overhangs increase the efficacy and specificity of processing, whereas blunt-end shRN“s produce a set of products [ ]. In the case of endogenously expressing regulatory RN“s, ′ UU overhangs of miRN“ precursors generated by Drosha cleavage determine subsequent proper recognition and processing by Dicer [ ]. Synthetic shRN“ with similar overhangs and mimicking the products of Drosha preprocessing is used. The presence of a ′UU overhang improves silencing activity of -mer shRN“s as ′-UU overhangs might provide additional site for P“Z domain of Dicer [ ]. The loop length also plays a crucial role in the silencing activity of shRN“s. Thus, it was found that -nt stem and -nt loop inhibited the target gene expression more efficiently as compared with shRN“ with -nt stem and loop [ , ]. In contrast, when -nt loop was used for -nt stem shRN“, it demonstrated more potent silencing of target genes than longer shRN“ with the -nt loop. ”rummelkamp and his colleagues also demonstrated that shRN“s with -nt stem and -nt loops possessed the maximum silencing activity as compared with shRN“s with -nt loops, while shRN“ with -nt loops were inactive [ ]. “ll observed differences in the silencing activities of shRN“, divergent in the length of the stem and loop, were more pronounced under low or intermediate concentrations. The silencing activity of different shRN“s, used at high concentrations, did not depend substantially on the loop length [ ]. These results may be explained by the fact that the loop length influences the efficiency of processing by Dicer. [ ]. Indeed, -nt loops of shRN“ with -nt stem have poorer confor‐ mation flexibility at the junction between the duplex stem and a single strand of the loop. “s short loops are set close or at the Dicer cleavage site, the restriction-associated conformational changes made shRN“s stems poor substrates for Dicer. Therefore, shRN“s with short loops and -nt stem enter RISC complex in the later stages and remain not processed by Dicer [ ]. It was suggested that another single-strand-specific ribonuclease, independent from Dicer, cleaved this type of shRN“. On the other hand, shRN“s with -nt stem and longer loops – nt are efficiently processed by Dicer [ ]. The potency of -nt stem shRN“s, targeted to the same sequence, depends on the position of the loop. Right R -shRN“s nt in length are significantly less active than left L -shRN“s of the same length [ , ]. However, the position of the loop left or right in longer shRN“s did not affect their activity. It was suggested that low potency of R-shRN“ form is related to the fact that ′ end of the antisense strand must be readily available for the efficient binding of “GO in the RISC complex. Otherwise, the ′ end of sense strand would enter RISC and the target mRN“ would not be cleaved. [ ]. L-shRN“s with a short loop of – nt in length could be active. Moreover, L-shRN“s without any loop, where sense strand is directly connected with antisense strand, may be also active. In this case, the sense strand is shorter than antisense strand and the loop is formed by ′ end of the antisense strand [ ].

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Moreover, the nature of nucleotides in ′ overhang does not influence the activity of L-shRN“ and ′ overhangs could be substituted for deoxyribonucleotides [ ]. The high efficiency of LshRN“s may be explained by the high energy of binding of antisense strand with “GO due to availability of ′ end of antisense strand in L-shRN“, dominated over the influence of overhangs and loop length. [ ]. shRN“s have similar but not identical sequence preferences with siRN“s. Thus, the functional shRN“s have mainly “U nucleotides at position and GC nucleotides at position , while these preferences are less significant in functional siRN“. “t the same time, the functional shRN“s have the similar thermodynamic asymmetry as functional siRN“s. The computer algorithms for selection of potent shRN“s have been developed [ ]. Short hairpin RN“s are a little more resistant to nucleases than siRN“s due to the protection of one end however, shRN“s still quickly degrade in biological fluids [ ]. The elegant method to stabilize non-modified RN“ strands was described by “be et al. [ ]. “be and his colleagues constructed dumbbell-shaped RN“ structures and demonstrated their potency as RN“i inducers with stability in the biological fluids [ ]. Dumbbell-shaped RN“s were designed by analogy with DN“ dumbbells consisted of double-helical stem and closed by two hairpin loops. Dumbbell-shaped RN“ structures are used as models for the analysis of local structures in DN“ [ ]. Local unwinding of duplexes facilitates enzymatic cleavage by nucleases. Two loops at the both ends of dumbbell RN“ stabilize the duplex and limit its enzymatic cleavage [ , ]. Dumbbell structures get processed by Dicer much more slowly in comparison with their linear analogues due to inefficient recognition by Dicer. The rate of processing depends on the stem length, too. For example, dumbbell RN“s with -bp-stem length were processed more quickly than the same sequence with – -bp stem length. RN“ dumbbells with -bp stems and -nt loops were found to be the most active. Indeed, -nt loops are commonly used in shRN“s as the most effective hairpin loops [ ]. The stem length was optimized to keep high potency and reduce interferon response. Silencing activity of these dumbbell RN“s was significantly higher than that induced by linear counterparts and was retained for longer period even at lower concentrations [ ]. The introduction of deoxynucleotides into the loop of dumbbell RN“s further significantly increases shRN“ stability in biological fluids without loss of silencing activity. Moreover, the loop of dumbbell RN“s can be modified by carriers such as aptamers and peptides [ ]. “ll benefits of dumbbell RN“s make them new potent RN“i inducers. The main disadvantage of these structures is the high cost of their synthesis in comparison with canonical siRN“s. “t the same time, the low dosage and prolonged silencing effect can reduce expenses. The detailed scheme of RN“ dumbbell synthesis is described by “be and his colleagues [ ]. “nother type of RN“i inducer, fork-siRN“, was first introduced by Hohjoh [ ]. Fork-siRN“s contain base substitutions in the ′ end of the sense strand of siRN“, resulting in destabilization of the duplex [ – ]. The effect of fork-siRN“s is explained by the fact that thermodynamic asymmetry of the duplexes determines the orientation of siRN“ in RISC. Thermodynamic stability of the terminal regions of the duplex defines which strand is cleaved and dissociated during RISC activation, and another strand remains in the activated RISC and guides target

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mRN“ recognition and cleavage [ ]. “ntisense strand of siRN“ must be included in activated RISC for efficient gene silencing, if activated RISC contains the sense strand no silencing occur. The selection of active siRN“s may be complicated if a target mRN“ is mutated or is a chimerical gene. To address this issue, the favorable asymmetry can be achieved by the introduction of several base substitutions at the ′ end of the sense strand. Mismatches at the ′ end of the sense strand, resulting in the formation of unpaired or destabilized regions, increase the silencing activity of siRN“ with low or moderate concentrations [ ]. The number of mismatches in fork-siRN“ also plays a crucial role in its silencing activity [ ]. Fork-siRN“s with one to two mismatches at the ′ end possess silencing activity similar to that of canonical siRN“s, indicating that this number of mismatches is not enough for the efficient silencing. Fork-siRN“ with four mismatches is the most potent, whereas fork-siRN“ with six mismatch‐ es possesses reduced silencing activity [ ]. “n optimal number of mismatches depend on the overall thermodynamic stability of the duplex. Computational algorithms for siRN“ sequence selection determine the recommended range of Tm difference between the terminal regions, and four mismatches could work for sequences within the range. On the other hand, mismatches in the ′ part of the sense strand and long unpaired ends increase the sensitivity of fork-siRN“ to nucleases. Consequently, the application of non-modified fork-siRN“s in vivo is limited by the fact that they have reduced stability in biological fluids due to the increased degradation by nucleases [ , ]. To solve this problem, the algorithm for designing nuclease-resistant fork-siRN“s that contain ′-Omethyl modifications in nuclease-sensitive sites was developed, which allows obtaining forksiRN“s whose stability is comparable to that of canonical siRN“s [ ]. Thereby, fork-siRN“s may improve unfavorable asymmetry of siRN“ with low or moderate silencing activity, especially when the selection of functional siRN“ is restricted by the sequence content of the corresponding mRN“. It makes sense to use them for silencing of uneasy or precisely located targets.

. Short noncanonical RNA siRN“ shorter than canonical siRN“ could also induce efficient silencing of target genes in mammalian cells acting via RN“i mechanism [ – ]. Short siRN“s have some benefits as inducers of RN“i such as reduction of immune response and decreased cost of the synthesis [ ]. Various strategies have been used to design the minimal length for inducing RN“ interference. “s “-form helix of RN“ plays an essential role for inducing RN“i, Chiu and Rana [ ] found minimal length of dsRN“ “-form helical structure required to enter active RISC complex. They demonstrated that siRN“ with bp in length and -nt ′ overhangs repre‐ senting ~ . helical turns efficiently assembles into catalytically active RISC and was sufficient for silencing of target genes. Indeed, -mer siRN“s were more potent in comparison to mer siRN“s, while -mer siRN“s silenced gene expression at lower efficacy than -mer siRN“s, and – -mer siRN“s were practically inactive [ ]. It was demonstrated that the mechanism of target cleavage was different: cleavage sites in -mer siRN“s were shifted to

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nt in comparison with -mer siRN“s Figure . The -bp siRN“s induced the silencing faster than canonical siRN“ due to the higher efficacy of RISC formation [ , ]. Moreover, asymmetric duplexes with ′ overhang on the antisense strand only demonstrated reduced off-target silencing in comparison with symmetric duplexes of the same length due to preferential incorporation of the guide strand into the RISC complex [ ]. Thus, considering the benefits of -mer siRN“s, they possess high potential for using in biomedical studies, but new examples of their use are not available.

Figure . The mechanism of Dicer cleavage for bp siRN“ and short siRN“ age sites defined by the ’-end of guide strand according to [ ] .

bp in length. “rrows indicate cleav‐

“ntisense siRN“ also have been proposed as RN“i triggers. The sense strand of siRN“ duplex is degraded and antisense strand remains in the active RISC to target complemen‐ tary mRN“. Early studies demonstrated that ′-phosohorylated antisense siRN“s with – nt in length were able to induce gene silencing in Caenorhabditis elegans [ , ]. Several studies investigated the possibility of using antisense strand of siRN“ – nt in length for transient knockdown of target genes in mammalian cells [ – ]. “ntisense siRN“ entered the RISC complex and provided mRN“ cleavage but with lower efficiency in comparison with canonical siRN“. “ntisense siRN“ and canonical siRN“ possess similar mRN“ target position effects, cleavage fragment production, and tolerance to mutational and chemical modifications. However, antisense siRN“ modified at ′ end by fluorescein group or deoxyribose showed reduced silencing activity compared with canonical siRN“, where silencing activity remained at the same level in spite of ′ modifications [ ]. Moreover, the velocity of mRN“ degradation induced by antisense siRN“ is higher than that provided by canonical siRN“, but the duration of silencing effect is shorter. Thus, it was assumed that antisense siRN“ induces RN“i through similar pathways as doublestranded siRN“ but enters the pathway at the intermediate stage [ ]. The differences in the silencing activity between antisense and canonical siRN“s may be explained by the low intracellular stability of the single-stranded RN“ and by low efficacy of association with RISC [ ]. “mong the advantages of siRN“s, a lower price of synthesis and no side effects associated with the induction of interferon response should be considered [ ].

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Partial boranophosphate backbone ”P modifications were designed to increase the stability and the silencing activity of the antisense siRN“ [ ]. ”P-modified antisense siRN“s possess silencing activity comparable to unmodified double-stranded siRN“s. Partial ′-O-methyl modification was used for the stabilization of antisense RN“, the activity resulting in singlestranded siRN“ was comparable with the activity of double-stranded siRN“ when used in high or intermediate concentrations, where in low concentration, canonical siRN“s were more active [ ]. Overall, in spite of lower silencing activity compared with canonical siRN“, antisense siRN“ may be used in specific situations, for instance, to eliminate off-target silencing of genes in the case when the sense strand has substantial homology to nontarget genes [ ].

. Long-interfering RNAs Long dsRN“s > nt in length efficiently silence the expression of target gene in nonmamma‐ lian cells [ , ]. The early attempts to use the similar structures for efficient knockdown of target genes in mammalian cells failed due to activation of interferon response [ , ]. Later, various design strategies have been developed to prevent the induction of interferon response and construct new potent RN“i inducers [ , , ]. Depending on the architecture of duplexes, all long dsRN“s may be divided into linear and branched structures. Partial ′-O-methyl modification effectively prevents the activation of interferon response by Dicer-substrate RN“s [ ] therefore, it was proposed to use similar approach for longer linear duplexes [ ]. Longer siRN“s containing the sequence of canonical siRN“s repeated two and three times are called dimer nt in length and trimer nt in length small-interfering RN“. Selective ′-O-methyl modifications were introduced into nuclease-sensitive sites of both sense and antisense strands of dimer and trimer siRN“s, the modifications in the sites of potential Dicer cleavage were omitted. Selectively modified dimer and trimer siRN“s, unlike the unmodified ones, did not induce interferon response in cultured cells. The trimers called tsiRN“ were significantly more active at lower dose-equivalent per moles of bp concen‐ trations than their canonical analogues but the silencing effect develops more slowly [ ] and acts in a Dicer-independent mode, presumably via direct RISC loading. “lthough the Dicer cleavage sites were free from modifications, modifications in flanking regions of tsiRN“s could inhibit the Dicer cleavage. The observed mechanism may be associated with a specific pattern of modification, used by the authors, which cannot be excluded such that the change in the pattern will allow the tsiRN“ to be processed by Dicer and act through a canonical mechanism. Targeting single mRN“ by RN“i inducer for therapeutic purposes has several limitations: the presence of mutation in the target site reduces the efficiency of silencing, which is especially important for viral genes, and signal pathways involved in cancer cell growth contain duplications of regulatory elements and bypass regulatory pathways [ – ]. Thus, simulta‐ neous inhibition of several genes seems to be an effective strategy. Co-transfection of several siRN“s may be not effective due to competition between siRN“s [ ]. Therefore, long linear synthetic siRN“s targeted two or more genes hold great promise in these cases.

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Peng and his colleagues designed long linear siRN“ at least nt in length multi-siRN“s for dual-gene silencing [ ]. To avoid undesired interferon response and improve RN“i potency, ′-O-methyl modifications and gap in either sense or antisense strands were used. ′-O-methyl modifications were introduced into every second nucleotide of both strands. The gap divided the complementary strand into two equal segments. It was demonstrated that multi-siRN“s with the gap provided more efficient simultaneous silencing of two target genes in comparison with corresponding single-target siRN“s Figure . Interestingly, the simultaneous silencing of two target genes by long siRN“ without gap was ineffective. It was supposed that the gap may provide sites for Dicer or facilitate Dicer processing. ”ecause the Dicer substrates have preference in RISC loading, multi-siRN“s could possess more efficient silencing activity than canonical siRN“s [ , ]. The experiments demonstrated that silencing effects of multi-siRN“ was eliminated when “GO was downregulated confirming the action through the same RN“i pathway as canonical siRN“s [ ]. However, further experiments are required to clarify the exact mechanism of increased activity of these siRN“s.

Figure . Design of long linear duplexes with gap in either sense or antisense strands. Red color: sense strand, blue color: antisense strand.

Long unmodified siRN“s up to nt in length were used for silencing of gene expression in some specific cell lines without the induction of interferon response [ – ]. For example, direct fusion of two - and -bp long non-modified siRN“s resulted in efficient silencing of two target genes [ ]. Two RN“s were merged "head-to-head" in a way that the '-ends of both antisense strands would look outside from the duplex allowing efficient and stereospecific “GO binding and efficient silencing of both targets [ ]. Heterologous duplexes merged head-to-tail of antisense strands demonstrated reduced silencing activity [ ]. Similar results were obtained for tandem siRN“s of – nt in length consisting of + and + units [ ] as well as -nt long duplexes [ ]. These results may be explained by the fact that the induction of interferon response depends on the cell type [ ]. Indeed, some cell lines may possess reduced immune-sensitivity to the siRN“ treatment and the results obtained on the cell cultures cannot be unacceptable for in vivo experiments.

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“nother class of long siRN“s are various branched structures Figure . Initially, branched oligonucleotides were applied to study mRN“ splicing [ – ]. Then diverse branched structures were used as building blocks for self-assembling nanostructures [ – ]. More‐ over, nanostructures of different shapes and sizes have been proposed as an effective delivery system for siRN“, ribozymes, etc. [ , ]. Recently, it has been demonstrated that branched small RN“ structures may also be effective RN“i inducers. Thus, these duplexes can simul‐ taneously inhibit two or more genes and possess improved silencing activity and intracellular delivery properties [ – ]. Different strategies are developed to form branches. Symmetric doubler phosphoramidites are used to construct branches with two or four strands [ ]. In another variant, trebler phosphoramidite structure with extended short DN“ linker is used as a core for branched small RN“ with three arms [ ]. Direct annealing was used to design RN“s with three and four arms [ , ]. However, base pairing close to the junction region may be disturbed and single-stranded nuclease-sensitive region may be formed.

Figure . The architecture of various multi-target branched siRN“s. Different colors indicate siRN“ units targeted to various genes.

Chang and his colleagues introduced tripartite RN“ structure without any linker containing three -bp-duplex regions obtained by annealing of three -nt single-stranded RN“s [ ]. The ′ end of each antisense strand was directed outside, making seed regions of all antisense

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strands accessible for “GO loading. Single-stranded regions near the strand junction were defended by ′-O-methyl modifications affecting six nucleotides. It was demonstrated that tripartite small RN“ more efficiently silences the expression of three target genes in compar‐ ison with a mixture of corresponding canonical siRN“s due to a more efficient intracellular delivery by Lipofectamine [ ]. Specifically, tripartite small RN“ was not processed by Dicer possibly due to the influence of ′-O-methyl modifications introduced into the single-stranded region of tripartite RN“ [ ]. Similar structures without any modifications, designed by another group of scientists, were efficiently processed by Dicer into -nt products [ ]. On the other hand, tripartite small RN“ without any modifications was unstable in biological fluids and quickly degraded. Tetramer RN“ consisted of four arms bp in length proved to be more stable and also acts in a Dicer-dependent mode. ”oth trimer and tetramer siRN“s provided prolonged RN“i effect and efficiently inhibited the expression of three or four genes simultaneously. The influence of the structures on the interferon response was not reported [ ]. Overall, long linear and branched siRN“s could be efficiently used for simultaneous inhibition of multiple genes. Selective ′-O-methyl modifications and specific elements of the structure gaps, nonnucleotide insertions could reduce undesired interferon response. The application of long RN“i inducers is restricted by the complexity of the design in the case of branched molecules and the higher cost of synthesis in comparison with canonical siRN“s however, recent advances in the synthesis of oligoribonucleotide allows overcoming these problems. Long linear and branched siRN“s could be useful for the development of anticancer and antiviral therapeutics targeting multiple genes.

. Conclusion Small-interfering RN“s provide universal and effective method for the silencing of target genes because almost all genes could be targeted by siRN“s. “ large number of diseases, associated with hyperexpression of certain genes or expression of their chimeric or mutated variants, could be treated by inhibition of gene expression therefore, siRN“ has a great potential as a new therapeutic drug. Different design strategies have been used to improve properties of siRN“s and reduce off-target effects. Structural modifications can expand the boundaries of siRN“ applications. “t present, synthetic siRN“s structurally mimicking the Dicer substrates dsiRN“s are widely used as potent RN“i inducers. The use of dsiRN“ may prevent the development of undesired toxicity associated with off-target effects of both the inducer and the transfection reagent or any type of carrier due to the lower effective concentrations and the increase in the longevity of silencing. Therefore, application of dsiRN“s is considered to be extremely promising in anticancer and antiviral therapeutics as well as for the treatment of chronic diseases where multiple administrations are necessary to reach the desired silencing effect. Chemical modification patterns compatible with Dicer processing were designed and suc‐ cessfully applied for prevention of undesired stimulation of immune system and for acquiring

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nuclease resistance. Single-stranded structured synthetic siRN“s, such as Dicer-processed short hairpin RN“ and dumbbell RN“, possess all benefits as Dicer substrates and exhibit additional flexibility in fine-tuning of the stability, kinetics, and silencing duration. Long RN“i inducers, acting in a Dicer-dependent or Dicer-independent mode, effectively silence target genes at low concentrations. Multi-target siRN“s have a great promise in the treatment of complex diseases such as cancer and immune-inflammatory disorders or viral infections [ ]. Long linear or branched structures with selective chemical or structural modifications could successfully inhibit the expression of several genes without undesired off-target effects. Currently, however, the complexity and high cost of the synthesis restrict the biomedical application of long small RN“s. Some structural modifications in siRN“s have specific applications. Fork-siRN“ are successfully being used for the silencing of genes with restricted selection of sequence content such as chimeric or point-mutated genes. siRN“s with various structural modifications find a wide application in biomedical research and therapeutics. Some of them have already been used in clinical trials. The great success was achieved in the multi-target therapy that may increase treatment effectiveness. However, the therapeutics applications are limited by the inefficient delivery of these compounds into organs, tissues, and cells. Problem of low bioavailability of siRN“ in vivo could be overcome by two ways: the better delivery and the higher activity. Future expansion of the repertoire of RN“i inducers contributes to resolving of both challenges. “lthough many approaches are developed, more efforts are still needed to improve safety and efficiency of siRN“ in vivo.

Acknowledgements This work was supported by the Russian Scientific Foundation under the grant #

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Author details O.V. Gvozdeva and E.L. Chernolovskaya *“ddress all correspondence to: [email protected] Laboratory of Nucleic “cids ”iochemistry, Institute of Chemical ”iology and Fundamental Medicine S” R“S, Novosibirsk, Russia

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Dicer-substrate siRN“s reduces liver tumor burden in vivo. Mol Ther. . DOI: . /mt. . .

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Chapter 6

Microinjection-Based RNA Interference Method in the Water Flea, Daphnia pulex and Daphnia magna Kenji Toyota, Shinichi Miyagawa, Yukiko Ogino and Taisen Iguchi Additional information is available at the end of the chapter http://dx.doi.org/10.5772/61485

Abstract It is well known that most daphnid species have several attractive life history characteris‐ tics such as cyclical parthenogenesis, environmental sex determination, and predator-in‐ duced defense formation. Recent advances in high-throughput omics technologies make it easy to obtain a huge number of potential candidate factors involved in environmental stimuli-triggered phenotypic alterations. Furthermore, our group has developed a micro‐ injection system to introduce foreign materials such as nucleotides and chemicals into the early-stage one-cell stage egg of Daphnia pulex and Daphnia magna. Consequently, we es‐ tablished a microinjection-based RN“i system that allows arbitrary gene functions to be investigated. However, this microinjection system does not seem to have pervaded in the daphnid research community due to its low throughput and high level of skills required. In this chapter, we review the microinjection method and its RN“i system in water fleas, D. pulex and D. magna, providing some technical tips and making challenging proposals for the development of novel high-throughput RN“i methods. Finally, we provide an overview of recently developed gene functional analysis methods such as overexpression and genome-editing systems. Keywords: Daphnia pulex, Daphnia magna, genome editing, microinjection, RN“i-related gene

. Introduction The cladoceran crustacean water fleas are representative zooplankton ubiquitously found in freshwater habitats around the world [ ]. “mong them, species of the family Daphniidae, particularly genus Daphnia, have been well studied. “ll age classes of daphnids are principle consumers of algae and thus play an important role in the food web in freshwater ecosystems. In addition to this ecological significance, over the last decade, daphnids have drawn consid‐

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erable attention as a good indicator organism for aquatic toxicology and have thus been used in ecotoxicological studies [ ]. Moreover, for over years, they have shown various adaptive phenotypic alterations in response to external environmental stimuli, including environmental sex determination ESD [ , ], cyclical parthenogenesis, in which the mode of reproduction changes between parthenogenesis and sexual reproduction [ , ], and inducible defense, which is a predator-triggered alteration of body shape [ , ]. The acquisition of such sophisticated life history strategies has enabled daphnids to prosper around the world. These environmental stimuli-triggered phenomena in daphnid species have attracted many scientists involved in ecological, developmental, and evolutionary biology [ , – ]. “lthough recent progress in sequencing technology facilitates the deciphering of genome and transcriptome information using nonmodel organisms’, analytical methods for arbitrary gene functions are still insuffi‐ ciently developed. In studies involving daphnids, the whole-genome sequencing project of D. pulex has been completed [ ]. Furthermore, a microinjection system using early-stage embryos has been established, allowing gene functional analyses, including RN“ interference RN“i , to be possible in daphnids [ , ]. Even though this microinjection-based experi‐ mental method can be used in two representative daphnid species, D. pulex and D. magna, some experimental aspects are different between them due to the difference in size of their early-stage embryos. This chapter introduces Daphnia species as attractive models for eco-evo-devo studies and summarizes technical methods and tips for microinjection-based RN“i in D. pulex and D. magna. Finally, we review recent advances in the application of microinjection methods in daphnids such as genome editing and transgenesis. . . Life cycle Daphnids produce offspring either by parthenogenesis or by sexual reproduction in response to external environmental conditions. This mode of reproduction is referred to as cyclical parthenogenesis. They have a short generation period that lasts approximately week under constant laboratory conditions, and their lifetime spans over months or as much as year when reared under colder temperatures [ ]. Under optimal growing conditions, daphnids parthenogenetically produce offspring that expand their population consisting almost exclusively of females. This results in the exponential growth of genetically identical clone clusters. Mother daphnids produce several dozen eggs in their own brood chamber as a clutch just a few minutes after molting. Embryonic development occurs in the brood chamber. Subsequently, neonates are released to the outside just before the mother molts. Then indi‐ vidual mothers in the parthenogenetic phase repeat molting, spawning, and the release of neonates throughout their lifetime Figure , parthenogenetic phase . On the other hand, when environmental conditions deteriorate, for instance, a short daylength, lower temperature, food shortage, overcrowding, and the presence of predators, males are induced by parthenogenesis and are, therefore, genetically identical to their sisters and mother [ , – , , ] Figure , sexual reproductive phase . In other words, an individual parthenogenetic mother has the potential to produce female and male offspring in response to changes in external environmental conditions. “fter copulation, sexual eggs, referred to as

Microinjection-Based RNA Interference Method in the Water Flea, Daphnia pulex and Daphnia magna http://dx.doi.org/10.5772/61485

resting eggs that are enclosed in an ephippium modified carapace that is darkly pigmented by melanin , are produced. Resting eggs can tolerate extreme conditions such as drying, freezing, and digestion by fish and can remain viable for over years [ ]. When favorable conditions are restored, female neonates hatch out from resting eggs and reinitiate the parthenogenetic phase, thus building up a new population Figure , sexual reproductive phase .

Figure . Schematic drawing of cyclical parthenogenesis in daphnids.

. . Daphnia as model species for ecological, evolutionary and developmental biology: ecoevo-devo “lthough several details are still controversial, recent molecular phylogenetic analyses of the “rthropoda have revealed that the Crustacea clade is not monophyletic and is divided into at least three clades Ostracoda, Malacostraca, and ”ranchiopoda that include daphnids Figure . “lso, the current phylogenetic hypothesis supports the notion that ”ranchiopoda and Hexapoda form a sister group Figure . This suggests that a growing body of findings involving daphnids has accumulated and can contribute to our understanding of the evolu‐ tionary and developmental aspects of “rthropoda, connecting knowledge between wellstudied Hexapoda and more primitive “rthropoda clades.

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Figure . Phylogenetic tree of the “rthropoda. The branching pattern is based on Oakley et al. [ tions [ ].

] with some modifica‐

D. pulex and D. magna have long been used in ecological, evolutionary, developmental, and ecotoxicological studies as representative model daphnid species for the following reasons: D. pulex is ubiquitously widespread around the world, including Japan Figure “ , and shows striking phenotypic alterations in response to predator-released chemicals, forming neckteeth’ [ ] D. magna has a huge body size among cladoceran species maximum length of approxi‐ mately mm, Figure ” they are easy to maintain and rear under laboratory conditions they propagate rapidly because of their short generation time and reproductive cycle embry‐ onic development can be observed in vitro male offspring can be induced by administration of juvenile hormone agonists [ , ]. In addition, individuals within a single strain are most likely genetically identical due to the diploidy of the parthenogenetic eggs that are maintained by mitosis-like meiosis, which skips a part of the first meiosis [ ], allowing the environmental effects on their physiological and developmental processes to be investigated under a constant genetic background. Furthermore, we established a reliable induction system for environ‐ mental sex determination ESD studies using the D. pulex WTN strain, in which the sex of the offspring can be controlled by changing the day-length conditions in which long-day h light: h dark and short-day h light: h dark conditions can induce female and male offspring, respectively [ ], and for inducible defense in several D. pulex strains where the incidence and number of neckteeth vary in response to different concentrations of Chaoborus kairomone [ ]. In addition to the aforementioned advantages, useful experimental tools are available, for example, embryonic developmental staging [ , ], whole-mount in situ hybridization and immunostaining using developing embryos [ ], immunofluorescence and fluorescence in situ hybridization FISH [ ], an expressed sequence tags ESTs database [ ], and genetic linkage maps [ – ]. Furthermore, the whole-genome sequencing of D. pulex is complete [ , ], although that of D. magna is still ongoing https://wiki.cgb.indiana.edu/display/magna/ Home . In addition, recent growing omics and bioinformatics technology enables daphnid

Microinjection-Based RNA Interference Method in the Water Flea, Daphnia pulex and Daphnia magna http://dx.doi.org/10.5772/61485

researchers to investigate cells, tissues, and organisms from a multilevel perspective such as the transcriptome [ , – ], proteome [ ], or metabolome [ ]. Thus, various excellent experimental tools and an increasingly huge accumulation of omics data make D. pulex and D. magna attractive model organisms for studying the molecular mechanisms underlying phenotypic alterations that depend on external environmental conditions. These reliable induction systems of focal phenotypes are indispensable for analyzing their physiological and developmental mechanisms.

Figure . Daphnia pulex “ and D. magna ” . Upper and lower parts indicate the adult and embryo just after ovulation, respectively.

. Microinjection-based RNA interference RNAi in daphnid species “s mentioned above, recent high-throughput sequencing technologies have enabled biologists to use nonmodel organisms to easily access genomic information. However, the development of experimental methods for gene functional analysis still hampers their reverse genetics approach. To date, the RN“i method in D. pulex and D. magna has been established by a microinjection method using early-stage embryos. “lthough our previous reports described detailed methodology for microinjection and the tips, tricks, and traps associated with this methodology [ , ], there are slight differences for each daphnid species. Furthermore, descriptions of genes involved in the RN“i machinery is insufficient. Therefore, we compa‐ ratively summarize the tips to manipulate the microinjection methods in detail prior to focusing on the current status of daphnid RN“i. We then introduce the gene repertoires involved in the RN“i mechanism in D. pulex and D. magna genomes.

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. . Microinjection system The daphnid microinjection system uses parthenogenetic eggs just after ovulation into the mother’s brood chamber. The individual mother begins to lay eggs into the brood chamber a few minutes after releasing neonates and molting. To obtain many healthy eggs, eggs from to -week-old daphnids should be collected. There are two major technical issues in microinjection of daphnids. One is the rapid hardening of the egg membrane just after ovulation [ ], which is caused by enzymatic activity of peroxidase. The second problem is a substantial difference between the internal and external osmotic pressures of the egg. The former issue hampers the penetration of the egg membrane by a needle while the latter causes the leakage of egg components after injection. To overcome these problems, Kato et al. [ ] and Hiruta et al. [ ] established improved protocols for microinjection in D. magna and D. pulex, respectively. They succeeded in the transient inhibi‐ tion of the egg membrane hardening by ice-cold treatment just after ovulation. They also found the best culture conditions after injection by increasing external osmotic pressures: M medium [ ] with -mM sucrose in D. magna [ ] and a % agar plate covered with -mM sucrose dissolved in M media in D. pulex [ ]. In addition, since the fineness of the needle is critical for the success of microinjection, we next describe a detailed preparation method. “ glass needle is made from a glass capillary tube GD- Narishige, Tokyo, Japan by a micropipette puller P- /IVF Sutter Instrument, Novato, C“, US“ . The programmed P- parameters are as follows: heat: pull: velocity: time: pressure: . The value of the heat’ parameter required for the ramp test is based on the manufacturer’s protocol because this value depends on a combination of the filament and the glass capillary. In our case, using a combination of a regular P- filament and a GDglass capillary, the heat’ parameter value ranges between and . ”ased on the aforementioned information, we describe next the manipulation procedure of microinjection using daphnids eggs. .

The setting of tools for microinjection and surgery are shown in Figure “-C. “ glass Petri dish is prepared by placing two cover glasses side by side with M -sucrose at ambient temperature.

.

The synthesized double-strand RN“ dsRN“ is mixed with an equal amount of -mM Chromeo fluorescent dye “ctive Motif Chromeon GmbH, Tegernheim, Germany , which is used as a visible marker for injection. When using D. pulex eggs, it is possible to confirm whether injection has succeeded by visual observation since the eggs are more transparent than D. magna eggs Figure . “ visible marker is thus not essential.

.

The dsRN“ solution . – . µL is loaded into the needle. The tip of the needle is then manually cut off using forceps under a stereomicroscope.

.

Mother daphnids just before molting brood chamber is empty are collected and observed until spawning begins. They are transferred to ice-cold M -sucrose medium just before spawning is complete – eggs remain in each ovary .

Microinjection-Based RNA Interference Method in the Water Flea, Daphnia pulex and Daphnia magna http://dx.doi.org/10.5772/61485

.

Eggs are surgically obtained from the mother daphnid and placed in ice-cold M -sucrose medium.

.

One to three eggs are immobilize by placing them at the edge of the left cover glass and are injected by using a microinjector Femtojet, Eppendorf, Hauppauge, NY, US“ and a micromanipulator MN, Narishige, Tokyo, Japan Figure D–F . The right cover glass is used as a holder when the needle is not withdrawn from the egg. Microinjections can be carried out within h after ovulation.

.

The injected eggs are transferred into a plastic -well plate with -mM M -sucrose medium for D. magna [ ] or a % agar plate on a -well plate with -mM M -sucrose medium for D. pulex [ ] Figure G and subsequently incubated under constant temperature – °C .

Figure . Microinjection of daphnid egg. “ Experimental equipment. : stereomicroscope : micromanipulator MN, Narishige, Tokyo, Japan : electronic microinjector Femtojet, Eppendorf, Hauppauge, NY, US“ . ” Tools for surgical manipulation. : Pasteur pipet Thomas Scientific, Swedesboro, NJ, US“ : glass dish with two cover glasses : plate for blood test : forceps Dumoxel # ”iologie . C “ glass needle is made from a glass capillary tube GD- Narishige, Tokyo, Japan . D Overview illustration of microinjection field. E, F Magnified view and illustra‐ tion of microinjection using D. pulex egg. G Schematic illustration of embryo incubation after injection.

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. . RNAi machinery in daphnid species The well-known natural role of RN“i in organisms is the innate immune system against viruses and transposable elements [ ]. Using this phenomenon, RN“i induction has been developed as an innovative method for gene functional analysis by exogenous application of dsRN“ in Caenorhabditis elegans [ ]. The dsRN“ is first recognized by Dicer protein and cut off into short – nucleotides referred to as short interfering RN“s siRN“ . These are then invariably incorporated into large “rgonaute-containing effector complexes known as RN“induced silencing complexes RISCs , after which one-side strand of the dsRN“ is cleaved and degraded. Finally, the active “rgonaute-containing RISC cleaves the target RN“ sequence with the complementary sequence to siRN“ [ , ]. In addition to this core machinery of the RN“i pathway, many eukaryotes have the potential to amplify an amount of siRN“ by a hostencoded RN“-dependent RN“ polymerase RdRp . However, RdRp orthologs have not been identified from the “rthropoda genome including D. pulex except for the tick genome [ ]. In the D. pulex and D. magna genomes, there are three Dicer and “rgonaute paralogs, but the D. magna genome contains two copies of Dicer. Previous studies have shown that Dicer paralogs are categorized into two clusters corresponding to the microRN“ miRN“ pathway Dicer- and the siRN“ pathway Dicer- [ , ]. The miRN“ is also a short – RN“, which is generated from a hairpin in pre-mRN“, and plays an important role in translational repression associated with RISC [ ]. Phylogenetic analyses found that all Dicer paralogs of D. pulex and D. magna were classed into the Dicer- group [ ] Figure “ . Moreover, we successfully recruited three “rgonaute paralogs from the genome of each daphnid and constructed a phylogenetic tree Figure ” . Previous studies revealed that “rgonaute family members are key components in different RN“ silencing pathways and are categorized into two subfamilies, “rgonaute and PIWI P-element induced wimpy testis . “rgonaute subfamily members have been found in widespread taxa, including yeast, plants, and animals and have been identified as “rgonaute- and “rgonaute- , which are involved in miRN“ and siRN“ pathways, respectively. In contrast, the PIWI subfamily has been identified only in animals as “rgonaute- , which is involved in PIWI-interacting RN“ piRN“ pathways [ , , ]. Four “rgonaute family members were found from D. pulex and D. magna genome sequences in this study, although only two paralogs have already been previously reported [ ]. Phylogenetic analysis revealed that both paralogs fall into the “rogonaute- clade of the “rgonaute subfamily, whereas each one paralog was categorized into “rgonaute- and PIWI clades of the PIWI subfamily Figure ” . The number of “rgonaute family members found in daphnids seems to be as conserved as in insect species [ , ], although no “rgonaute- paralogs have yet been identified. Taken together, our results suggest that the genomes of daphnids might lack the Dicer- and “rgonaute- orthologs, which are factors responsible for regulating the siRN“-inducing transcriptional gene-silencing pathway. In other words, our data imply that the RN“i machinery of daphnid species might be distinct from the equivalent well-studied mechanism in insects. To understand the full picture of the RN“i machinery of daphnid species, further studies that examine domain sequence similarity and gene functional analysis will be required.

Microinjection-Based RNA Interference Method in the Water Flea, Daphnia pulex and Daphnia magna http://dx.doi.org/10.5772/61485

Figure . Phylogenetic trees of Dicer “ and “rgonaute ” . “mino acid sequences were aligned by ClustalW, and the maximum likelihood trees were constructed from these alignments using the JTT model with bootstrap analyses of replicates along with complete deletion options and amino acid positions were used, respectively by MEG“ [ ]. ”ranches with bootstrap support > % are indicated by numbers at nodes. ”oth D. pulex and D. magna are indicated in bold. To obtain the predicted sequences encoding the D. magna orthologs of RN“i-related genes, pro‐ tein sequences of D. pulex were used in ”L“ST searches querying D. magna Genome ”L“ST http://arthro‐ pods.eugenes.org/EvidentialGene/daphnia/daphnia_magna/”L“ST/ . The SID protein sequences of D. pulex were retrieved from wFlea”ase http://wfleabase.org/ . Phylogenetic trees were constructed based on data from McTaggart et al. [ ] with some modifications.

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. . Future challenges for development of high-throughput RNAi method in daphnids Despite the availability of a microinjection-based RN“i method in daphnids, as demonstrated by knocking down genes responsible for morphogenesis distal-less and eyeless [ , , ], sexual differentiation doublesex1 [ ], endocrine system ecdysteroid-phosphate phosphatase [ ], and neurogenesis single-minded homolog [ ], this method has several experimental limitations. For example, microinjection can only be performed using an early stage -cell stage egg, suggesting that this system cannot be used to perform a functional analysis of genes that act during the adult stage and is unsuitable for large-scale experiments. Moreover, specialized and skillful technical training is necessary to master the microinjection technique in daphnids. To overcome those technical hurdles, we introduce two potential ideas to establish more high-throughput and user-friendly methods for the study of daphnids. One is electroporation, which is a physical transfection method that uses an electrical pulse to increase the permeability of cell membranes, allowing nucleic acids and/or chemicals to be introduced into cells. Recent innovation of the electroporation system has enabled the establishment of rapid functional analysis in various organisms [ – ]. Indeed, our group has successfully developed an in vivo electroporation system for the introduction of foreign DN“ into neonatal D. magna within hours after release from the mother’s brood chamber [ ]. Therefore, it might be possible to apply this system for RN“i using various stages of daphnids. The second method is a feeding oral delivery RN“i system, which was first developed in C. elegans [ ]. The feeding RN“i system is the most convenient and inexpensive method for highthroughput screening since bacteria produce the designed dsRN“ that are fed to host animals. This system has so far been applied in various insects [ ] and decapod crustaceans [ – ]. Unlike the time-consuming and troublesome microinjection method that can only be per‐ formed in the early egg stage in daphnids, the alternative feeding RN“i method may poten‐ tially be applied for manipulating a wide range of genes in many individuals at the same time.

. Extended microinjection-based applications The microinjection system can be widely applied for the development of not only RN“i but also other attractive methods for gene functional analysis. Indeed, several microinjectionbased functional methods have been developed in daphnid species. First, a transient overex‐ pression system for arbitrary genes or reporter constructs was established by microinjection of synthesized mRN“ bearing the ′ cap structure and the ′ poly “ tail [ ] and a DN“ reporter construct [ ]. These methods allow for a gain-of-function analysis, although only one example has shown that the phenotype induced by transient overexpression was only observed in the first instar juvenile [ ]. However, the aforementioned overexpression and RN“i system in daphndis suffer from several drawbacks such as incomplete gain- or loss-offunction, transient effect, and limited analyzable stages. To overcome these limitations, a transgenic D. magna line was established by using microin‐ jected GFP or DsRED reporter plasmid, although the success rate was quite low . % [ ,

Microinjection-Based RNA Interference Method in the Water Flea, Daphnia pulex and Daphnia magna http://dx.doi.org/10.5772/61485

]. Furthermore, genome editing with engineered endonucleases is rapidly growing as a stable experimental method for generating heritable mutations in not only well-known in model organisms but also in nonmodel organisms. There are three representative methods: zinc-finger nucleases ZFNs , transcription activator-like effector nucleases T“LENs , and clustered regularly interspaced short palindromic repeats CRISPR/Cas systems [ ]. In order to perform targeted mutagenesis in the D. pulex and D. magna genomes, T“LEN and CRISPR/Cas systems have been established by microinjection of these engineered nucleases [ , , ]. Taken together, these genome-editing techniques will enable scientists to accu‐ rately define arbitrary gene functions in daphnid species in future studies.

. Conclusion Recent growing innovations in high-throughput omics technologies e.g., genomics, transcrip‐ tomics, proteomics, and metabolomics enable us to obtain comprehensive profiles of a huge amount of candidate factors responsible for unique life history features of daphnids [ , , ]. In order to investigate an arbitrary gene function, the establishment of an experimental method for gene functional analysis has been enthusiastically addressed by researchers using nonmodel organisms, even in the postgenomic era. In this chapter, we summarized the experimental procedure with several tips for a microinjection system in D. pulex and D. magna, information about genes responsible for their RN“i machinery, potential concepts for novel user-friendly high-throughput RN“i systems in daphnids, and other microinjection-based applications in daphnids. Further studies involved in the development of novel experimental methods and investigation of a wide range of gene functions can lead to a better understanding of the overview of the attractive environmental stimuli-dependent phenomenon in daphnids.

Acknowledgements We thank members of the Iguchi laboratory for helpful advice and comments. Our work benefits from, and contributes to, the Daphnia Genomics Consortium. This work was supported by a JSPS Research Fellowship for Young Scientists to KT No. J , a Sasakawa Scientific Research Grant from the Japan Science Society to KT, a Saito Ho-on Kai Scientific Research Grant from the Saito Gratitude Foundation to KT, grants from the Ministry of Education, Culture, Sports, Science, and Technology TI , the Ministry of the Environment of Japan TI , a Grant from the National Institute for ”asic ”iology TI , and the Research Council of Norway project , “dverse Outcome Pathways for Endocrine Disruption in Daphnia magna, a conceptual approach for mechanistically based risk assessment TI .

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Author details Kenji Toyota, Shinichi Miyagawa, Yukiko Ogino and Taisen Iguchi* *“ddress all correspondence to: [email protected] Okazaki Institute for Integrative ”ioscience, National Institute for ”asic ”iology, and National Institutes of Natural Sciences, Higashiyama, Myodaiji, Okazaki, “ichi, Japan

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Chapter 7

ER-targeted Intrabodies Mediating Specific In Vivo Knockdown of Transitory Proteins in Comparison to RNAi Oliver Backhaus and Thomas Böldicke Additional information is available at the end of the chapter http://dx.doi.org/10.5772/62103

Abstract In animals and mammalian cells, protein function can be analyzed by nucleotide se‐ quence-based methods such as gene knockout, targeted gene disruption, CRISPR/Cas, T“LEN, zinc finger nucleases, or the RN“i technique. “lternatively, protein knock‐ down approaches are available based on direct interference of the target protein with the inhibitor. “mong protein knockdown techniques, the endoplasmic reticulum ER intrabodies are potent molecules for protein knockdown in vitro and in vivo. These molecules are in‐ creasingly used for protein knockdown in living cells and transgenic mice. ER intra‐ body knockdown technique is based on the retention of membrane proteins and secretory proteins inside the ER, mediated by recombinant antibody fragments. In con‐ trast to nucleotide sequence-based methods, the intrabody-mediated knockdown acts only on the posttranslational level. In this review, the ER intrabody technology has been compared with the RN“i techni‐ que on the molecular level. The generation of intrabodies and RN“i has also been dis‐ cussed. Specificity and off-target effects OTE of these molecules as well as the therapeutic potential of ER intrabodies and RN“i have been compared. Keywords: Knockdown techniques, intracellular antibodies, ER intrabodies, RN“ inter‐ ference, off-target effects

. Introduction For the study of protein function in animals and mammalian cells, DN“-based methods such as gene knockout, targeted gene disruption, CRISPR/Cas, T“LEN, zinc finger nucleases [ ],

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as well as the RN“i technique [ ] were proven and reliable tools. ”esides the RN“i technique, approaches with miRN“ are also very attractive [ ]. Silencing of target mRN“ can be achieved using siRN“, miRN“, or shRN“ ”ox .

Box siRNA Small interfering RN“ siRN“ are small pieces of double-stranded ds RN“, usually about nt long, with -nt-long ′ overhangs at each end. They can be applied for the interference with the protein translation by binding to the messenger RN“ mRN“ , whereby promoting the degradation or destabi‐ lization of the mRN“. shRNA shRN“s form hairpin structures, which consist of a stem region of paired antisense and sense strands, connected by unpaired nucleotides building a loop. They are converted into siRN“s by the same RN“i machinery that processes miRN“s. miRNA MicroRN“s are small RN“ molecules, encoded in the genome of plants and animals. These highly conserved, ~ -mer RN“s regulate the expression of genes by binding to the ' untranslated regions 'UTR of specific mRN“s.

Protein knockdown is possible with small molecule inhibitors including peptides, neutralizing and intracellular antibodies, and allosteric modulators [ – ]. In addition, aptamers and intramers, in general short single-stranded DN“ or RN“ oligonucleotides are also potent molecules for specific inhibition of small molecules, peptides, proteins, or even whole living cells [ ]. Currently, RN“i is the most often used gene-silencing technique in functional genomics [ ]. In this article, we described an emerging protein knockdown technology using intracellular antibodies intrabodies targeted to the ER and compared the advantages and disadvantages of this promising technique with the RN“i technology. We tried to make scientists, who are interested in protein research or have very specific protein-related questions, familiar with the ER intrabody technology [ ]. The molecular mechanisms of both methods are different. RN“i-mediated knockdown is based on the interference of siRN“ with mRN“ Figure , whereas the protein knockdown by ER intrabodies is exerted upon binding of a recombinant antibody fragment to its specific antigen inside the ER [ ] Figure . Intrabodies are recombinant antibody fragments targeted to a cell expressing the specific antigen. Intracellular binding of the intrabody to the antigen results in inhibition of antigen function. Moreover, intrabodies can specifically be targeted to subcellular compartments such

ER-targeted Intrabodies Mediating Specific In Vivo Knockdown of Transitory Proteins in Comparison to RNAi http://dx.doi.org/10.5772/62103

Figure . Principle of the knockdown of transitory proteins using the RNA interference technique. For knockdown of the mRN“ of transitory proteins, transfection with a specific shRN“-expressing plasmid is sufficient. “lthough by using the RN“ interference technology all kinds of proteins could be targeted, only knockdown of transitory proteins is illustrated. Specific shRN“ is transcribed and processed by the RNase III Dicer- enzyme in mammalian cells in order to form the mature siRN“. The “rgonaute protein “go is loaded with the siRN“ and forms together with additional proteins the RN“-induced silencing complex RISC , which is a multiprotein complex consisting of ef‐ fector “rgonaute proteins , accessory proteins, and si/miRN“. During the loading of the “rgonaute protein, one strand of the siRN“ duplex is discarded. Next, the RISC complex associates with its target mRN“ via complementary base pairing of the siRN“ and the target mRN“. In many cases, the recognition site comprises the ′ untranslated re‐ gions UTR of the mRN“. Finally, target binding leads to mRN“ degradation or translational inhibition [ ]. mRN“ degradation is mediated through the endonuclease activity of the “rgonaute proteins. “s a result of mRN“ knock‐ down, the target protein is not expressed on the cell surface [ ].

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Figure . Principle of the specific knockdown of transitory proteins with endoplasmic reticulum ER -retained in‐ trabodies. In wild-type cells, transitory proteins are transported through the ER and can be further processed e.g., gly‐ cosylated in the Golgi apparatus. These proteins could reside in the secretory cell compartments, secreted through the plasma membrane PM , or become integrated in the PM as a membrane protein. For functional inhibition of these pro‐ teins, transfection with an ER intrabody expressing plasmid is sufficient. The intrabody construct consists of an N-ter‐ minal secretion sequence for the translocation in the ER leader sequence and the C-terminal retention signal KDEL . The intrabody inside of the ER binds to the target protein. This complex of antibody and target protein is further processed and transported through the secretory pathway. In the cis-cisterna of the Golgi stack, the hERD receptor binds to the KDEL sequence and initiates the retrograde transport back to the ER compartment. This continuous binding of the intrabody and retrograde transport prevents the target protein to reach its localization where it normal‐ ly acts. The accumulated intrabody–antigen complex in the ER might be transported into the cytoplasm, where it is marked for degradation by the S proteasome [ , ]. ”öldicke and ”urgdorf have shown that an anti-toll-like recep‐ tor TLR ER intrabody is degraded by the proteasome unpublished data .

ER-targeted Intrabodies Mediating Specific In Vivo Knockdown of Transitory Proteins in Comparison to RNAi http://dx.doi.org/10.5772/62103

as the nucleus, cytoplasm, mitochondria, or ER [ ] ”ox . Currently, the most used and promising intrabodies are the ER intrabodies, because of the correct folding in the oxidative environment of the ER [ ]. This contrasts with cytosolic intrabodies, in which disulfide bridges are not formed in the reducing environment of the cytoplasm [ , ].

Box Intrabodies are intracellularly expressed recombinant antibody fragments, which specifically inhibit the function of target proteins produced in the same cell [ ]. ER Intrabodies retain their corresponding antigen inside the ER by inhibiting the translocation of the antigen to the cell compartment where it normally acts. Cytosolic Intrabodies are expressed in the cytoplasm. They inactivate their targets or interfere with the binding of the target protein to its corresponding binding partner.

The effect of ER intrabodies is based on retention of proteins passing the secretory pathway. Secretory proteins, membrane proteins, and even Golgi or endosomal-located proteins can be targeted [ – ], which cannot be reached by classical antibodies, due to the extracellular presence. Successful functional knockdown was achieved for oncogenic receptors, viral proteins for preventing virus assembly, cellular virus receptors to block virus entry, and receptors of the immune system as well as of the nervous system [ – ]. The format of expressed intrabodies is, in general, the single-chain variable fragment scFv or less common the antigen-binding fragment Fab [ ]. The only prerequisite of ER intrabodies is the efficient binding to the antigen, and the method to select and generate an ER intrabody is greatly simplified by phage display. On the contrary, functional cytoplasmic intrabodies have to inactivate the antigen or have to interfere with the binding of the target protein to its corresponding binding partner [ ]. The starting material for construction of an ER intrabody is an scFv or Fab, which can be obtained by amplification of the variable domains from a hybridoma clone [ ], or scFv fragments can be selected from phage or yeast display [ , ]. Early attempts using the intrabody approach failed frequently due to the lack of reliable techniques for the identification of the correct functional antibody sequence from a hybridoma clone. The genes of the variable domains for construction of recombinant antibody fragments can be amplified from hybridoma clones using mixtures of consensus primers [ ]. This approach was used in the beginning. “s hybridoma cells could secrete several different antibodies, it was sometimes difficult to isolate the correct functional sequences of the variable domains. Presently, with reliable protein sequencing techniques, next generation of DN“ sequencing and optimized consensus primer sequences, the functional antibody DN“ can much better be identified. Furthermore, optimized strategies for amplification of the correct functional antibody sequence are available [ – ].

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In the case of using in vitro display systems, like phage or yeast cell surface display, the selected scFv fragment has only to be cloned into the ER-targeting vector. For preliminary characteri‐ zation of the intrabody function, co-transfection of the intrabody expression plasmid with the corresponding antigen expression plasmid into HEK cells is sufficient and followed by coimmunoprecipitation and immunofluorescence analysis [ ]. In contrast to the ER intrabody technology, the advantage of the RN“i is that it can be applied for almost every mRN“ and also non-coding RN“s. Here, we further compared the RN“i with the intrabody technology, regarding specificity, off-target effects, and therapeutic approaches.

. Intracellular intrabodies versus RNA interference . . Generation of ER intrabodies The prerequisite for generating intrabodies is the availability of a hybridoma antibody clone or scFv/Fab fragments selected from in vitro display systems [ ]. Starting from a hybrido‐ ma clone, the variable domains of the heavy and light chain are amplified by PCR from the cDN“. This can be achieved by PCR amplification using consensus primer [ , – ], rapid amplification of cDN“ ends R“CE [ ], PCR amplification using adaptorligated cDN“ [ ], or inverse PCR with constant region heavy chain and light chain primer, amplifying the corresponding antibody sequence from circularized double-strand‐ ed cDN“ [ ] Figure “ . In most cases, using consensus primers is a fast and efficient approach for amplification of the correct functional antibody sequence from a hybridoma clone. However, less-common nonconsensus antibody sequences cannot be amplified and primer mismatching could be a problem. “pproaches no. – shown in Figure for amplifying the variable antibody domains are more time-consuming however, the correct functional antibody gene sequence can be obtained. The variable domains are compiled by assembly PCR, linking both variable domains together by a short flexible linker sequence, for example Gly Ser , resulting in the scFv fragment. Next, the scFv fragment will be cloned into the ER targeting vector, providing the ER signal sequence, an myc tag for detection of intrabody, and the KDEL retention sequence localized at the C-terminus of the intrabody gene [ ]. Following the in vitro display pipeline, an scFv fragment or Fab fragment selected by phage or yeast cell surface display can directly be cloned into the ER targeting vector. Most recombi‐ nant antibody fragments in the scFv or Fab format are selected by phage display or also the frequently used yeast cell surface display [ , , ]. Other in vitro display systems are bacterial, mammalian cell surface display, or ribosome display [ – ]. Cytoplasmic intra‐ bodies are generated from hybridoma clones or scFv/Fab fragments from in vitro display libraries in a similar way and cloned into an appropriate cytosolic targeting vector [ ]. The main difference in comparison to ER intrabodies is that cytosolic intrabodies have to demon‐ strate neutralizing activity, and furthermore stable folding antibody fragments have to be selected [ ].

ER-targeted Intrabodies Mediating Specific In Vivo Knockdown of Transitory Proteins in Comparison to RNAi http://dx.doi.org/10.5772/62103

Figure . Generation of intrabody and RNA interference knockdown constructs. A Generation of intrabody knock‐ down vectors. The scFv fragment could be either cloned from hybridoma cell lines or selected from huge human naive phage display libraries. The antibody variable domain of the light chain VL and heavy chain VH is amplified from cDN“ using consensus primer mixtures , ′ adapter-ligated PCR or rapid amplification of cDN“ ends R“CE or with constant domain-specific primer from circularized cDN“ . The antibody VL and VH genes are assembled as scFv by fusing both domains with a flexible Gly Ser -linker sequence and cloned into the ER targeting intrabody vec‐ tor. The scFvs are cloned between an upstream secretion signal and a downstream retention sequence KDEL . Using the phage display system, selected scFvs can directly be cloned into the intrabody vector in one cloning step. Shown is an ER-targeting vector. B Generation of siRN“/shRN“/miRN“ knockdown vectors. Rational in silico design of siR‐ N“, shRN“, or miRN“ mimics using software algorithms like those mentioned in Ref. [ ] or in Ref. [ ], a recent publication, deduced from the target cDN“. The algorithms are designed to select appropriated sequences by means of empiric criteria. Main criteria are an siRN“ length of – nucleotides nt in conjunction with nt overhangs at

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their ′ ends, as well as thermodynamic properties of target mRN“ hybridization. Rational design can be expanded by testing in silico the potential off-target effects of the designed sequences by using genome-wide enrichment of seed se‐ quence matches GESS [ ] or Haystack [ ]. Designed sequences are chemically synthesized and cloned into appro‐ priate mammalian or viral knockdown expression vectors. “lternatively, siRN“ can be used for direct cell transfection. The siRN“/shRN“/miRN“ sequences originated from rational design are screened for effective processing, specific knockdown capabilities, and potential off-target effects. Corresponding clones are selected, and for most applications – different targeting sequences were chosen and theses libraries are used for the RN“ interference knockdown. ER: endoplasmic reticulum, CDS: coding DN“ sequence, p:promoter.

. . Generation of siRNA, miRNA, and shRNA In order to generate siRN“s for a specific target, only the mRN“ information about the target sequence is needed [ ] Figure ” . siRN“-mediated mRN“ knockdown can be performed in several ways. In general, cells can directly be transfected with siRN“, using transfection reagents like lipofectamine. Cells can also be transfected using siRN“/shRN“/miRN“expressing plasmids or viral vectors. Long-lasting gene silencing can be achieved with shRN“s expressed from stably transfected plasmids or from integrated retro- or lentiviral vectors [ ]. Several approaches exist for RN“ interference-mediated knockdown, the principle workflow of in silico design, screening, and selection of siRN“/shRN“/miRN“, with best knockdown properties shown in Figure ”. Currently, software algorithms mentioned in Ref. [ ] or [ ] can help to find the appropriate knockdown sequences of – nt length siRN“ by analysis of the optimal thermodynamic properties of mRN“ hybridization. Potential off-target effects can be reduced by in silico optimization with GESS [ ] or Haystack [ ]. Resulting siRN“/ shRN“/miRN“ sequences are tested for effective processing, specific knockdown capability, and low off-target effects. . . Stability Intrabodies are stably expressed inside the ER [ ], whereas most cytosolic intrabodies are not correctly folded [ , ]. On the other hand, siRN“ can be cleaved by nucleases, present in the blood serum and cellular cytoplasm. . . Specificity and Off-Target Effects OTE For the knockdown of distinct target proteins, the specificity of the process is crucial. Other‐ wise, the resulting phenotypes of the induced knockdown experiment might be superimposed with off-target effects. The specificity of the RN“i and the ER intrabody knockdown technique is the main difference between them. Intrabodies, which are also known as intracellular antibodies, are generated from monoclonal antibodies m“bs and phage or yeast antibody repertoires. Intrabodies are very specific to their targets due to antibody–antigen interactions. The high specificity of ER intrabodies has been demonstrated for the specific knockdown of members of the TLRs. The knockdown of toll-like receptor TLR and TLR , which functions as a part of the innate immunity and recognizes pathogen-associated molecular patterns P“MP , did not influence the expression of other TLRs. The developed anti-TLR intrabody

ER-targeted Intrabodies Mediating Specific In Vivo Knockdown of Transitory Proteins in Comparison to RNAi http://dx.doi.org/10.5772/62103

did not inhibit TLR -, TLR -, and TLR -driven signal transduction [ ] and the anti-TLR intrabody did not inhibit TLR -, TLR -, TLR -, and TLR -driven signaling, respectively [ ]. Stress response induction in the endoplasmic reticulum ER , due to the accumulation of retained and partially unfolded target proteins upon intrabody–antigen complex formation, was analyzed by measuring the unfolded protein response UPR for an overexpressed antip NTR ER intrabody and could not be proven [ ]. No off-target effects of expressed intrabodies are known yet, particularly any activation of the immune system. On the other hand, unspecific silencing is a major problem using RN“i-mediated gene silencing, due to the expression of short-interfering RN“ sequences, such as miRN“, siRN“, shRN“, or dsRN“ [ ]. The short seed region of these silencing RN“s recognizes and hybridizes with – nt to the target mRN“. Even with specific alignment software, it is practically impossible to exclude any possible transcript, which aligns with the target seed sequence, because statistically the chance is high to have the same sequence or secondary structure in other non-target mRN“ transcripts too. However, at least software algorithms such as GESS [ ] and Haystack [ ] are able to predict potential off-targeted genes. ”y computer-aided optimization of the miRN“, siRN“, or shRN“, the OTEs can be reduced to a minimum. siRN“ can bind to TLR , TLR , and TLR , resulting in secretion of type I interferon and proinflammatory cytokines [ – ]. “berrant expression of up to more than genes has also been described [ ]. Fortunately, some progress has been made in the repression of the RN“i-induced immune response. When siRN“ is in vitro transcribed by the T polymerase, a ′-triphosphate group is added. The ′ triphosphate is recognized by the innate immunity, and it activates the type I interferon response. This can be prevented by chemical synthesis of siRN“, which misses the ′-triphosphate group. Furthermore, the siRN“ molecules can be modified by adding ′-Omethyl groups, in order to reduce the recognition by toll-like receptors TLRs [ ]. Interest‐ ingly, this modification additionally hampers degradation of the siRN“ by RNases, leading to an increase in serum half-life [ ]. Finally, strong destabilizing unlocked nucleic acids UN“s , which were altered to have an acyclic ribose, also reduce the recognition by TLRs [ ]. The specific suppression of one allele in heterozygous genes is of concern in dominantly inherited genetic disorders. Huntington’s disease HD is caused by a dominant mutation of the huntingtin protein Htt and an excellent target for the examination of allele-specific knockdown of the mutated Htt, with high therapeutic potential. Huntington’s disease is based on a long stretch of C“G triplets on one disease-caused allele [ ]. Most of the patients are heterozygous for the htt gene mutation and % of the “merican and European HD patients are heterozygous at a single nucleotide polymorphism SNP site, making this genetic disease a bono fide target for specific protein knockdown. “pproaches to inhibit the appearance of Huntington’s disease is silencing of wild type and mutant Htt or silencing of only the diseasecausing allele. “lthough it was found in HD mice that co-silencing of wild type and mutant Htt provides therapeutic benefit, nothing is known of such a long-term suppression of huntingtin [ ]. Thus,

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the effect and safety over decades have yet to be proven in clinical trials. Therefore, there is still a need for high allele-specific inhibition of the mutant Htt protein, which is toxic due to an expanded polyglutamine polyQ motif C“G motif . Targeting of the C“G motif is not selective for the mutant allele and affected both alleles. Genotyping of the Huntington’s disease patients resulted in three single nucleotide polymorphisms SNP in huntingtin [ ]. Therefore, an alternative strategy when using RN“i is targeting a single nucleotide mutation localized in the disease-caused sequence [ ]. Furthermore, targeting of the mutant huntingtin SNPs or the expanded C“G motif by designed artificial miRN“s was recently demonstrated in vitro, using an allele-specific reporter system and in vivo in a transgenic mouse model [ ]. For the RN“i technique, it is possible to discriminate between very similar targets with a specific reduction on the RN“ and protein level [ , , ]. However, there are still some concerns and limitations. The RN“i-mediated allele-specific knockdown may result in a broad off-targeting and therefore has to be further evaluated in appropriate preclinical model systems [ ]. Using both target strategies, C“G motif and prevalent mutant SNPs, in the case of huntingtin, the wild-type allele is also affected by the knockdown, and the knockdown ratio between the wild type and mutant allele remains unsatisfactory. Furthermore, the shift to in vivo delivery systems can have a substantial impact on the specificity, as was demonstrated in the mouse model [ ]. Next, a limited expression of the miRN“ vectors is important to avoid saturation of the miRN“ processing machinery, as the selectivity seems to be reduced when miRN“s are highly expressed in vivo [ ]. Different alleles can also be targeted and discriminated using specific intracellular antibodies intrabodies and represent a valuable alternative to RN“ interference. Intrabodies targeting, for example, huntingtin have to recognize an epitope common in most disease-associated huntingtin SNP forms, which also has to be different in the translated amino acid between the mutant and wild-type allele. “lternatively, they could target the expanded polyglutamine polyQ motif associated with misfolding and aggregation [ ]. Furthermore, cytoplasmic intrabodies have been developed, which efficiently inhibited aggregation of mutant HD [ ]. Interestingly, a disulfide bond-free single-domain intracellular antibody with high affinity was developed after affinity maturation [ ] from a specific anti-HD scFv fragment, demonstrating the power of antibody engineering. For the allele-specific knockdown, the intrabody technology utilizes the high specificity of monoclonal antibodies, with no or low concerns about off-target effects and activation of the immune system. In the case of huntingtin, no RN“i approach was able to discriminate effectively between the wild-type and mutant expanded polyglutamine stretch [ ]. Here, intracellular antibodies could, in principle, recognize different conformational epitopes formed by polyglutamine and might be able to discriminate between the length of the polyQ motifs [ ]. However, in the case of the cytoplasmic huntingtin protein, it is more difficult to generate and select cyto-intrabodies, due to the reducing environment of the cytoplasm. In general, the allele-specific knockdown strategy should be also applied with ER intrabodies. The kind of mismatches introduced into siRN“s or artificial miRN“s, in order to increase allele specificity for preference of the mutant allele, can differ. Purine-to-purine mismatches, for example, are more effective than purine-to-pyrimidine mismatches. This limitation can be

ER-targeted Intrabodies Mediating Specific In Vivo Knockdown of Transitory Proteins in Comparison to RNAi http://dx.doi.org/10.5772/62103

overcome by introduction of a second mismatch, preferentially into the seed or cleavage region of the siRN“/miRN“ [ ]. Using a set of SNP sites, common in disease-associated alleles, might enable reaching many patients [ ], but it is hard to access the whole population. For those genotypes that could not be cured by using mutant SNP-targeting siRN“, intrabody-mediated protein knockdown, recognizing a prevalent mutant epitope could be superior. Whereby, in the case of SNPs due to the posttranslational targeting, the intrabody technology demonstrates one of its weaknesses. Discrimination between mutant and wild-type SNP could only be achieved when the mutant SNP induces a change of the encoded amino acid. In addition, mutant SNPs in introns and untranslated regions UTR cannot be addressed, as it is in the case of HD.

Features

Intrabodies

siRNA, shRNA, miRNA

Monoclonal antibody Requirements

or scFv/Fab selected by phage or yeast cell Sequence of the mRN“ surface display

Very high specificity to the antigen Stability

+

Stable in the ER

Off-target effects Susceptibility to nucleases

Inhibition of post-translational

+

-

+

+

+

+

+

+

-

+

+

+

modifications Inhibition of splice variants Inhibition of several protein isoforms with one intrabody or siRNA Targeting of specific protein domains High-throughput screening In vivo knockdown Table . Intrabodies versus siRN“

. . High-throughput screening Oligonucleotide and cDN“ microarrays can be applied for simultaneous quantitative moni‐ toring of gene expression of thousands of genes [ ]. “ combination of cDN“ microarrays and

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RN“ interference was used to validate upregulated genes, playing an important role in cancer development [ ]. In this case, a pre-screening with cDN“ microarrays is performed followed by silencing of selected upregulated mRN“s using RN“i. This might also be possible with intrabodies. “lthough high-throughput RN“i screening is very useful in order to validate new genes involved in cancer pathogenesis or infection processes [ , ], such high-throughput screen‐ ing is not possible with intrabodies. . . Therapeutic potential of siRNA and ER intrabodies The therapeutic potential of siRN“ and ER intrabodies has been shown in different mouse models [ – ]. It has been shown that siRN“ protected mice from fulminant hepatitis [ ], viral infection [ ], sepsis [ ], tumor growth [ ], and macular degeneration [ ]. In these mouse models, synthetic siRN“ was delivered systemically, peritoneally, or subretinally. Furthermore, in an “lzheimer’s and spinocerebellar ataxia disease-related mouse model, RN“i suppresses the expression of amyloid-β peptide or ataxia, respectively [ , ]. In these mouse models, target-specific RN“i was virally delivered using adeno-associated virus or Herpes simplex virus. Interestingly, the knockdown of angiopoietin- mRN“ in a mouse model with pancreatic carcinoma and xenotransplantation suppresses metastasis and down‐ regulates metalloproteinase- [ ]. Many ER intrabodies have shown therapeutic potential against relevant targets in cancer, infection, and brain diseases, for example, Erb”- , EGFR, VEGFR- , Tie- , VEGFR- × Tie- , metalloproteinases MMP- , MMP- , E oncoprotein of human papillomavirus, CCR , TLR , TLR , and amyloid-β protein [ , , – ]. Nevertheless, only four of these antigens have been applied in xenograft tumor mouse models so far, using an anti-Tie intrabody [ ], a bispecific VEGFR- × Tie- intrabody [ ], an anti-amyloid-β protein intrabody in an “lz‐ heimer’s disease mouse model [ ], and an anti-E oncoprotein intrabody in a mouse infection model with human papillomavirus [ ]. Intrabody delivery was performed via adenovirus, adeno-associated virus, and retrovirus, respectively. 2.6.1. Transgenic mice Transgenic RN“i mouse against p -Ras GTPase-activating protein [ ] and cytokineactivated Iκ” kinase IKK has been established [ ]. Furthermore, RN“i transgenic mice and non-germline genetically engineered RN“i cancer mouse models were established [ ]. In contrast to constitutive RN“i transgenic mice, generation of conditional RN“i in mice is also possible [ ]. Recently, two transgenic ER intrabody mice have been generated against VC“M and gelsolin [ , ]. In addition, a transgenic mouse expressing an anti-EVH intrabody has been pub‐ lished [ ]. However, the inhibitory results obtained with these mice have been criticized because the intrabody was directed to the secretory pathway, but confusingly recognized a cytosolic protein [ ]. Interestingly, the transgenic VC“M intrabody mouse was viable in

ER-targeted Intrabodies Mediating Specific In Vivo Knockdown of Transitory Proteins in Comparison to RNAi http://dx.doi.org/10.5772/62103

contrast to the lethal knockout mice generated by targeted homologous recombination [ ]. The intrabody mice were deficient in VC“M- cell surface expression. 2.6.2. Clinical approaches Different clinical approaches have been performed with siRN“. RN“i-based clinical trials are ongoing phase I–III [ , ]. For example, a ”evasiranib RN“i targeting VEGF has been applied to heal macular degeneration [ ] and RN“i targeting the RSV nucleocapsid SPC has shown significant anti-viral activity [ ]. In comparison to the RN“i, only one example of an ER intrabody targeting erb”- has been applied in a clinical phase I study [ ]. “s demonstrated, none of the patients treated in this study exhibited a dramatic clinical benefit. ”oth methods share the limitations of viral and non-viral delivery methods. Using integrating vectors, insertional mutagenesis is still the main problem [ ]. Concerning non-viral delivery methods, lipid-based and peptide polymer-based delivery systems have been applied [ ]. However, for some diseases like HD, the non-neurotropic feature of many delivery systems and the lack of passing the blood–brain barrier ””” remain problematic. Cell- and tissue-specific targeting is also always a concern however, transductional and transcriptional targeting is promising [ ]. Tissue-specific carrier for siRN“ includes aptamers, antibodies, peptides, proteins, and oligonucleotide agonists [ ]. Referring to ER intrabodies, the use of mRN“ in clinical approaches is promising [ ]. . . Other features Intrabodies are able to inhibit posttranslational modifications, such as phosphorylation sites [ , ]. This is not possible using RN“i. ”esides the high specificity of intrabodies, this is an important advantage of intrabodies over RN“i. Recently, single-stranded siRN“ was used to suppress the spliced variants of proteins [ ]. This might also be possible with specific intrabodies Table . In addition, targeting of specific protein domains and isomers of a protein might also be feasible. For example, miRN“ suppresses specifically an oncogenic isoform [ ]. Intriguingly, the suppression of different protein isoforms with only one intrabody or one siRN“, recognizing a common epitope within all isoforms, might be possible, for example, the knockdown of all interferon alpha isoforms different subtypes in human . . . miRNA It is known that miRN“ influences tumorigenesis [ ], and therefore miRN“ and combined miRN“/siRN“ pharmacological approaches are attractive [ ]. miRN“ has been applied in cancer mouse models as for lymphoid malignancies [ ]. Furthermore, important studies using miRN“ has been performed for diagnosis, prognosis, and prediction of cancer [ ]. One of the most developed microRN“-based candidates is MRX , a miR- mimetic that restores the function of miR- in cancer cells [ ], which is applied in an ongoing multicenter

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phase I clinical trial. The repression of expression of several potential miR- target oncogenes was demonstrated [ ]. Finally, miRN“s can be used to reprogram somatic cells into pluripotent stem cells [ ]. However, siRN“ and miRN“ share the same silencing machinery and microRN“ causes also off-target effects [ ].

. Conclusions and perspectives siRN“ and ER intrabody technology are both efficient knockdown techniques. siRN“ is acting on the mRN“ level, whereas ER intrabodies are acting on the protein level. The strength of the RN“i technology results from the possibility that nearly all mRN“s of a cell can be targeted. Currently, the knockdown of proteins mediated by intrabodies is most promising with ER intrabodies, because they are correctly folded inside the ER and can be generated more easily than in the past. ”ecause of the availability of many new scFv fragments, generated by research consortia, one cloning step is sufficient to convert selected scFv fragments into ER intrabodies. Stable cytosolic intrabodies have to be selected with considerable effort. Two approaches are successful and reliable: the intracellular antibody capture technology, based on an antigendependent two-hybrid system [ ] and single-domain antibodies [ ], which are stably folded in a reducing environment for inhibition of cytoplasmic proteins. Single-domain antibodies comprise only one V region, the variable domain of the heavy or light chain. Most successfully applied are camelid single-domain antibodies VHHs [ – ]. “lternatively, human VL and VH domains are also potent molecules and their successful construction is ongoing [ , ]. The number of ER intrabodies will increase due to the fact that international research consortia as the “ffinomics initiative [ ] in the European Union and similar initiatives in the United States have already generated several thousands of recombinant antibodies, including the Vregion genes, which can be used to build up a new repertoire of intrabodies. Using this pipeline, the duration for development of intrabodies is similar to that of siRN“/shRN“/miRN“s. In the future, scFvs against very valuable disease-related targets have to be provided. The main advantage of intrabodies is their specificity, no off-target effects, and posttransla‐ tional modification inhibition. The specificity of an intrabody can be estimated by immuno‐ assays such as ELIS“, flow cytometry, and immunoprecipitation. On the contrary, the specificity and off-target effects of RN“i are often more difficult to predict. Conferring to in vivo application, RN“i has been currently applied predominantly in phase and studies [ , ]. In the future, the success of clinical approaches using RN“i and ER intrabodies is dependent on the development of safe viral vectors and the development of nonviral vectors possessing high transfection efficiency [ ]. Two attractive applications of RN“i, hardly to perform with ER intrabodies, are genome-wide screening [ , ] and reprogramming of somatic cells into pluripotent stem cells [ ]. Thus, the ER intrabody approach has demonstrated its huge potential for in vitro and in vivo analysis of protein function [ ]. The ER intrabody technique can complement the RN“i

ER-targeted Intrabodies Mediating Specific In Vivo Knockdown of Transitory Proteins in Comparison to RNAi http://dx.doi.org/10.5772/62103

technique in cases where siRN“, shRN“, and miRN“ molecules demonstrate unwanted unspecificity and off-target effects.

Acknowledgements The authors thank Prof. Peter M(ller for critical reading of the manuscript.

Author details Oliver ”ackhaus and Thomas ”öldicke* *“ddress all correspondence to: [email protected] Helmholtz Centre for Infection Research, Department of Structural and Functional Protein Research, ”raunschweig, Germany

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Chapter 8

RNAi Therapeutic Potentials and Prospects in CNS Disease Kyoung Joo Cho and Gyung Whan Kim Additional information is available at the end of the chapter http://dx.doi.org/10.5772/62896

Abstract Over the past years, RN“ interference RN“i technology has provided a new regula‐ tory paradigm in biology. This technique can efficiently suppress target genes of interest in mammalian cells. Small non-coding RN“s play important roles in gene regulation, in‐ cluding both in post-transcriptional and in translational regulation. For in vivo experi‐ ments, continuous development has resulted in successful new ways of designing, identifying, and delivering small interfering RN“s siRN“s . Proof-of-principle studies in vivo have clearly demonstrated that both viral and non-viral delivery methods can pro‐ vide selective and potent target gene suppression without any clear toxic effects. There are also the persistent problems with off-target effects OTEs , competition with cellular RN“i components, and effective delivery in vivo. “lthough recent researches and trials from a large number of animal model studies have confirmed that most OTEs are not dangerous, other important issues need to be addressed before RN“i-based drugs are ready for clinical use. Currently, RN“i may be harnessed as a new therapeutic modality for brain diseases. Finally, there are already several RN“i-based human clinical trials in progress. It is hoped that this technology will have also effective applications in human central nervous system CNS -related disease. Keywords: RN“i therapy, brain, neurodegenerative disease, allele-specific, neurovascu‐ lar

. Introduction During developmental stage and in response to internal and external cellular stresses, small RN“ molecules regulate gene expression [ ]. Specialized ribonucleases and RN“-binding proteins govern the production and action of small regulatory RN“s [ ]. In most eukaryotic cells, RN“ interference RN“i is a regulatory mechanism using small double-stranded RN“

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dsRN“ molecules to direct homology-dependent control of gene activity [ , ]. Small size [ – nucleotide nt ] non-coding RN“s and associated proteins regulate the expression of genetic information [ ]. The discovery of RN“i phenomenon widened our understanding of gene regulation and revealed related pathways in small RN“s [ ]. “s it processes, RN“i has been finding widespread in plants [ ] and animals [ ]. Each small RN“ associates with an “rgonaute “GO family protein to form a sequence-specific complex. “fter then, genesilencing ribonucleo-protein complex with specificity conferred by base pairing between the small RN“ guide RN“ and its target mRN“ [ ]. The pathway is well known as the RN“induced silencing complex RISC , which gives a target mRN“ silencing by degradation or transcriptional regression [ ]. Small interfering RN“s siRN“s loaded into RISC are doublestranded, and “GO- , which having an active catalytic domain in human, cleaves and releases the passenger strand. RISC is activated with a single-stranded guide RN“ molecule to impose the specificity recognizing the target by intermolecular base pairing [ ]. MicroRN“s miRN“s are other endogenous substrates for the RN“i machinery, but the cellular origins of miRN“ and siRN“ are distinct. miRN“s are derived from the genome, whereas siRN“s may be endogenous or arise through viral infection or other exogenous sources [ ]. Typically, miRN“s are initially expressed in the nucleus with a transcript as long as primary miRN“ pri-miRN“ , and the transcripts are at least over nt. Pri-miRN“s are processed by the microprocessor complex histone deacetylase proteins consisting in DroshaDGCR [DiGeorge critical region a double cysteine-ligated Fe III heme protein DGCR ] in the nucleus [ , ]. They are cleaved in the nucleus into – base pair bp hairpins, which are consisted in single-stranded ′- and ′-terminal overhangs and about -nt distal loops [ ]. In cytoplasm, the loop is further processed by the RN“se III Dicer, and one strand is loaded onto RISC. The mature miRN“s bind to the ′ UTR of target mRN“s and then degrade the target [ ]. Despite their differing origins, these RN“ processing pathways converge once either type of RN“ assembles into the RISC. With development of an efficient delivery system in various diseases, RN“i has been an emerging therapeutic approach for in vivo studies with specific synthetic siRN“s against each disease. It should be considered as novel and interesting therapeutic challenge with the major concern how to administer the siRN“s with specific, efficient, and targeted way. Despite some hurdles for applying to clinical challenges such as anatomical barriers, drug stability and availability, various delivery routes, and different genetic backgrounds, an application of siRN“s has become extremely attractive in development of new drugs. Currently, one of the important challenges in siRN“ bioinformatics is target prediction, when there is still no proper tool with certain drug design grade. ”esides specific challenges in siRN“ therapeutics, an efficient delivery method, targeting a specific tissue or cell, is another fundamental challenge. This chapter introduced two of main themes. The first is the possibilities of therapeutics using RN“i principles and technique. The second is the challenges with siRN“s or miRN“s specifically in the area of brain disease. In addition, this chapter provided some prospects of siRN“s or miRN“s on disease prognosis, progress, and therapeutics in the present and future.

RNAi Therapeutic Potentials and Prospects in CNS Disease http://dx.doi.org/10.5772/62896

. Principles of RNAi therapy “s far as it is true that siRN“ has promising benefits, and, concomitantly, siRN“ has still some of technological barriers to be widely used in clinical therapy, which generally due to the lack of efficient delivery tools. To success with siRN“ therapies, an effective and safe carrier system is required that would overcome the inherent defects of siRN“ and achieve maximum genesilencing effect. There are many approaches that are being developed to achieve the efficient delivery of siRN“. In that, non-viral vectors have advantages of reproducibility, low immu‐ nogenicity, and relatively low production cost [ ] therefore, non-viral vectors made siRN“ to be a potential therapeutic and nucleic acid–based drugs, such as plasmid DN“s or antisense oligonucleotides “SOs [ ]. . . Advantages of RNAi Theoretically, all disease-associated genes could be amenable to antisense-mediated RN“i suppression. RN“i can be a strategy for silencing of virtually all annotated protein-encoding genes in the human genome in large scale. The high specificity of siRN“ lets targeting of disease-specific alleles that differ from the normal allele by only one or few nucleotide substitutions. This high fidelity and specificity of siRN“s are useful for targeting for some oncogenes, too. The first advantage is the powerfulness of RN“i when compared with other antisense strategies, such as antisense DN“ oligonucleotides and ribozymes [ ]. It is important fact that the effector molecules work at much lower concentration than any other antisense oligomers or ribozymes, suggesting that RN“i has higher potency. This is a critical point to set thera‐ peutics. The second is efficacy. The efficacy is generally presented by the half level of maximal inhibition or the value of IC against target site. The efficacy level is crucial for determining thermodynamic stability [ ], targeted gene accessibility [ ], or structure [ ] of designed siRN“. For designing siRN“, the most important thing is end stability that is different from each end and is also meaning asymmetry and consistent with selected miRN“ [ ]. However, to date, our knowledge of siRN“ and the selection of targets are incomplete and being explored. The identification of hyperfunctional siRN“s, functioning at sub-nanomolar concentration, remains an elusive task. . . Basic strategies for targeting-specific molecules RN“i can be triggered by two different pathways: a RN“-based approach, where the nt long duplexed siRN“ effectors are delivered to target cells, and a DN“-based strategy, where the siRN“ effectors are produced by intracellular processing of longer RN“ hairpin transcripts [ ]. DN“-based strategy is based on short hairpin RN“ shRN“ synthesis in nucleus and transportation to the cytoplasm through miRN“ machinery, which subsequently is processed by Dicer. “lthough the direct use of siRN“ effectors is simple and effective way for gene silencing, the effect is transient. Therefore, it is costly for clinical usage due to the need

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of multiple large-scale application. In contrast, DN“-based RN“i drugs have the potential and stably introduced for application in a gene therapy. In principle, DN“-based RN“i allows a single treatment of viral vector that delivers shRN“ genes to the targeted cells/or tissues. . . Delivery routes for targeting The effective delivery of siRN“s acts to be significant step in accelerating RN“i-based treatments. The instability of RN“ and the relatively inefficient encapsulation process of siRN“ remain critical issues toward the clinical translation of RN“i as a therapeutic tool. There are several obstacles for extracellular introduction of siRN“ to deliver the target. Under normal physiological condition, the introduced molecules ought to have a positive charge to diffuse to cell membrane [ ]. It is the simplest way of naked nucleotides or transfecting siRN“s to deliver into cells [ ]. “nother technique is microinjection and electroporation for direct delivery, but it has higher level of cellular toxicity [ ]. The delivery routes can be intraperitoneal, intra-vascular, intra-muscular, intra-splenic, intra-cranial, and intra-tumoral injection. In addition, siRN“s can be delivered through subretinal, subcutaneous, mucosal, topical application, and oral ingestion to improve delivery [ ]. However, these transfection processes should be optimized for siRN“ concentration, cell density, and ratio of transfection reagent to siRN“ [ ]. Carriers for delivery of siRN“ with cationic environment surrounding of siRN“s can be liposomes and dendrimers. These carriers reduce the nuclease activity and improve siRN“ delivery into cells [ ]. Microsponge is one of the mediators for siRN“ delivery. Carrier and cargo combine and selfassemble into nanoscale pleated sheets of hairpin RN“. Subsequently, this complex forms sponge-like microspheres [ ]. The complex of siRN“ and microsponges consists in cleavable RN“ strands, and the stable hairpin RN“ converts into working siRN“ once cells uptake the complex. Therefore, it can provide a protection for siRN“ during delivery and transport it to the cytoplasm. Single microsponge complex can deliver more than half a million copies of siRN“ when uptaken into a cell [ ]. . . Stabilizing the siRNA delivery The stability of the siRN“ complexes, penetrating into target cells without stimulating immune responses, is one of the limiting factors and the major bottleneck for developing siRN“ therapeutic tools. It restricts the delivery of siRN“ macromolecular complexes to the desired cell types, tissues, or organs. Usually, siRN“s do not easily penetrate the cellular membrane because of their negative charge and macromolecular size. Manipulation of nucleotide bases is needed to increase stability and protein interactions, which can harness to increase the structural improvement of siRN“s [ ]. The delivery systems for siRN“ consist of four main methods, namely naked, lipid-based, peptide-based, and polymer-based delivery [ ]. ”asically, polymer-based methods are similar to lipid-based methods in targeting, except some special triggers, such as temperature, pH, or pulse release [ ]. Initial efforts to improve stability addressed above were focused on incorporating chemical modifications into the sugar backbone or bases of siRN“ duplexes [ ]. The modified siRN“

RNAi Therapeutic Potentials and Prospects in CNS Disease http://dx.doi.org/10.5772/62896

molecules increased stability, which effectively lowered the dose to achieve measurable and reproducible gene silencing [ ]. Several modifications were introduced. The thio −SH , hydroxyl −OH , or iodo −I can modify bases in specific sites or utilize the pseudouracil base in siRN“, which would augment potency of naked siRN“ [ ]. There are three most popular chemical modification sites on siRN“ structure containing the phosphodiester backbone, ribose ′-hydroxyl group R- ′-OH , and ribose ring. Endogenous cellular endonucleases can easily digest phosphodiester bond in RN“ backbone [ ]. “lternative modification is oxygen bridges of RN“ backbone that can be replaced with phosphorothioate, although it would increase toxicity and reduce silencing activity [ , ]. “nother alternative is boranophosphate linkages. These are more nuclease resistant and less toxic compared to phosphorothioate [ ]. Phosphonoacetate linkages are other candidates [ ]. The linkage is completely resistant to nuclease and is electrochemically neutral when they are esterified [ , ]. “nother modifica‐ tion is ′-O-methoxyethyl ′-O-MOE , ′-O-alkyl, and other bulky groups. These modifica‐ tions can improve anti-nuclease shield of siRN“ that simultaneously makes them less tolerable when they are positioned on ′ overhangs [ ]. Despite disturbing thermodynamic asymmetry of siRN“ by addition of ′-aminoethyl at ′ end of passenger strand, this modification improves efficiency of target silencing [ ]. On the other hand, alterations in sugar compartment of nucleotides reduced flexibility and nuclease sensitivity of siRN“ structure [ , ]. ”inding of ribose ′O into ′C with methylene bridges, which finally produces oxetane, forms a locked conformation nucleic acid locked nucleic acid LN“ [ ]. In vivo nuclease resistance of this structure is enhanced [ ]. In contrast to LN“, derivatives of RN“ without C ′–C ′ sugar bonds unlocked nucleic acid UN“ destabilize a sequence structure [ ]. Substitution of pentose with hexose monosac‐ charides, such as cyclohexenyl, anitrol, and arabinose, was applied to develop CeN“, “N“, and ′-F-“N“ [ ], subsequently resulting in enhanced stability of siRN“ in vivo [ ]. During systemic delivery, however, internal modifications failed to improve central nervous system CNS entry and uptake. Researchers put new efforts to move toward using liposomes, nanoparticles, and cell-penetrating peptides CPPs , among others, to stabilize and navigate siRN“s into and throughout the brain [ ]. 2.4.1. Liposomes Generally, liposomes are classified into three classes: multilmellar vesicle . ~ µm , small unilamellar vesicles ~ nm , and large unilamellar vesicles ~ nm [ ]. Liposomes are developed for passive or active targeting mechanisms in different complexes of liposome and other interacting molecules, namely lipoplex cationic liposome-pDN“ complex , liposome polycationic DN“, mannose liposome, and so on [ , ]. The siRN“ with mannose Man -coated liposomes would be useful for treatment of some cancers, especially liver and brain cancers [ ]. 2.4.2. Dendrimers Dendrimers are hyper-branched, tree-shaped, and -D structures [ ]. Dendrimer can utilize broad spectrum, and the broad range of functional groups makes it possible to introduce

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dendrimers with extensive applications. There are different classes of cationic and anionic dendrimers, such as polyamidoamine P“M“M , polypropylene imine PPI , and polyethy‐ lene glycol PEG -grafted carbosilane [ ]. Specific dendritic polymers like P“M“M have been widely utilized in in vivo drug delivery [ ]. Conjugation of Tat peptide GRKKRRQRRRPQ with P“M“M-G can efficiently inhibit multi-drug resistance- MDR- gene expression in vitro [ ]. Capping poly-l-lysine PLL dendrimers with methotrexate enhances stability and decreases toxicity [ ]. 2.4.3. Cationic polymers Cationic polymers include chitosan, gelatin, cationic dextran, cationic cellulose, and cationic cyclodextrin and some synthetic biocompatible polyethyleneimine PEI , PLL, poly amidoa‐ mine s P““s , poly amino-co-ester , and poly -N,N-dimethylaminoethylmethacrylate . Moreover, they are less immunogenic response because these polymers are natural biode‐ gradable [ ]. 2.4.4. Cationic peptides CPPs are cationic peptides. CPPs interact covalently or non-covalently through disulfide or electrostatic–hydrogen interactions with siRN“s [ ]. Viral protein VP [ ], MPG a peptide vector [ ], amphipathic peptide [ ], and poly-arginine [ ] were reported the same abilities. In addition, small cationic polypeptides poly His, Lys, and “rg coat and neutralize siRN“ helping to pass through membrane [ ]. 2.4.5. Nanoparticles For systemic delivery, a targeted nanocarrier-siRN“ complex has been used. There are some studies that have experimentally condensed DN“ or RN“ into cancer-targeted nanoparticles with PEI, PLL, and cyclodextrin-containing polymers [ ]. PEI–PEG–arginine–glycine– aspartic acid RGD fusion was used to inhibit vascular endothelial growth factor receptorVEGFR- expression [ ]. “ngiogenesis can be inhibited by downregulation or silencing of VEGFR- expression [ ]. PEGylation of nanoparticles causes muco-inert properties, which enhances diffusion process through mucus and peptidoglycan barriers [ ]. 2.4.6. Aptamer siRN“s can be coupled with aptamers or oligodeoxynucleotide through a disulfide bond. This releases actively into targeted cells siRN“s before cytosolic uptake. Conjugate of aptamer siRN“ has suggested a novel therapeutics with widespread applications in medicine [ ]. . . Limitations 2.5.1. Competition with endogenous RNAs In human brain diseases and normal brain development, RN“i potentiates the important role in normal neuronal function, although it is underestimated. When exogenous shRN“ is

RNAi Therapeutic Potentials and Prospects in CNS Disease http://dx.doi.org/10.5772/62896

introduced into the neuron, it might be considered whether the RN“i machinery perturbs normal physiologic condition of the system. ”ioactive drugs that rely on cellular processing to exert their action face the risk of saturating such pathways and hence perturb the natural system. Sometimes, ectopically introduced RN“i does not trigger the silencing process because siRN“/shRN“ activity may depend on the endogenous miRN“ to achieve efficient target silencing. Mice that received liver-directed associate adeovirus ““V -encoded shRN“s were damaged in liver with dose-dependent manner. Within months, the mice were killed by introducing high doses of ““V-encoded shRN“s. It was interpreted that the liver-specific miRN“ was unexpectedly down-regulated by introducing shRN“ [ ]. The enhanced expression of Exportin , the nuclear export component, increased RN“i efficacy, which was shown by competition assay [ ]. 2.5.2. Stimulation of innate immune responses RN“i therapy is importantly considered because of its potential for generating an adverse immune response, particularly in neurodegenerative diseases with affected brain. It has been already known as heightened state of alert to start chronic pro-inflammatory signaling cascades [ ]. “ll evolutionary conserved mechanisms aimed at combating against invading viral pathogens [ ]. In general, innate immune responses to non-virally delivered siRN“s are mediated by members of the toll-like receptor TLR family or by the two different dsRN“sensing proteins: retinoic acid-inducible gene- and dsRN“-binding protein kinase [ ]. Certain siRN“ sequence motifs invoked TLR -dependent immune stimulation [ ]. The particular sequence motif ′-GUCCUUC““- ′ seems to be recognized by TLR in plasma‐ cytoid dendritic cells and activates immune responses. The GU-rich regions, so-called danger motifs, stimulated innate immune responses and lead to secretion of inflammatory cytokines in a cell type and sequence-specific manner. “s siRN“-mediated immune induction seems to rely on endosome-located TLR receptors TLR and TLR [ ], the delivery and compart‐ mentalization of the siRN“ significantly influence the cellular responses [ ]. These interactions can occur during endosomal or lysosomal compartments’ internalization or intracellular release of the siRN“ molecule. It has a manner of dose and sequence dependence. Importantly, the chemically modified or nanoparticle-encased siRN“ complexes avoid stimulation of immune response. 2.5.3. Suppression of off targets Harmfulness of RN“i is OTE. Genome-wide sequencing analyses have clearly demonstrat‐ ed that siRN“-treated cells show off-target silencing of a large number of genes [ ]. The research result suggests that siRN“s with a ′-O-MOE modification at the second base can significantly reduce off target without compromising the degree of silencing target [ ]. Experimentally, it has been verified that off targets have ~ -nt long matching to the siRN“, and it is called seed region [ ]. When the siRN“ guide strand contains seed-sequence matching to mRN“ ′-UTR regions, the siRN“ guide strand functions as a miRN“, which might lead to harmful OTEs by translational repression [ ]. To avoid siRN“ seed matching with mRN“ ′ UTRs, the use of online ′-UTR search algorithms would potentially reduce the detrimental OTEs [ ].

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The OTEs can also derive from non-specific changes in gene expression due to the activation of the interferon response IR [ ]. The OTEs can change another gene by binding either strand of the shRN“ to partially complementary sequences rather than binding to the intended target gene [ ]. In case of dsRN“, it can result in a signaling cascade that culminates with the activation of interferon responsive genes and global translational repression [ ]. Neverthe‐ less, IR activation was variable among the siRN“s used for each of these studies, and one recent report did not detect IR activation by siRN“s [ ]. In mice, injection of naked siRN“ did not show detectable induction of an IR in one study while another study showed sequencedependent induction of innate immunity [ , ].

. Applications of RNAi RN“i has been used to generate model systems to identify novel molecular targets [ ], to study gene function [ ], and to create a new niche for clinical therapeutics [ ]. Many researchers reported that siRN“s have successfully been tested in various disease animal models. Recent reports reviewed the therapeutic potential of synthetic siRN“s in various human diseases and disorders [ ]. . . Application for therapy with RNAi in vivo “pplications, such as gene function analysis, target identification and validation, and thera‐ peutic agents, are the main spots of this new technology [ ]. “lthough RN“i is an efficient technique for in vitro studies, there are some challenges for in vivo applications. siRN“s have undesired characteristics, such as non-specific silencing of non-targeted genes and dosedependent immunogenic response [ ]. In addition, it is extremely complicated to avoid the OTEs due to spatiotemporal gene expression pattern of these molecules [ ]. Furthermore, age, sex, tissue, organ, tumor, and individual-specific specificity should be also considered as other variables [ ]. Prediction of susceptible off-target domains that can influence silencing efficiency is the first step for applying in vivo therapy [ , ]. Some studies recommend utilization of more sensitive alignment algorithms or siDirect instead of ”L“ST database [ , ] to predict a target for siRN“ matching without cross-reactivity [ ]. The administration route for siRN“, such as oral or intravenous, is not feasible and not efficiently delivered the siRN“ into target cells. “ single injection of naked siRN“ into the brain parenchyma failed to good efficacy [ ]. “ study reported that continuous infusion of siRN“ into the ventricular CSF success with very high concentration [ ]. To penetrate the blood–brain barrier ””” and reach the target cells in the interesting site, receptor-specific pegylated immunoliposome PIG is used. PIGs encapsulate the plasmid vector–encoding siRN“ or shRN“ and are administered with peripheral route to the brain. This tool has been tried in brain cancer animal model and successfully worked [ ]. “nother study showed effective and long-term knock down of endogenous tyrosine hydroxylase TH in rodent brain using shRN“-expressing adeno-associated virus ““V [ ]. There have been many successful in vivo studies with using viral vector. They are included two models of autoimmune hepatitis

RNAi Therapeutic Potentials and Prospects in CNS Disease http://dx.doi.org/10.5772/62896

[ ], hepatitis ” virus [ ], respiratory viruses such as influenza virus [ ], respiratory syncytial virus [ ], parainfluenza virus, and sexually transmitted disease such as herpes simplex virus[ ]. ”oth non-viral and viral shRN“ delivery systems have been trailed. . . Application for therapy with RNAi in brain diseases Many works using RN“i to suppress dominant disease genes have occurred primarily in cell culture models [ , ]. “llele-specific silencing aims to suppress the disease gene without affecting any other normal genes. The possible therapeutic applications of RN“i for neuro‐ logical diseases are broad, ranging from acquired diseases, such as viral infections, to purely genetic disorders. Particularly, one attractive group of candidate diseases for RN“i therapy is the dominantly inherited neurodegenerative diseases, including polyglutamine disorders such as Hunting‐ ton’s disease HD [ ], amyotrophic lateral sclerosis “LS [ ], familial “lzheimer’s disease “D [ ], and frontotemporal dementia caused by tau mutations [ ]. HD has been approaching with animal model mimicking the human disease to provide some therapeutic clues with various ways. In the new preclinical study, single injection of a cholesterol conju‐ gated-siRN“ was targeting mutant Huntingtin mhtt , and, subsequently, the pathologic symptoms containing behavioral dysfunction were improved [ ]. The exciting recent works have taken place in vivo in mouse models of neurodegenerative brain disease. The best example of RN“i-mediated therapy to date is in spinocerebellar ataxia typeSC“- [ ]. “s another case, RN“i-mediated therapy was tried on DYT dystonia with animal disease model. DYT dystonia is another inherited dystonia. DYT dystonia is caused by deletion of G“G that is coding TOR “, which results in one of a pair of glutamic acid from the carboxyl terminal of the torsin “ T“ protein-coding region [ ]. Prion disease is one of the brain diseases that is invariably fatal, and no therapy is available. Once serious damage to the brain has already occurred, clinical symptoms manifest after the untreatable brain damage. Causing this reason, prion disease treatments have aimed not to cure the disease but to slow disease progression [ ]. Prion disease is caused by prions, in which a self-replicating, infectious protease resistant form of PrP termed PrPSc , is the only essential component identified to date. PrPSc multiplies through conversion of the normal cellular PrP PrPC [ ]. Some reviews are presenting that lentivector-mediated anti-PrPC shRN“ expression effectively suppressed prion replication in a murine neuroblastoma cell line, and researchers created chimeric mice using embryonic stem cells, which were transfected with a lentiviral vector carrying an anti-PrPC shRN“. Results showed that the survival time after prion inoculation was markedly prolonged [ , ].

. Prospects of RNA therapeutics in CNS disease The current phamaceuticals required more knowledge to decipher potentials of the RN“i in spite of flourishing future. It is crucial that each disease has not only a unique pattern but also

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the understanding for pathogenesis relating pathways and activating or inhibiting factors [ ]. To introduce the DN“ therapeutics into the CNS is much more complicated due to the ”””, which can be only permeable to lipophilic molecules of less than Da [ ]. Using human viruses, DN“ delivery system has been extensively trailed for over three decades. However, the results have been not satisfactory. Therefore, a critical goal for clinical neuro‐ science is to develop the efficient RN“i therapy to prevent the neuronal damage [ ]. We categorized the neurological disease containing cancers in below sections. . . Genetic neuronal disease-familial neurological disease The application of siRN“ has been advanced in development of various incurable disease therapies, apart from the widespread usage of RN“i in fundamental biological application. Particularly, dominant inherited disorders are major application field. “mong familial neurobiological diseases, HD has been tried to lots of therapies based on RN“i and may be beneficial effect from the therapy using siRN“. In the N - Q transgenic HD mouse model, a study using shRN“ showed a – % decrease in the N - Q mRN“ when injected to striatum and a complete elimination of mHtt protein inclusions from the neuronal cells [ ]. There was also a rescue of motor dysfunctions. siRN“s against the R / huntingtin htt mRN“ reduced brain atrophy and neuronal inclusions in the R / transgenic mouse model [ ]. With using a r““V vector and administrating to the striatum, long-term expression of a mHtt-siRN“ partially reduced in neuropathology condition [ ]. ”esides ““V, there is lentiviral vector that can be applied after onset of symptoms [ ]. Using lentivirus vector decreased htt protein expression by up to % and altered htt-related pathways but did not reduce cellular viability for at least months after treatment. To enhance cellular uptake of siRN“, cholesterol-conjugated duplexes cc-siRN“ have been applied to target htt mRN“ [ ]. “llele-specific targeting of mhtt helped to overcome the side effects of RN“i where “SO or single nucleotide polymorphisms SNPs in the mHtt allele have been used to specifically target only the mutant gene product [ ]. Intra-cellular antibody frag‐ ments bind to abnormal aggregations, and allele-specific siRN“ disrupts mhtt gene [ , ]. Targeting of just three SNPs with five siRN“s covered most of the HD patients in the population studied [ ]. Tuberous sclerosis is a common, dominantly inherited disorder caused by mutations in the tumor suppressor complex- TSC or tumor suppressor complex- TSC genes [ ]. The proteins hamartin encoded by TSC and tuberin encoded by TSC form a complex. This protein complex represses mTOR-S K- E-”P signaling pathway [ ]. Mutated TSC and TSC lead to loss of activity resulting in unchecked cell growth and hamartoma formation in the CNS. Recent studies propose that the target may be the GTPase Rheb [ ]. RN“i sup‐ pression of Rheb might respond the dysregulated cell proliferation in tuberous sclerosis. Particularly, allele-specific silencing is apt for inherited neurological diseases. DYT is the most commonly inherited dystonia [ ]. “lthough the pathogenesis of DYT is unclear, several facts make DYT a good candidate to explore the therapeutic potential of RN“i [ ]. The three nucleotide difference between the wild type and the mutated gene has been enough to allow

RNAi Therapeutic Potentials and Prospects in CNS Disease http://dx.doi.org/10.5772/62896

allele-specific silencing against mutant T“ the mutated protein in DYT using in vitro synthesized siRN“ [ ].

in cultured cells

“llelic discrimination has also been demonstrated for superoxide dismutase SOD mutations responsible for familial “LS [ ], and also a mutation in an acetylcholine receptor subunit causes congenital myasthenia [ ]. In a tau mutation responsible for fronto-temporal dementia, siRN“s can act by discriminating between sequences differing by a single nucleo‐ tide [ ]. “n important role for RN“i in the brain is also presented for Fragile X syndrome FXS in human [ ]. FXS is the one of the most common forms of inherited mental retardation caused by mutations in Fragile X Mental Retardation Protein FMRP , a protein influencing synaptic plasticity [ ]. FXS is stemmed from mutations in FMRP and is supported by the involvement of the RN“i process in human neurological disease [ ]. Increasing evidences from different studies support the view that FMRP regulates protein translation by regulating RN“i in neurons [ , ]. . . Sporadic neurodegenerative diseases Neurodegenerative diseases are age dependent, and many of them are inherited. However, non-genetic neurological diseases, such as sporadic “D or migraine, are much more common than diseases due to single-gene mutations. The most common sporadic neurodegenerative disease, “D, is also the best studied with siRN“ therapy. Many studies of “D pathogenesis investigate an essential role for β-amyloid “β in familial and sporadic forms of “D [ ]. Different RN“i strategies have been applied to regulate this pathogenic cascade. Researchers tried by directly silencing of amyloid precursor protein “PP [ ], by silencing of β-secretase ”“CE that is one of two proteases required for “β production but not essential gene in mice [ ], or by silencing of tau expres‐ sion that is a component of the neurofibrillary tangles of “D neurons. Therapeutic use of RN“i is now being tested in animal models of “D targeting these proteins. Migraine, one of the most common neurological disorders, is caused by diminished production of calcitonin gene-related peptide CGPR in the trigeminal system. CGPR can protect from migraine attacks [ ]. The CGPR-limited animals are normal, but the paroxysmal nature of this disorder necessitates to use promoters for CGPR. From the beginning of the pathogenic cascade, expression of the shRN“ targeting CGPR can terminate the growing pain of this disease. This pain alleviating therapy for migraine is limited because of high threshold dose needed for RN“i [ ]. . . Motor dysfunction disease “ viral delivery of shRN“ was used to achieve a long-term RN“i in the CNS. In some reports, the delivery of shRN“-expressing lentivirus showed a rescue of spinal motor neurons with behavioral and histopathological phenotypes in a mouse model having dominant familial “LS [ ].

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Parkinson’s disease PD is the second most common neurodegenerative disease. Patient brain of PD is often littered with Lewy body, which is abnormal protein aggregate primarily of alphasynuclein α-syn [ , ]. parkinsonism is linked to hereditary to a single-point mutation in the α-syn as well as genetic duplication or triplication of the α-syn SCN“ [ ]. The studies targeting the α-syn expression revealed RN“i as a therapeutic approach to PD [ , ]. To date, conflicting results were reported. Regarding the effectiveness and tolerability, there is a report that nigrostriatal degeneration was detected after depleting the α-syn level in the brain [ ]. It can be inferred that RN“i approaches can be used to validate them in genetic and sporadic models of PD. . . Neurovascular disease RN“i can be applied to cardiovascular and cerebrovascular diseases. Cardiovascular disease results from the progressive occlusion of arteries, and it is most common in a process called atherosclerosis, which can ultimately culminate in a myocardial infarction or stroke [ ]. It may be a trigger for the death of cardiac muscle cells or neurons [ ]. “lthough some of the cells die rapidly by necrosis, many other cells die more slowly by apoptosis in such cardiac myocytes and brain neurons [ , ]. RN“i technology may be used to intervene in athero‐ sclerosis or to reduce the damage of heart tissue and brain cells following a myocardial infarction or stroke [ ]. “nother vascular disease is an ocular disease. Representatively, there were two RN“i clinical trials. The trials performed direct intra-vitreal injection of siRN“s that are targeting VEGF or the VEGFR to test for the safety and efficacy in ocular diseases [ ]. siRN“s, targeting VEGF and VEGFR , are currently in the early stages of clinical trials. The direct injection approach can also prove its usefulness for the other ocular diseases. . . Cancer “ chemo-resistance or radio resistance is a major obstacle in cancer treatment. Targeted therapies that enhance cancer cell sensitivity have the potential to increase drug efficacy while reducing toxic effects on untargeted cells . “ctually, oncogenes expressed at abnormally high levels are attractive targets for RN“i-based therapies against cancers [ ], and such approaches have effectively inhibited tumor growth in vivo in mouse models. In nasopharyngeal carcinoma, hyaluronan receptor CD gene silencing resulted in pro‐ found reduction of malignant potential of the cells: tumorigenesis and metastasis of tumors in nude mice [ , ]. It is also suggested a possible therapeutic effect of direct introduction of siRN“ to CD into some human solid tumors with high expression of the CD gene [ ]. “lthough the role of epidermal growth factor receptor EGFR in altering tumor chemosensi‐ tivity has not yet been fully elucidated, selectively targeting EGFR supplies the reversal possibility of chemoresistance in many tumor types [ ]. Reduction of EGFR expression and increased chemosensitivity to docetaxel are emerging an effective strategy for the sensitization of cancer cells to taxane chemotherapy [ ]. siRN“-PG-“mine polyplexes can be systemically

RNAi Therapeutic Potentials and Prospects in CNS Disease http://dx.doi.org/10.5772/62896

delivered to tumors in mice [ ], and siRN“-nanocarrier system can efficiently inhibit expression of a specific gene in tumor cells. Once the intact siRN“ molecule moves to the target, the gene of interest gets silenced. The PG-amine-based delivery system actually combines both tumor passive targeting with the sequence selectivity of siRN“ [ ]. The limiting point of targeted therapy is alternative pathway compensation by gene amplifi‐ cation. The synthetic lethality is proposed idea to overcome the above problem [ ]. This concept suggests that two genes may be considered to have a synthetically lethal relationship [ ]. When a mutation is existed either of the two genes alone has no effect on cell survival, but when mutations in both genes cell death is triggered at the same time. ”y genome-wide RN“i library screening, some synthetic lethal molecules have been discovered. “naphasepromoting complex/cyclosome “PC/C and polo-like kinase PLK are synthetically lethal with the R“S oncogene in colorectal cancer [ ]. The STK gene is also synthetic lethal interacting with a R“S mutation in multiple cancer cells from different tumor types [ ]. Modified EGFR amplification or truncation and hyperactivation of “KT play a major role in the development of glioblastoma, one of the extreme malignancies [ ]. There are approaches to develop the siRN“ delivery efficiency such as the use of dsRN“-binding domain DR”D with a T“T peptide transduction domain PTD delivery peptide [ ]. These facilities are stable and efficient delivery of siRN“s into cells [ ].

. Conclusions Small RN“s and non-coding small RN“s were important discovery for molecular cell biology these small RN“s have a vital role in gene regulation that can be controlled by RN“ interfering technology. Presently, attempts to integrate gene expression profiling and protein interaction mapping are the main research objectives. The proof-of-principle studies in vivo have clearly demonstrated that both viral and non-viral delivery methods can provide selective and efficient target gene suppression without any clear toxic effects. Initial results have been very promising, and many pharmaceutical companies are already focusing on commercialization of various disease-specific RN“i drugs. Despite successful trials in a large number of animal model studies including brain diseases, to develop an efficient therapeutic application, there are numerous hurdles and concerns regarding targeted delivery of siRN“s into brain subre‐ gions that must be overcome before wide clinical application of RN“i as a new therapeutic solution. The OTEs, competition with endogenous cellular RN“i components, and effective delivery in vivo remain to be optimized. “lthough recent research has improved the safety and toxicity from the OTEs, it still remains a crucial issue and needs to be addressed before RN“ibased drugs are ready for clinical use. Translational research using RN“i has taken place with an unprecedented speed, and already there are several RN“i-based human clinical trials in progress that will provide breakthrough therapeutic tools for effective treatment human CNSrelated disease.

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Author details Kyoung Joo Cho and Gyung Whan Kim* *“ddress all correspondence to: [email protected] Department of Neurology, Severance Hospital, Yonsei University College of Medicine, Seoul, South Korea

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Chapter 9

RNAi-based Gene Therapy for Blood Genetic Diseases Mengyu Hu, Qiankun Ni, Yuxia Yang and Jianyuan Luo Additional information is available at the end of the chapter http://dx.doi.org/10.5772/61644

Abstract Therapies for blood genetic diseases can be divided into different categories, including chemotherapy, radiotherapy, gene therapy, and hematopoietic stem cell transplantation. “mong these treatments, gene targeting is progressively becoming a therapeutic alterna‐ tive that offers the possibility of a permanent cure for certain blood genetic diseases. In recent years, gene therapy has played a more important role in curing genetic blood dis‐ orders. RN“ interference RN“i is one of the directions for gene therapy, which was in‐ tensively studied in the past decades for its potentials in the treatment of diseases. In order to provide useful references and prospective directions for further studies concern‐ ing RN“i-based gene therapy for blood genetic diseases, current RN“i-based gene thera‐ pies for several typical blood genetic diseases have been summarized and discussed in this chapter. Keywords: RN“ interference, gene therapy, mechanism, therapeutic strategy, blood ge‐ netic diseases

. Introduction Conceived in the s, gene therapy did not produce any meaningful results until recent reports of success appeared in clinical studies. Gene therapy attempts to treat inherited diseases using normal copies of the defective genes. Insertion and expression of specific exogenous genetic materials via the transfer of nucleic acids directly in vivo, or through modified cells in vitro, correct a cellular dysfunction or provide a new cellular function. It has the potential to cure any genetic disease with long-lasting therapeutic benefits [ , ]. Gene therapy can be classified into i germ line and ii somatic line gene therapy types. In the former, genomes of germ cells sperms or eggs are integrated by exogenous functional genes, which can be carried onto the patient’s offsprings. In the latter, therapeutic genes are intro‐ duced into somatic cells and the effects will only be limited to the individual patient [ ].

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“ccording to the mechanisms, gene therapy may include three major categories: a direct modulation of the disease-causing gene, which can be applied to monogenic hereditary diseases b indirect treatment through gene modulation, which can be applied to multifac‐ torial diseases and c immunotherapy by gene modulation DN“ vaccines , which leads to the synthesis of the relevant antigen or an adjuvant [ ]. RN“ interference RN“i can be used for gene therapy. It was intensively studied in the past few decades for its potential in the treatment of blood genetic diseases. RN“i-based gene therapy possesses several therapeutic advantages such as high efficiency, sequence specificity, and potentially less immunogenicity. Less immunogenicity is largely due to the use of nonprotein-coding gene products to trigger RN“i, which makes gene therapy less likely to be potentially hampered by the host immune system [ , ]. Compared to traditional small molecules and protein drugs, the target specificity and universal treatment spectrum make RN“i-based gene therapy an ideal treatment for blood genetic diseases. However, there are still obstacles that remain, such as barriers in the blood circulation system and the diseased tissues that block the actualization of the RN“i effects [ ]. RN“i technology is a relatively new discovery, and it has already become a potent method for gene regulation. In order to provide useful references and prospective directions for further studies, current RN“i-based gene therapies for blood genetic diseases have been summarized and discussed in this chapter.

. The mechanism of RNAi-based gene therapy ”eing a well-described gene regulatory mechanism, RN“i not only suppresses transcription by transcriptional gene silencing TGS but also activates a homology-based mRN“ degrada‐ tion process by post-transcriptional gene silencing PTGS . ”oth silencing pathways resulted in the decrease of the coding transcript level mRN“ [ ]. We will focus on PTGS due to its important role in RN“i-based gene therapy. Two distinct mechanisms regulate PTGS. The first one is the repression and degradation of mRN“s with imperfect complementarity. Endoge‐ nous microRN“s miRN“s belong to this category. They induce translational repression and mRN“ degradation when the guide antisense strand has limited complementarity to the target mRN“. The second one is the sequence-specific cleavage of perfectly complementary mRN“s. Exogenous small interfering RN“s siRN“s or short hairpin RN“s shRN“s belong to this category. They have perfect or near-perfect base-pairings with the intended target mRN“. The miRN“s production and processing rely on host machinery that is guided by complementary miRN“ strands to the target mRN“ [ , ]. During the process, doublestranded RN“s dsRN“ of the target gene are produced and then processed into – noncoding small RN“ duplexes with the help of RNaseIII enzyme dicer and its homologs. These siRN“s are then incorporated into a multi-subunit endonuclease silencing complex called RN“-induced silencing complex RISC . siRN“s are associated with the defense against parasites, heterochromatin formation, transposon and transgene silencing, and PTGS. These siRN“s, loaded into the RISC, are used as the guide to recognize and degrade or suppress the complementary gene or mRN“ utilizing the endonucleases activity of RISC. Gene silencing by RN“i can be used in different biological situations when sequence-specific knockdown of

RNAi-based Gene Therapy for Blood Genetic Diseases http://dx.doi.org/10.5772/61644

gene expression is required, thus providing a convenient tool for analysis of gene function as well as gene therapy [ , , ]. Recent reports have shown that off-targeting can commonly occur during RN“i, despite the belief that initial gene silencing through RN“i was thought to be specific. ”ecause nonspecific hybridization of siRN“s with non-target transcripts can induce undesired effects, it should be guaranteed that the dsRN“ and corresponding siRN“ sequences do not exert off-target effects that negatively influence host physiology. Off-target silencing is determined by the similarity of sequences between siRN“ and non-target genes, or mRN“ sequences not selected for RN“i, as well as the size of siRN“ and transitive RN“i. Thus, off-targeting effects can be avoided by using highly specific sequences in siRN“ expressed under specific and inducible promoters. In addition, it is important to examine the off-targeting effects of RN“i at multiple levels, since undesired effects on the host may occur through the silencing of genes associated with regulatory functions and multiple metabolic pathways, such as transcription factors or signaling molecules [ ].

. RNAi-based gene therapy for blood genetic diseases . . Blood diseases ”lood diseases refer to the disorders in the hematopoietic system or plasma components. The development of blood disorders is always thought to be related with inheritance, the envi‐ ronment, drugs, and biological factors where the changes in chromosome and/or genes play a critical role in some specific hemopathies. Therapeutic approaches for blood diseases can be divided into different categories, including chemotherapy, radiotherapy, RN“i-based gene therapy, and hematopoietic stem cell transplantation. “mong these, RN“i-based gene therapy is progressively becoming a therapeutic alternative which offers the possibility of a permanent cure for some blood diseases [ ]. . . Hemophilia Hemophilia “ and ” are X-linked monogenic bleeding disorders resulting from deficiencies of factor VIII and IX, respectively. Gene therapy, utilizing both viral and non-viral delivery vectors in vivo and ex vivo, has been attempted for the treatment of both hemophilia “ and ” [ , ]. Given its recent clinical success, adeno-associated vector ““V -mediated hepatic gene transfer could be primarily used for the treatment of hemophilia ”. However, a number of problems, such as current immunosuppressive regimen and pre-existing neutralizing antibodies, limit the broad applicability of this approach. For hemophilia “, while ““Vmediated gene therapy has potential, a number of limitations reduce its desirability, such as packaging capacity and inefficient expression. While a number of transgene modifications have increased the expression levels, the vector doses require the corrective F.VIII expression to remain significantly higher than the F.IX. These expression limitations lead to further concerns about immune responses to both the capsid and, if expression levels are not sufficient, the transgene. “s such, ex vivo gene transfer may be more effective for hemophilia “ due to its

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ability to enhance expression through cellular division. While a number of promising gene therapies for hemophilia have been elucidated, there are clearly numerous problems that still need to be addressed to develop approved gene therapies, especially RN“i, for both hemo‐ philia “ and ” in humans [ ]. RN“i-based gene therapy for hemophilia is still in its early stages of development. . . β-Thalassemia The globin chains have an extremely precise structure, ensuring their function of loading, delivering, and unloading oxygen. The globin chains are coded by genes in the chromosome α-gene and β-gene . The normal structure of globin is based on the balanced match between α-chains and β-chains. When the condition is not met, there will be a complete or partial defect in one or both allelic globin genes, such as β-thalassemia [ ]. β-Thalassemia is a worldwide-distributed inherited hemoglobin disorder resulting in severe, chronic anemia [ , ]. It is a heterozygous condition in which only a single β-globin gene is affected and results in the absence or reduced β-globin chain synthesis. The defects of β-globin synthesis lead to an excess of unmatched α-globin, which release free iron, non-heme iron, or hemi‐ chrome. These iron species promote a severe red cell membrane oxidative stress and lead to abnormal β-thalassemic red cell features. The abnormal red cells are finally removed by the macrophage system and results in anemia [ , ]. ”lood transfusion is a primary way to treat the most severe forms of β-thalassemia. “ppro‐ priate goals and optimal safety of transfused blood are necessary for routine administration of red blood cells to patients. The problem is that the high frequency of blood transfusion can lead to iron overload. In its less severe form, chronic transfusions are not required, but iron overload may still develop due to the chronic suppression of the synthesis of the iron regula‐ tory hormone hepcidin by ineffective erythropoiesis. Untreated iron overload can be fatal, resulting in cardiac complications [ , ]. Therefore, handling iron overload is a key factor for the successful treatment of this disease. TMPRSS , a serine protease expressed predominantly in the liver, can inhibit an iron-responsive bone morphogenetic protein-mother against the decapentaplegic ”MP-SM“D signaling pathway, resulting in the downregulation of hepcidin transcription. Researchers found that therapeutics with the lipid nanoparticle LNP formulated RN“i targeting of TMPRSS , in conjunction with oral deferiprone therapy, is superior to monotherapy with dietary iron deficiency and iron chelate for reducing hepatic iron storage [ ]. . . B-lineage lymphoid malignancies ”-precursor acute lymphoblastic leukemia ”PL is the most common form of cancer in children and adolescents. “ dysfunctional CD is expressed in ”PL cells due to the deletion of Exon CD ΔE resulting from a splicing defect related to homozygous intronic mutations. CD is a negative regulator of multiple signal transduction pathways critical for the proliferation and survival of ”-lineage lymphoid cells, and CD ΔE leads to uncontrol‐ led proliferation and the survival of ” cells. Recently, researchers have found that CD ΔE is especially associated with therapy-refractory clones in pediatric ”PL, thus implicating the

RNAi-based Gene Therapy for Blood Genetic Diseases http://dx.doi.org/10.5772/61644

impact of the CD ΔE genetic defect on the aggressive biology of relapsed or therapyrefractory pediatric ”PL. “t the same time, forced expression of CD ΔE in transgenic mice causes fatal ”PL, demonstrating that CD ΔE alone is sufficient as an oncogenic driver lesion for malignant transformation and clonal expansion of ”-cell precursors [ ]. Recent studies have demonstrated that ”-lineage lymphoid malignancies in children and adults are characterized by a high incidence of the CD ΔE genetic alterations. Moreover, the relationship between CD ΔE and aggressive biology of ”PL cells is also reported through the demonstration that siRN“-mediated knockdown of CD ΔE in primary ”PL cells is associated with a remarkable inhibition of their carcinogenicity. “ unique polypeptidebased nanoparticle formulation of CD ΔE -siRN“ is described as a first-in-class RN“i therapeutic candidate targeting of CD ΔE . This formulation is capable of delivering siRN“ cargo into the cytoplasm of leukemia cells, leading to a remarkable inhibition of leukemic cell growth [ ]. It is expected that further development of this nanoparticle may promote an effective therapeutic RN“i strategy of aggressive or chemotherapy-resistant ”-lineage lymphoid malignancies [ ]. . . Myeloid leukemia Leukemia arising from genetic alterations in normal hematopoietic stem or progenitor cells results in the impaired regulation of proliferation, differentiation, and apoptosis, as well as the survival of malignant cells. Overall, the relative -year survival rate for various leukemias is only around % [ ]. Leukemia is still a worldwide health problem, although various therapies have been explored to cure the disease. “mong them, chemotherapy is always considered to be a frontline treatment, mainly containing a broad spectrum of cytotoxic agents and therapeutic molecules. “lthough leukemic cells respond well to chemotherapy at the onset of treatment, over a period of – months the drugs might lose effectiveness in a considerable fraction of patients. Moreover, significant side effects of traditional cytotoxic agents are inevitable at efficacious doses, which limit its function with the progression of the disease. For example, in chronic myeloid leukemia CML , resistance to current frontline therapy of imatinib and failure to a complete cytogenetic response may occur in % of patients within months. With a better understanding of molecular changes in leukemia, the treatment targeting tumor-specific changes, such as RN“i, is expected to make a difference in the therapeutic effectiveness of the disease [ ]. Researchers have explored the suitability of RN“i in suppressing the growth and proliferation of myeloid leukemia cell lines including HL- , U , THP- , and K , which express c-raf and bcl- genes. The results were exciting, as the siRN“ duplexes succeeded in significantly decreasing the level of target proteins by eliminating the expression of c-raf and bcl- genes. This led to the inhibition of the differentiation and programmed cell death suppression of myeloid leukemia cells. These results demonstrated the possibility of RN“i as a novel therapeutic approach to myeloid leukemia [ ]. In recent years, based on the molecular alteration of CML, two clinical trials of RN“i therapy have been conducted to target the aberrantly expressed isoforms of the ”CR-“”L fusion protein. In one case, there are no publishable outcomes. In the other case, silencing aberrant proteins with RN“i have been

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found to be less prone to drug resistance [ ]. In another in vivo application of targeted and non-virally delivered synthetic bcr-abl siRN“, a remarkable apoptosis of CML cells was found in a female patient with a recurrent Philadelphia chromosome with positive CML. The patient was resistant to imatinib and chemotherapy after hematopoietic stem cell transplantation. There were no clinically adverse events, implying feasibility and safety of the application of RN“i-based gene therapy for CML [ ]. Furthermore, RN“i-based gene therapy also proves to be effective in acute promyelocytic leukemia “PL . “PL is the M type of acute myeloid leukemia characterized by a clonal proliferation of abnormal promyelocytes in bone marrow and has a severe bleeding tendency. In % of “PL patients, a balanced translocation between chromosomes and [t q q ] was found, which leads to the formation and fusion of promelocytic leukemia protein PML and retinoic acid receptor alpha R“Rα [ , ]. “ variety of chromosomal aberrations have been identified in “PL including t q q , t q q - , t q q , and der , in which the R“Rα gene is fused to the PLZF, NPM, NuM“, and ST“T b genes, respectively [ ]. The differentiation of leukemic cells and complete remission of “PL may occur after treatment with “TR“ all-trans retinoic acid . However, the alternative strategies of specific targeting of “PL are required because of the “TR“ resistance in patients with PLZF-R“Rα fusion mutation and a treatment of relapse. It has been found that the siRN“ targeted knockdown of fused PML-R“Rα mRN“ can induce differentiation and apoptosis of human “PL cells moreover, an injection of pretreated “PL cells with antiPML-R“Rα siRN“ greatly inhibits the progression of “PL in mice. Therefore, a targeted RN“i for PML-R“Rα fusion might be a promising treatment strategy for “TR“-resistant “PL patients [ ]. “t present, several technological requirements and mechanistic challenges i.e., targeted delivery of siRN“s for efficient clinical trials have to be explored and overcome to make RN“ibased gene therapy readily applicable for the treatment of myeloid leukemia [ ]. . . Multiple myeloma Multiple myeloma MM of clonal plasma cells in bone marrow can cause damage to multiple organs. MM is the second most common hematological malignancy. Chromosomal abnor‐ malities, oncogene activation, and growth factor dysregulation contribute to the development of MM. Like leukemia, chemotherapy is the most common treatment for MM right now. However, it is remarkably resistant to chemotherapy [ ]. Molecular therapy became an alternative treatment, and the introduction of bortezomib has contributed to the improved survival of patients with MM. Resistance to this therapy inevitably occurs, and the clinical efficacy of bortezomib is significantly diminished. “t present, RN“i is found to be helpful in sensitizing tumor cells to chemotherapy and radiation. ”mi- , an oncogene, has been impli‐ cated in the pathogenesis of MM and might influence the response to bortezomib in MM patients. ”mi- has been silenced in two MM cell lines using shRN“ targeting ”mi- sh”mi- . The cell cycle progression and apoptosis of MM cells are evaluated. The prolonged G phase and enhanced apoptosis were observed that suggest RN“i-derived knockdown of ”mireducing the resistance to bortezomib. Therefore, ”mi- -specific RN“i may serve as an important treatment strategy for MM [ ].

RNAi-based Gene Therapy for Blood Genetic Diseases http://dx.doi.org/10.5772/61644

. Conclusions and future perspectives “s a powerful and novel treatment of blood genetic diseases, RN“i-based gene therapy has propelled the clinical testing of siRN“s for a variety of diseases at early stages. It is still too early to determine whether the RN“i-based therapeutics will be an efficient tool for the treatment of blood genetic diseases. Therefore, understanding the basic mechanisms of a targeted gene RN“i and its interconnection with genetic, biochemical, and physiological pathways, as well as off-targets and side effects, are the most important factor for developing an efficient therapeutic RN“i strategy. It is highly possible that the RN“i-based gene therapy can be readily and efficiently used for clinical treatment in conjunction with other therapies. The power of sequence-specific suppression of gene expression without offtargets and undesired side effects makes RN“i-based gene therapy very promising. “lthough progress has been made [ ], major obstacles, such as the development of methods for efficient targeted delivery of siRN“s in patients, still remain a critical task. In addition, uncovering target domains of genes related to blood genetic diseases and the discovery of more blood genetic disease-causing genes are also key research areas for developing RN“i-based gene therapy for important blood genetic diseases.

Acknowledgements This study was supported by the Natural Science Foundation of ”eijing [

].

Author details Mengyu Hu #, Qiankun Ni #, Yuxia Yang * and Jianyuan Luo ,

*

*“ddress all correspondence to: [email protected] [email protected] Department of Medical Genetics, Peking University Health Science Center, ”eijing, China Department of Medical & Research Technology, School of Medicine, University of Maryland, ”altimore, US“ #

These authors contributed equally.

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] Kalpravidh RW, Tangjaidee T, Hatairaktham S, Charoensakdi R, Panichkul N, Sirita‐ naratkul N, Fucharoen S. Glutathione redox system in beta-thalassemia/Hb E pa‐ tients. Sci World J. : . DOI: . / /

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] Schmidt PJ, Racie T, Westerman M, Fitzgerald K, ”utler JS, Fleming MD. Combina‐ tion therapy with a Tmprss6 RN“i-therapeutic and the oral iron chelator deferiprone additively diminishes secondary iron overload in a mouse model of β-thalassemia in‐ termedia. Exp Toxicol Pathol. : – . DOI: . /ajh.

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] Cioca DP, “oki Y, Kiyosawa K. RN“ interference is a functional pathway with thera‐ peutic potential in human myeloid leukemia cell lines. Cancer Gene Ther. : – .

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Chapter 10

Non-viral siRNA and shRNA Delivery Systems in Cancer Therapy Emine Şalva, Ceyda Ekentok, Suna Özbaş Turan and Jülide Akbuğa Additional information is available at the end of the chapter http://dx.doi.org/10.5772/62826

Abstract RN“ interference represents a promising therapeutic strategy for the silencing of specific target genes in cancer therapy. Small interfering RN“s and DN“-based vectors encoding short hairpin RN“s provide sequence-specific post-transcriptional gene silencing by binding to its complementary RN“. For the therapeutic use of siRN“ in cancer, efficient intracellular delivery is necessary. The efficient cancer therapy with RN“i is not still ac‐ complished because of internalization and intracellular trafficking problems such as low transfection efficiency, enzyme degradation, inappropriate subcellular localization, and endosomal trapping of siRN“s in cells. Cancer is a complex disease including multiple genes and pathways. The most important benefits of siRN“ therapy are high target spe‐ cificity and non-toxicity compared with chemotherapy. The uptake of siRN“ by cells without a carrier system is possible, but naked siRN“ is mostly degraded with nucleases and activates the immune responses. Development of appropriate delivery systems is an important issue in the use of siRN“-based therapeutics. Non-viral delivery systems have great potential for safe and effective delivery of siRN“ therapeutics to tumor cells. Nano‐ carriers such as nanoplexes, lipoplexes, nanoparticles, and liposomes have been common‐ ly used for siRN“ delivery. This chapter highlights the importance of non-viral delivery systems in vitro and in vivo cancer therapy. Keywords: Cancer, non-viral vectors, RN“i, siRN“, shRN“

. Introduction RN“ interference RN“i is a conserved endogenous cellular process for post-transcriptional regulation in sequence-specific gene silencing. The regulatory RN“ molecules include small interfering RN“s siRN“s , and short hairpin RN“s shRN“s provide the specific degrada‐ tion of target mRN“ in mammalian cells [ ]. siRN“s are the products of long double-stranded

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RN“ dsRN“ molecules in cells, which are expressed transgenically or delivered exogenous‐ ly. Synthetic siRN“s can be transfected into cells that specifically silence the expression of target genes. In the RN“i pathway, dsRN“ over nt molecules are cleaved into - to nucleotide duplexed RN“s, termed as siRN“ duplex, by endoribonuclease Dicer or RN“se III-type enzyme. The cleaved siRN“ duplexes contain ′-phosphate and two-base ′ over‐ hangs. siRN“s are incorporated into the endogenous RN“-induced silencing complex RISC . One of the two strands of siRN“ duplex is guide antisense strand, and the other is passenger sense strand. siRN“ duplex is unwinded by RN“ helicase activity. While the guide strand binds to the RISC, the passenger strand is degraded. The activated RISC binds to mRN“ with base-pairing for sequence-specific degradation of complementary mRN“. The mRN“ fragments cleaved by “rgonaute “go proteins are released from RISC and degraded by other endogenous nucleases. “fter mRN“ degradation, the active RISC is rebuilt and can participate in another RN“i pathway [ , ]. In the event, RN“i process decreases specific mRN“ levels, and thus decreases target gene expression. “mong the nucleic acid–based drugs, siRN“s as potential novel drug candidates are offered a highly promising strategy in cancer therapy. The knockdown of abnormal gene overexpres‐ sion, occuring in cancer by using siRN“, has been used in therapeutic applications [ ]. Targets of chemical drugs are limited to certain classes of receptors, ion channels, and enzymes. In the treatment with siRN“, known sequence of any gene of interest is sufficient and its target choice is unlimited [ ]. The potential advantages of siRN“ treatment compared with other treatment methods are that siRN“s [ – ] i provide sequence-specific gene therapy ii can specifically target many undruggable genes or downregulate gene products iii are considered the safe therapeutics iv are potent and efficient molecules and possess high gene silencing activity and iv can be easily designed for any disease. RN“i might be a promising new pharmaceutical area for treatment of incurable and severe diseases such as cancers and infections. RN“i applications have been recently achieved by using synthetic siRN“s and vector-based siRN“ expression systems or short hairpin RN“s shRN“s synthesized within the cells by vector-mediated production. The expressed shRN“s from plasmid and viral vectors in nucleus are cleaved by Dicer in cytoplasm and siRN“s are formed. There both strategies have advantages and disadvantages. Vector-based siRN“ expression systems have several advantages for applying RN“i compared to synthetic siRN“s. ”oth permanent and transient transfection with vector-based systems can be ach‐ ieved, and thus vector-based system increases the period of siRN“-mediated inhibition of gene expression [ ]. In addition, shRN“ constructs are more stable than siRN“s [ ]. Low amount nM of siRN“ and less than five copies of shRN“ are sufficient for stable transfection and for acheiving gene silencing effect [ ]. The synthetic siRN“s can be easily synthesized in large amounts and chemically modified to improve stability, permeability, efficacy, and transfection control however, the modified siRN“s are highly expensive [ ]. siRN“s are not integrated into host genome. The modification of vector-based shRN“ systems is difficult, but shRN“ expression systems can be regulated or induced by appropriate promoters and termination sequences. Choice of promoter, loop structure of shRN“, length and arrangement of sense and

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antisense strands, and orientation of restriction enzyme regions are important for shRN“ expression cassette preparation. Similar to the various RN“i applications for targeted gene silencing, the chimeric expression cassettes of siRN“ and shRN“ in the same expression unit might also be made [ ]. Design of optimal siRN“ sequence plays a key role for successful siRN“ therapy. The choice of potent and specific siRN“ sequences is important for minimization of immune responses and off-target effects. siRN“s are – base pairs in length, but mostly preferred to be nt of siRN“s with a structure of nt duplex region and two nucleotide overhangs at the ′ end, usually TT and UU, which are important for recognition by the RN“i machinery. Increasing the length of the dsRN“ may enhance its potency, dsRN“s with nucleotides are up to times more potent than the siRN“s containing nucleotides. The longer – nt duplexes act as a substrate for Dicer Dicer-subtrate siRN“s . This Dicer-substrate siRN“s are more efficiently loaded into the RISC over the siRN“s with bp and newly produced siRN“s from the long dsRN“s directly incorporated into the RISC complex. Thus, gene silencing mecha‐ nism can be facilitated [ , ]. On the other hand, dsRN“s longer than about bp can lead to interferon response, which is the defense mechanism against viral infection. The activation of interferon pathway causes non-specific mRN“ degradation and apoptosis [ , ]. The long dsRN“s activates innate immune response by interaction with protein kinase receptor PKR and Toll-like receptors TLR and are activated by ssRN“ TLR activated by unmethylated CpG and TLR activated by dsRN“ . The activation of these receptors induces interferon IFN and proinflammatory cytokines [ , ]. In addition to immune responses, another important problem for efficient RN“i therapy is offtarget effects of these molecules. The cause of off-target effects are the suppression of undesired or unpredicted genes other than the desired target genes. The absence of homolog sequences between siRN“ and its target mRN“ can cause cleavage of non-targeted mRN“ regions. Offtarget effects of siRN“ can lead to problems in the interpretation of gene silencing studies, serious and unwanted side effects such as potential toxicity and even cell death [ ]. There are many factors and mechanisms leading to occurence of off-target effects. These factors are the length of dsRN“ or siRN“, the length and position of siRN“-target sequence mismatch, and coding sequences and untranslated regions in genes [ ]. The mechanisms leading to off-target effects of siRN“s are i the regulation of unwanted transcripts by seed sequence homology to the ′ UTR of cellular mRN“s, ii the saturation of RISC by affecting cellular miRN“ activity of siRN“s in large amounts, iii the function of miRN“s as siRN“s or shRN“s because of similarities in gene silencing pathway, iv non-specific distribution by non-targeting systemic delivery, and v immunostimulatory motifs in siRN“ sequences [ , , ]. Many preclinical studies with siRN“ indicated that it is a hopeful molecule for clinical research of various diseases. Up to date, at least RN“i-based drugs have been evaluated in clinical trials [ ]. The first clinical trial with siRN“ was made in by Opko Health. The clinical studies of ”evasiranib, that is, siRN“ targeting vascular endothelial growth factor VEGF to suppress ocular neovascularization in patients with age-related macular degeneration “MD , continued to the phase III trial, but the clinical trials was terminated in because of its poor efficacy and causing vision loss. “llergan company has terminated the phase II clinical trials

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of siRN“ “GN- , targeting VEGF because of its off-target effects [ ]. The clinical translation of RN“i can be possible with development of safe and efficient RN“i delivery systems that lack of off-target effects [ , ]. “lthough siRN“s are used as the potential therapeutic molecules in cancer and other diseases, the most important challenge in the development of RN“i-based drugs is efficient and safe delivery of appropriate doses to target cells and tissues. Therefore, the development of siRN“based viral and non-viral delivery systems are required to have an enhanced efficacy, im‐ proved stability, and minimized non-specific gene silencing such as off-target effects and immune responses. This chapter focuses on recent improvements in the non-viral siRN“ delivery systems in cancer therapy.

. RNAi in cancer therapy Cancer is a multistep genetic disease, which develops as a consequence of changes in the control of cell proliferation and differentiation. In the transformation of normal cells to tumor cells, the affected cells undergo mutations such as downregulation of tumor suppressor genes and overexpression of oncogenes. RN“i-based therapeutics have been extensively used for knockdown of cancer-associated genes. The in vitro and in vivo studies with siRN“ and shRN“ have shown that silencing of genes related to tumor cell growth, invasion, angiogenesis, metastasis, and chemoresistance in various types of cancer [ ]. RN“i technology, as a new approach for cancer therapeutics, offers many advantages over conventional cancer treatment strategies. “dvantages of gene silencing by siRN“ and shRN“ are the high degree of specif‐ icity to target tumor cells and tissues, a capacity to inhibit target gene expression, a simple and rapid design, and synthesis [ ]. While non-specific chemotherapy leads to death of cancer cells, it significantly damages the surrounding healthy tissues and organs, causing extensive systemic toxicity. The side-effects of chemotherapeutic drugs can be minimized with siRN“ treatment. Cancer cells have the ability to develop a resistance to chemotherapeutic drugs. RN“i-based therapeutics can simultaneously target multiple genes in cancer signaling pathway. The simultaneous silencing of multiple genes in cancer therapy have importance in terms of minimizing the multiple drug resistance caused by small chemical molecules given in high dose [ , ]. This therapy inhibits survival signals and pathways that take part in the development of multi-drug resistance in cancer cells. Oncogenes, mutated tumor supressor genes, survival and apoptotic genes, causing tumor initiation and progression, are major targets for RN“i-based therapy. Simultaneous suppres‐ sion of one target gene or multiple genes has provided a significant advantage in cancer therapy. siRN“s can be designed for effective gene knockdown by targeting any gene or multiple genes in cells [ ]. siRN“s are likely to be more effective than other antisense approaches because of many properties such as a highly specific mRN“ degradation, cell-tocell spreading of gene silencing effect, long silencing activity, improved stability in vivo, and their efficiency in lower concentrations [ , ]. “ single therapeutic strategy is insufficient for

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the inhibition of cancer growth and progression RN“i as a new therapeutic strategy may be used as well as with chemotherapy, immunotherapy, anti-hormone therapy, and radiotherapy for achieving synergistic therapeutic effect. In clinical trials, the most of siRN“s have been given by local administration. When siRN“s are delivered to target tissue locally, lower siRN“ doses can be used for pharmacologic effect e.g., saline-based formulation, or excipiants such as % dextrose and any drug delivery approaches e.g., liposome, nanoparticle, and complexes [ ]. However, systemic drug administration by intravenous injection is required for cancer diseases [ ]. In systemic effect, siRN“s must encounter several extra- and intra-cellular barriers until it reaches the target cell and tissue. siRN“s cannot freely cross physiological and cellular barriers because of their high molecular weight and negative charge. The significant challenges of using siRN“ are their poor cellular uptake, degradation by serum nucleases, and rapid elimination. These factors and barriers reduce therapeutic effect of siRN“. Therefore, efficient in vitro and in vivo delivery of siRN“-based therapeutics in cancer is dependent on the development of appropriate delivery systems. siRN“ delivery systems should a protect siRN“s against degradation enzymes and serum proteins, b prolong the circulation time of siRN“, c provide siRN“ stability in blood serum, d avoid sequestration in the reticuloendothelial system RES , e avoid aggregation in serum, f minimize non-specific tissue and cellular uptake, g achieve target-specific siRN“ delivery, h allow for immune evasion, i resist rapid renal clearance, j enhance vascular permeability to reach cancer tissues, k promote trafficking to the cytoplasm and uptake into RISC, and l have low or non-toxicity [ , , ].

. Delivery strategies of RNAi-based therapeutics siRN“s have large molecular weight ~ kDa and are polyanionic nature ~ negative phosphate charge and are easily degraded by enzymes in cells, tissues, and bloodstream. In addition, siRN“s cannot easily cross the cell membrane [ ]. The naked siRN“s are readily degraded by serum endonucleases. The half-life of circulating naked siRN“ is less than minutes because of its rapid clearance by the kidneys, so that they cannot reach to target cell efficiently. The gene silencing activity of unmodified or uncomplexed siRN“s is little or absent [ ]. To solve this problem, two strategies are used: chemical modifications and conjugation of siRN“ molecules or use of gene delivery systems for increasing efficiency of RN“i-based therapeutics. Chemical modification is the major approach to overcome in vivo siRN“ delivery problems. Chemical modifications of naked siRN“s have been performed to i enhance siRN“ stability, ii protect siRN“ from degradation, iii avoid recognition by the innate immune system and minimize immunostimulatory responses, iv minimize off-target effects, v reduce required dose for gene silencing, vi improve pharmacodynamic properties, vii increase delivery to target cells, and viii allow the delivery by systemic administration. The sugar, backbone and nucleobase modifications of siRN“, can significantly protect siRN“ in both serum and cytoplasm. The commonly used chemical siRN“ modifications are the incorporation of locked

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nucleic acids LN“ , phosphorothioate linkages, and ′-o-methyl, ′-amine, ′-fluoro groups [ , , ]. Chemical modifications must increase the stability of siRN“ without affecting its gene silencing activity [ ]. However, these substitutions may lead to off-target effects, cytotoxicity, reduced RN“i activity, and impaired biological activity [ , ]. Other chemical strategies for siRN“ are cholesterol, folate, and aptamer conjugation and peptide modification. siRN“s can associate with aptamers, ligands, and antibodies by electrostatic interaction or direct conjugation. The conjugation of these functional groups provides cell- or tissue-specific targeting and efficient delivery. “s a result, the efficacy of silencing can be increased [ ]. Viral and non-viral vectors have been extensively used in the siRN“-based therapy. Viral vectors encoding shRN“ have a high gene transduction and gene silencing effects. “denoassociated viral vectors, lentiviral vectors, and adenoviral vectors have been extensively used in gene knockdown studies [ ]. The transfering of shRN“-encoding vectors into the nucleus of cells have obtained high and long-term shRN“ expression. In addition, viral vectors can integrate the host genome [ ]. “lthough viral vectors have a high gene transfection efficiency, the challenges such as inflammatory reactions, strong immunogenicity, insertional mutagen‐ esis, and oncogenic transformation of viral vectors can cause important safety concerns. In addition, some viral vectors have low capacity for transgene insertion. To overcome these problems, non-viral vectors have been developed and used in siRN“ delivery. Compared to viral vectors, non-viral vectors have several advantages such as lack of immunogenicity, low or no integration into genome, large-scale production, and use of wide variety of nucleic acids size [ ]. However, the transfection efficiency of non-viral vectors is not as high as the viral vectors. . . Non-viral vectors The non-viral delivery of siRN“ and shRN“ therapeutics to target tumor cells is a multi‐ step process. To achieve efficient delivery and therapeutic gene silencing, siRN“s should be stable in biological fluids and must have above mentioned properties [ , ]. The circulat‐ ing siRN“s after systemic administration must be evaded from the reticuloendothelial system RES . Negatively charged siRN“s gain the positive charge after complexed with cationic charged polymers. This positive charge facilitates cellular internalization of siRN“s however, the cationic charge increase non-specific interactions by non-target cells, negatively charg‐ ed serum proteins, and extracellular matrix. “s a consequence of these non-specific interac‐ tions, clot-like accumulations or aggregations are formed. Complexes are entrapped in the endothelial capillary bed or taken up by RES recognition. While RES organs such as spleen, liver, and bone marrow uptake the major part of injected dose, the minor part of this reaches to tumors [ , , ]. Non-viral delivery vectors prolong the biological half-life and mean residence time of siRN“, and they enhance accumulation of siRN“ molecules in tumor tissues. siRN“ therapeutics can be accumulated into cancer tissue by enhanced permeability and retention EPR effect as a result of discontinuous vasculature permeation and poor lymphatic drainage retention in

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the abnormal tumor blood vessels compared to the normal blood vessels. Tumor endothelium allows penetration of macromolecules [ , ]. The other challanges of RN“i-based therapeutics delivery to the tumor tissues after systemic circulation are crossing of cellular membrane, intracellular traffic into the cells with endoso‐ mal/lysosomal compartments, release of siRN“ or shRN“ from carriers, and nuclear transport for vector-based siRN“/shRN“ therapeutics and entry to cytoplasm for siRN“-based therapeutics. The cell membrane is an important extracellular barrier for siRN“ uptake. The average size of a single siRN“ molecule is less than nm. Despite their small size, polyanionic nature and hydrophilicity of siRN“ make crossing of biological membranes difficult [ ]. To overcome this problem, the complexation of negatively charged siRN“ with cationic polymers or lipids are performed. The net positive charge of this formulations facilitates binding to negatively charged cell membranes, following internalization by adsorptive pinocytosis. For cell-type specific delivery, targeting ligands, antibodies, and aptamer-binding non-viral vectors pass through the cell membrane by receptor-mediated endocytosis [ ]. “fter crossing from the cell membrane, siRN“s and vector-based siRN“s/shRN“s encounter several intracellular barriers that include the endosomal trafficking, unpackaging of siRN“, and nuclear traffic. The intracellular traffic of endosomal content is important for succesful siRN“ delivery. When siRN“ released from the carrier reaches cytosol, RN“i mechanism is induced inside the cells. However, for the onset of RN“i effect, transfer of vector-based siRN“/shRN“ to the nucleus is required. In the delivery process, early release of siRN“ from endosome is required. If siRN“ remains inside the endosome for long time, it will be degraded. Therefore, different agents fusogenic protein conjugated with polymers disrupt the endosomal mem‐ brane. In addition, polymers possess proton-sponge effect polyethyleneimine, PEI , which have been used to induce osmotic swelling and subsequent disruption of the endosome [ ]. 3.1.1. Polymer-based RNAi delivery system in cancer therapy Negatively charged siRN“s or shRN“s can readily bind to cationic polymers or load to the nanocarriers by ionic interactions. Nanosized complexes or polyplexes by electrostatic interactions and nanoparticle formulations by encapsulation have been developed for efficient siRN“/shRN“ delivery. Thus, siRN“s can be protected from nuclease attack and cellular uptake of siRN“s via endocytic pathway faciliated. Many natural and synthetic polymers are used for gene delivery, such as polyethyleneimine PEI , poly-l-lysine PLL , chitosan, protamine, gelatin, atelocollagen, cationic polypeptides, cyclodextran polymers, dendrimers, poly-lactide-co-glycolide PLG“ , and polydimethylaminoethylmethacrylate PDM“EM“ [ ]. In addition, polyethyleneglycol PEG is widely used as a linker between polymer and ligand or nucleic acid or for binding of siRN“ onto nanocarrier surface [ ]. 3.1.1.1. Chitosan “mong the non-viral vectors, chitosan or its derivatives are attractive where chitosan has been shown to be biodegradable, biocompatible, non-toxic, mucoadhesive, and non-inflammatory and has low cost of production. Chitosan is a cationic polysaccharide, consisting of N-acetyld-glucosamine and d-glucosamine units. In addition, chitosan has been designated as Gen‐

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erally Recognized “s Safe GR“S by the FD“ [ ]. It has been widely used in in vivo siRN“ and shRN“ delivery applications because of positively charged amines, allowing electrostatic interactions with negatively charged nucleic acids to form stable complexes. The protonated amine groups allow transportation to cellular membranes and subsequent endocytosis into cells. Moreover, the high amounts of chitosan in siRN“ complexes may lead to increase cellular accumulation of siRN“ molecules and facilitate release of siRN“ from endosomes to cytosol under high osmotic pressure in the endosomes of cells [ ]. Chitosan-based nanocarriers are prepared by three different methods. These include simple complexation, ionic gelation siRN“ entrapment , and adsorption of siRN“ onto the surface of chitosan nanoparticles [ ]. The molecular weight and degree of deacetylation of chitosan influence its solubility, hydrophobicity, charge density, and thus the interaction ability with nucleic acids. The N/P ratio ratio between chitosan nitrogen per siRN“ phosphate of chitosan/siRN“ nanoplexes is an important factor for optimization of complex properties size and zeta potential , transfection, and gene silencing efficiency. Increasing the N/P ratio not only helps to obtain a high transfection efficiency but also enhances toxicity. The excess of free chitosan in the formulations can interact with cell membrane and cellular process, and thus, may reduce cell viability [ ]. Chitosan has a great potential in siRN“-based cancer therapy studies, because it can be safely and efficiently delivered to cancer cells. It is reported that chitosan or modified chitosan nanoplexes and nanoparticles as delivery system exerted antitumoral effects in different cancers [ – ]. Studies with chitosan formulations in different cancers Howard et al. [ ] developed chitosan nanoparticles using polyelectrolyte complexation method. The size of nanoparticles was between and nm. The endogenous enhanced green fluorescent protein EGFP silencing efficiency with nanoparticles was found to be . and . % in human lung carcinoma cells H and murine peritoneal macrophages. The siRN“/chitosan nanoparticles reduced EGFP expression % compared to untreated control in transgenic EGFP mice. They suggested that this chitosan-based system can be used in the treatment of systemic and mucosal diseases. Salva and “kbuga [ ] studied silencing effect of chitosan/VEGF shRN“ nanoplexes in breast cancer cell lines. “ significant VEGF gene silencing % was obtained after nanoplexes application in MCF- cells. Salva et al. [ , ] demonstrated the successful application of chitosan/siRN“ or shRN“ VEGF nanoplexes in in vivo breast cancer models. “fter intratu‐ moral and intraperitoneal injection, comparison was made and higher tumor inhibition was obtained with intratumoral injection. qRT-PCR and Western ”lot analysis showed that VEGF mRN“ and protein expression was significantly reduced by chitosan nanoplexes. Salva et al. [ ] also studied the IL- encoded plasmid pIL- to improve the therapeutic efficacy of siRN“ targeting VEGF because of the anti-angiogenic effect of IL- molecule. Researchers prepared chitosan nanoparticles containing shRN“ VEGF and pIL- , and they have reported that co-delivery of shRN“ VEGF and pIL- into chitosan nanoparticles caused additive effect on breast tumor cell growth in rat model % inhibition [ ].

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In another study, Salva et al. [ ] obtained enhanced silencing effect by using siRN“s targeting to VEGF and HIF-1α in different breast cancer cell lines such as MCF- , MD“-M”, and MD“-M”. Two siRN“s were encapsulated into liposome coated with chitosan, and the co-delivery of siRN“ VEGF and HIF-1α into liposomal form have significantly inhibited VEGF % and the HIF-1α % [ ]. Yang et al. [ ] reported that chitosan/siRN“ VEGF nanoparticles prepared by complex coacervation method showed spherical morphology with a mean diameter of – nm and positively charged surface mV . Chitosan nanoparticles were effectively transfected to mouse melanoma cells ” -F , and they have investigated % of the VEGF gene silencing efficiency in cells without any cytotoxicity. Wang et al. [ ] prepared the chitosan-TPP tripolyphosphate nanoparticles by ionic gelation method for the delivery of shRN“ expressing vector to the human rhabdomyosarcoma RD cell line and for the inhibition of TGF-β1 expression. Suppression of TGF-β1 gene by chitosan nanoparticles containing shRN“ has resulted in decrease of RD cell growth in vitro and tumorigenicity in nude mice. Huang et al. [ ] studied the effect of chitosan/shRN“ VEGF nanocomplexes on angiogenesis and tumor growth in hepatocellular carcinoma HCC . The administration of low molecular weight chitosan/shRN“ VEGF complexes by intratumoral or intravenous injection demon‐ strated more effective suppression of tumor angiogenesis and tumor growth in the different HCC models. They showed that LMWC could effectively deliver shRN“ into tumor tissue. shRN“ VEGF concentrations in tumor tissue dramatically increased after intravenous administration of chitosan/shRN“ VEGF complexes. Studies with chitosan derivatives and conjugation with other polymers and ligands in different cancers In order to increase the transfection efficiency of chitosan, different modifications are made on the structure of chitosan. Modified forms of chitosan such as carboxymethyl or trimethyl chitosan, trisaccharide-substituted chitosan oligomers, and succinated or galactosylated chitosan are formed. Chitosan is also conjugated with folic acid or PEG [ ]. Jere et al. [ ] used chitosan-graft-polyethylenimine CHI-g-PEI copolymer for delivery of shRN“ Akt1 expressing plasmid in lung cancer cells. The formed complexes were silenced Akt1 onco-protein and significantly reduced the survival, proliferation, and growth progres‐ sion of lung cancer cell. Akt1 silencing induced apoptosis in cancer cells. The suppression of Akt1 oncoprotein decreased “ cell malignancy and metastasis. The therapeutic efficiency of CHI-g-PEI-shRN“ Akt was found higher than PEI K-shRN“ Akt compared to carrier. Noh et al. [ ] prepared a copolymer containing additional cationic moieties linked with chitosan to enhance the cationic charge of chitosan. Therefore, chitosan derivation with polyl-arginine PLR and polyethyleneglycol PEG PLR-grafted CS polyplexes were used for in vitro and in vivo delivery of siRN“ RFP. PLR alone can be cytotoxic, thus conjugation of PLR to chitosan both decreased cytotoxicity of PLR and enhanced siRN“ delivery efficiency. The pegylation of cationic polymers reduces the charge of polymers and limits the interaction with

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cell membranes. PEG-CS-PLR did not significantly reduce the cellular delivery of siRN“. Three intratumoral injections of µg of PEG-CS-PLR/siRN“ RFP complexes to ” F -RFP tumor-bearing mice had decreased RFP expression at % level in tumor tissues. It is indicated that PEG-CS-PLR can be a useful carrier for delivery of oncogene-specific siRN“s. Fernandes et al. [ ] investigated folate conjugation to improve gene transfection efficiency of chitosan. When chitosan was conjugated with folate, the folate-chitosan-siRN“ complexes have increased gene silencing efficiency because of promoted uptake in HeLa and OV- cell lines, which are known to have high folate receptor expression. Higher transfection efficiency and lower toxicity of folate-chitosan complexes are reported in folate receptor–positive cells. Cell penetrating peptide-based systems may improve cellular uptake and gene silencing efficiency of siRN“s without side effects. Protamine is a cationic, non-toxic polypeptide that has membrane translocation and nuclear localization activities because of its arginine-rich amino acid sequences. In addition to its stabilization enhancing properties, protamine is known to exhibit cell penetrating activity and is an important compound for several cancer targeting systems [ ]. Salva et al. [ ] have developed ternary nanoplexes of chitosan/protamine/siRN“ targeting VEGF in breast cancer cell lines for efficient siRN“ uptake and inhibition effect. Ternary nanoplexes showed the highest cellular uptake than binary nanoplexes. Erdem-Cakmak et al. [ ] reported that addition of protamine to chitosan complexes increased the silencing of VEGF genes after using chitosan/shRN“/protamine nanoplexes. In terms of the gene silencing and transfection, when the molecular weight of chitosans were compared at the different cell lines including HEK , HeLa, and MCF- , low molecular weight chitosan kDa proved more efficient than medium molecular weight chitosan. Gene inhibition values in cell lines after transfection of binary and ternary complexes followed the rank HEK >HeLa>MCF- . In addition, any cytotoxicity was not found after the complexes. Song et al. [ ] used protamine/antibody fusion protein to deliver siRN“s targeting c-my, MDM2, and VEGF specifically to HIV envelope-expressing ” melanoma cells and envelopeexpressing subcutaneous ” tumors. The positively charged protamine served as binding partner for negatively charged siRN“ and showed cell internalization and release of the siRN“ cargo. The antibody-protamine delivery system can target siRN“ specifically to cells. Choi et al. [ ] reported that complexes prepared with low molecular weight protamine LMWP inhibited cell growth by suppressing VEGF expression in hepatocarcinoma cancer cells. In tumor tissues, the expression of VEGF was inhibited through the systemic application of peptide complex, thereby suppressing tumor growth. 3.1.1.2. Polyethylenimine Polyethylenimines PEIs are water-soluble cationic synthetic polymers. They can be synthe‐ sized in different lengths and different molecular weights such as branched bPEI or linear lPEI and low molecular weight < Da or high molecular weight > kDa . PEI has a high cationic charge density because of the protonation of its primary, secondary, and tertiary

Non-viral siRNA and shRNA Delivery Systems in Cancer Therapy http://dx.doi.org/10.5772/62826

amine groups positioned at every third nitrogen [ ]. While in linear PEI all nitrogen atoms are protonable, in the branched form, two-thirds of nitrogens can be charged. PEI can lead to proton accumulation in endosome, which was brought in by endosomal “TPase with an influx of chloride anion. Proton accumulation in endolysosome counteracts pH decrease, inhibits nucleases and unbalances endosome osmolarity depend on CI concentration and results in osmotic swelling of endosome. This effect of PEI is named as proton sponge effect . PEI may enhance intracellular delivery by facilitating endosomal escape and induce lysosomal dis‐ truption, endosomal release, and DN“/siRN“ protection from lysosomal degradation by buffering endosomes [ ]. The molecular weight of PEI is important in the development of gene delivery and level of cytotoxicity in cells. The high molecular weight PEI has higher transfection efficiency than low molecular weight PEIs. PEI has a high electrostatic capacity, which can provide strong electrostatic interactions with the siRN“ and contribute to cell membrane binding and internalization. Especially, the kDa bPEI is one of the most effective non-viral vectors in gene silencing because of efficient endosomal escape. However, the high positive charge of bPEI leads to severe cytotoxicity and non-specific interactions with serum proteins [ , ]. The cytotoxicity of PEIs can be decreased with modification of free amine groups or conjuga‐ tion of cell binding and targeting ligands. Therefore, graft copolymers have been usually preferred as a delivery system. Schiffelers et al. [ ] prepared PEGylated PEI nanoplexes with “rg-Gly-“sp RGD peptide ligand containing siRN“ targeting VEGFR-2 and investigated the effect of angiogenesis and tumor growth in tumor-bearing mice. This study indicated that nanoplexes containing siRN“ VEGFR-2 reached tumor tissues after systemic administration. This delivery system has sequence-specific inhibition effect and reduced the tumor growth. Jiang et al. [ ] studied anti-VEGF siRN“/PEI-H“ complex prepared by PEI-hyaluronic acid PEI-H“ . Complexes at the dose of . µg of siRN“/mouse were applied intratumorally to C ”L/ mice by daily injection for days. “t hours post-injection, the siRN“ VEGF formulations were distributed mainly in the tumor, spleen, lung, heart, liver, and kidney. This study suggested that siRN“ VEGF/PEI-H“ complexes can be used for the treatment of cancer in the tissues having H“ receptors such as the liver and kidney. Park et al. [ ] synthesized siRN“/ PEI-SS -b-H“ complexes and, after characterization, applied to in vitro and in vivo gene silencing for target-specific tumor treatment. This delivery system demonstrated an excellent in vitro gene silencing efficiency – % . siRN“ VEGF/ PEI-SS -b-H“ complexes were administrated intratumorally to colorectal tumor bearing mice every days. “fter the treatment of tumor, VEGF gene silencing and reduction in tumor growth were seen. 3.1.2. Other non-viral delivery systems “mong cationic polymers, poly l-Lysine PLL is one of the mostly studied polymers used for nucleic acid delivery. It formed complexes with DN“ smaller than nm. Its complexes can target different cells after binding suitable ligands. PLL can be easily produced in large

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scale and is physiologically stable and biosafe [ ]. PLL may protect siRN“ from degradation effect of nucleases. However, PLL has some hurdless that impade its clinical application. PLL does not have the proton buffering ability to enhance lysosomal release of transported siRN“. It can be modified also by addition of ligands [ ]. The ternary copolymer mPEG-b-PLL-g ss-IPEI was used for siRN“ delivery to SKOVovarian cancer treatment. Nanocomplexes were administered to SKOV- -implated ”alb/c mice and tumor growth inhibition was observed [ ]. Dendrimers are highly branched spherical and synthetic multifunctional macromolecules. The surface functional groups of dendrimers can be modified to enhance biocompatibility and decrease toxicity. Polycationic dendrimers such as poly amidoamine P“M“M and poly propyleneimine PPI dendrimers, because of the high density of positive charges on the surface, are highly attractive for delivery of negatively charged pDN“, antisense oligonucleo‐ tide “sODN , and siRN“s. P“M“M dendrimers have primary amine groups on their surface and tertiary amine groups inside. Their amine groups are complexed with siRN“s. Thus, compact structure promote cellular uptake of siRN“ and tertiary amine groups initiate the proton sponge effect to enhance endosomal release of siRN“ [ , ]. Waite et al. [ ] conjugated cationic P“M“M dendrimers with RGD targeting peptides to enhance the delivery efficiency of siRN“ to glioma cells. They suggested a promising strategy of RGD-conjugated dendrimers for siRN“ delivery to solid tumors. Liu et al. [ ] investigated in vitro characterization and anticancer effect of P“M“M dendrimermediated shRN“ against human telomerase reverse transcriptase hTERT in oral cancer. Dendriplexes had nm size and + mV zeta potential which were favorable parameters for escape from the vasculature and intracellular delivery. shRN“ hTERT dendriplexes were applied by intratumoral administration to tumors. Dendrimer-mediated shRN“ TERT resulted in cell growth inhibition and apoptosis in vitro and tumor growth inhibition in vivo in the xenograft model. In addition, expression of HTERT and PCNA proteins was reduced in tumors. “telocollagen, which is produced from bovine type I collagen, has biomaterial properties such as high biocompatibility, biodegredability, and low immunogenicity. “telocollagen forms a helix of three polypeptide chains and has positive charge, which enable its binding to nucleic acid molecules [ ]. “t low temperature, atelocollagen exists in liquid form – °C , therefore, it can be easily mixed with nucleic acid solutions [ , ]. Thus, atelocollagen can increase cellular uptake, nuclease resistance, and prolong release of nucleic acids. The size, charge, and sustained release of atelocollagen/siRN“ complexes can be altered by ratio of siRN“ to atelocollagen [ , ]. Takei et al. [ ] first studied anti-tumoral effect of atelocollagen complexes containing siRN“ VEGF in vitro and in vivo. They showed that siRN“ VEGF with atelocollagen inhibited tumor angiogenesis and tumor growth in PC- cell lines in vitro and xenograft tumor in vivo model. Koyanagi et al. [ ] reported that siRN“ targeting vasohibin- VASH-2 using atelocollagen complexes siginificantly inhibited ovarian tumor growth and angiogenesis in ovarian cancer

Non-viral siRNA and shRNA Delivery Systems in Cancer Therapy http://dx.doi.org/10.5772/62826

xenograft model. The knockdown of VASH2 with atelocollagen/siRN“ VASH2 complexes exerted a significant antitumor effect and helped in tumor vascularization. PLG“ has been widely used as gene delivery system because of its biodegredability, biocom‐ patibility, and non-toxic properties. FD“ has approved PLG“ as a pharmaceutical excipient. PLG“ nanoparticles enter the cells efficiently by specific and non-specific endocytosis. Nanoparticles can release the encapsulated drug slowly leading to sustained drug effect [ ]. Murata et al. [ ] investigated anti-tumor effect of long-term sustained release of PLG“ microspheres encapsulating anti-VEGF siRN“. The release of siRN“ from microspheres was sustained for over one month. Intratumoral injection of PLG“ microspheres containing siRN“ VEGF inhibited tumor growth. Su et al. [ ] prepared PEI-coated PLG“ nanoparticles loaded with paclitaxel and Stat3 siRN“. PLG“-PEI nanoparticles more rapidly released Stat3 siRN“ than paclitaxel. Thus, decrease of Stat3 expression by siRN“ in human lung cancer cells “ and “ -derived paclitaxelresistant “ /T cell lines reduced resistance of cell to paclitaxel. The released paclitaxel from nanoparticles killed the cancer cells that induce microtubule aggregation. In summary, inhibition of Stat3 expression decreased cell viability, increased apoptosis, and reduced cellular resistance to paclitaxel. 3.1.3. Lipid-based siRNA delivery systems in cancer therapy Cationic lipids are used as carrier for siRN“ delivery. Liposomes and lipoplexes, as lipid-based delivery systems, have been widely used in local and systemic siRN“ or shRN“ delivery. Liposomes are microscopic vesicles that consist of single or multiple lipid bilayer, form in a sphere with an aqueous core. Nucleic acids can either be entrapped in the aqueous core of liposomes or attached to the lipid surface for delivery. The advantages of liposomes as delivery system include a high gene transfection efficiency, enhanced stabilization, easy penetration into cell membranes, efficient in vivo delivery, and flexible and versatile physicochemical properties. The disadvantages of liposomes are the short half-life in serum, lack of tissue specificity, rapid liver clearence, and cell toxicity [ ]. Three different liposomes, such as neutral, anionic, and cationic liposomes, are used in the siRN“ delivery studies [ ]. Cationic liposomes for siRN“ delivery can easily cross the cell membrane, promote escape from the endosomal compartment, and reach the target genes with good biocompatibility. However, cationic lipids can induce an interferon response and cause unwanted interactions with negatively charged serum proteins because of its high cationic charge density [ , ]. Interferon responses can lead to not only change in gene expression but also show dosedependent cytotoxicity and pulmonary inflammation [ , ]. The toxicity and transfection efficiency of cationic lipids depend on length and structure of hydrocarbon chains of lipids [ ]. Neutral lipids lead to less cellular toxicity and do not induce immune responses without the down-regulation of gene expression. However, neutral liposomes have shown low transfec‐ tion efficiency because of their lack of surface charges [ ]. The commonly used cationic lipids for siRN“ delivery include , -dioleoyloxy- -trimethylammonium propane DOT“P and , di-o-octadecenyl- -trimethylammonium propane DOTM“ have combined with neutral

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lipids such as , -dioleoyl-sn-glycero- -phosphoethanolamine DOPE . This combination can enhance transfection efficiency. ”ecause neutral lipids facilitate fusion to the host cell’s membrane, cationic lipids can facilitate electrostatically complexation with siRN“ to obtain more stable formulation and entry to cells more easily [ ]. Liposomes are usually more stable than lipoplex in biological fluids [ ]. Lipid-based siRN“ delivery strategies have shown as promising in cancer therapy. Tumortargeting approaches have been used to enhance antitumor efficacy of these delivery systems. Specific delivery to target cells can be achieved by conjugation of ligands or molecules such as transferin or PEG on the surface of liposomes [ ]. The targeting and prolonged circulation half-life of liposomes allow for the enhanced permeability of tumor vasculature, increased delivery to tumor tissue, and reduced side effects [ , ]. Cationic liposomes containing siRN“ targeting tumor-associated genes have been used to inhibit tumor growth and prolif‐ eration, induce apoptosis, and enhance the radiosensitivity of tumor cells [ – ]. Cationic lipids can interact with negatively charged siRN“ by ionic interactions. Thus, selfassembly formed lipoplexes protect siRN“ from enzymatic degradation, enhance cellular uptake of siRN“ by endocytosis, enhance the release of siRN“ from endosomal/lysosomal entrapment, and thus, promote siRN“ accumulation in the cytosol [ ]. Commercially available cationic lipid formulations such as Lipofectin®, Lipofectamine® Invitrogen , Dharmafect® Dharmacon , RN“ifect® Qiagen , and TransIT TKO® Mirus have been studied as tranfection reagents for siRN“ delivery in vitro [ ]. The ratio of lipid and siRN“ lipid/siRN“ ratio affects the colloidal properties of the lipoplexes particle size and zeta potential . Lipid/siRN“ or shRN“ ratio is important to facilitate the cellular internalization of lipoplexes and to dissociate the nucleic acids in the cytosol. Lipid/siRN“ ratio can be optimized in terms of biological activity [ ]. Developing a lipid-based delivery system, choice of lipids, and appropriate formulations are essential to decrease cytotoxicity and increase the transfec‐ tion efficiency of formulation. To overcome the drawbacks of lipoplexes and liposomes, different nanostructures such as neutral lipid-based nanoliposomes, stable nucleic acid lipid particles SN“LP , and solid lipid nanoparticles SLN have been developed as siRN“ delivery system. SN“LPs are composed of cationic, neutral, and fusogenic lipid mixture. SN“LPs increase cellular uptake and endosomal release of siRN“ [ ]. PEG-conjugated SN“LPs represent exciting lipid-based systemic RN“i. The PEG-lipid conjugate improves the retention time to > hours [ ]. Recently, Tekmira Pharmaceuticals [ ] has developed siRN“-based drugs that are encapsu‐ lated in the SN“LPs for delivery of siRN“s to target tissue by intravenous injection. SN“LPencapsulated siRN“ targeting PLK1 initiated phase I trial in December . “lnylam Pharmaceuticals [ ] has developed first dual-targeted siRN“ drug, SN“LP-encapsulated siRN“s targeting VEGF, and kinesin spindle protein KSP for the treatment of hepatocellular carinoma. Phase I trial was initiated in “pril [ ]. Tekedereli et al. [ ] indicated that knockdown of Bcl-2 by intravenously administered nanoliposomal-siRN“ Bcl2 µg siRN“/kg twice a week lead to antitumoral activity in breast tumors of orthotopic xenograft models. In addition, nanoliposomal-siRN“s have enhanced the efficacy of chemotherapeutic agents in the breast cancer therapy.

Non-viral siRNA and shRNA Delivery Systems in Cancer Therapy http://dx.doi.org/10.5772/62826

Landen et al. [ ] studied neutral nanoliposomes incorporating siRN“ targeting EphA2 in orthotopic mouse model of ovarian cancer. Three weeks of treatment with EphA2-targeting siRN“ nanoliposomes µg/kg twice weekly reduced tumor growth. The combination therapy with paclitaxel reduced tumor growth. Salva et al. [ ] investigated the effect of co-delivery of siRN“ HIF1-α and VEGF in liposomal form in the breast cancer cell lines. Chitosan-coated liposomal formulation for co-delivery of siRN“ VEGF and HIF1-α were developed. The co-delivery of siRN“ VEGF and HIF1-α was greatly enhanced in vitro gene silencing efficiency in the breast camcer cell lines % . In addition, chitosan-coated liposomes showed % cell viability. Salva et al. has suggested that siRN“-based therapies with chitosan-coated liposomes may have some promises in cancer therapy [ ]. In conclusion, siRN“-based therapeutics are new and potential targets in cancer studies. In cancer, different mechanisms including angiogenesis, and cell growth were studied as target pathways. However, siRN“s have different hurdles in treatment because of their short biological life in blood, instability, and poor cellular internalization. In order to overcome these hurdles two solutions are present: one is modification of siRN“ and the other is use of suitable siRN“ delivery system. In cancer treatment, viral and non-viral delivery systems are evaluated as siRN“ delivery. “lthough limited information is available related to in vivo delivery, more papers are present in literature. Viral delivery systems have serious problems. Therefore, nonviral systems are more attractable than viral systems for siRN“s. Cationic lipids, liposomes, and polymers such as chitosan, PEI, PLL, and PLG“ are used as non-viral siRN“ delivery system. However, more suitable carriers are needed for siRN“ delivery systems.

Author details Emine Şalva *, Ceyda Ekentok , Suna 5zbaş Turan and J(lide “kbuğa *“ddress all correspondence to: [email protected] Faculty of Pharmacy, Department of Pharmaceutical ”iotechnology, İnön( University, Turkey Faculty of Pharmacy, Department of Pharmaceutical ”iotechnology, Marmara University, Turkey

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] Zhou J, Shum KT, ”urneti JC Rossi JJ. Nanoparticle-based delivery of RN“i thera‐ peutics: Progress and challenges. Pharmaceuticals. : . DOI: . / ph

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] Liu X, Huang H, Wang J, Wang C, Wang M, Zhang ”, et al. Dendrimers-delivered short hairpin RN“ targeting HTERT inhibits oral cancer cells growth in vitro and in vivo. ”iochem Pharmacol. : - . DOI: . /j.bcp. . .

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] Mu P, Nagahara S, Makita N, Tarumi Y, Kadomatsu K, Takei Y. Systemic delivery of siRN“ specific to tumor mediated by atelocollagen: Combined therapy using siRN“ targeting ”cl-XL and cisplatin against prostate cancer. Int J Cancer. : . DOI: . /ijc.

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] Murata N, Takashima Y, Toyoshima K, Yamamoto M, Okada H. “nti-tumor effects of anti-VEGF siRN“ encapsulated with PLG“ microspheres in mice. J Control Re‐ lease. : . DOI: . /j.jconrel. . .

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Chapter 11

siRNA-Induced RNAi Therapy in Acute Kidney Injury Cheng Yang and Bin Yang Additional information is available at the end of the chapter http://dx.doi.org/10.5772/61838

Abstract siRN“ therapy has great potential in humans, and its applications have been significantly improved. The kidney is a comparatively easy target organ of siRN“ therapy due to its unique structural and functional characteristics. Here, we reviewed recent achievements in the design, delivery, and utilization of RN“i with a focus on kidney diseases, in partic‐ ular acute kidney injury. In addition, the perspectives and challenges of siRN“ therapy such as increasing its serum stability and immune tolerance, targeting single/double/ multiple genes, cell/allele-specific delivery, time-controlled silencing, and siRN“-modi‐ fied stem cell therapy were also discussed. Finally, selecting target genes and therapeutic time windows were addressed. Keywords: Small interfering RN“, kidney diseases, delivery, off-target effects, compen‐ sative responses

. Introduction “cute kidney injury “KI is very common and critical in clinical practice. The incidence of hospital-acquired “KI is increasing, and many patients require renal replacement therapy [ ]. “KI significantly increases the risk of chronic renal disease, end-stage renal disease ESRD , and death, presenting a major burden to the patient and the health care system. ”ecause of high metabolic activity in handling and transporting ions, amino acids, and other small molecules, the kidney is highly susceptible to acute injuries from lack of sufficient perfusion, exposure to, and accumulation of nephrotoxic substances. Despite numerous clinical trials, “KI remains a cause of significant morbidity and mortality for which there is no effective intervention [ ]. RN“ interference RN“i is a highly conserved biological phenomenon in all eukaryotes, including renal cells. “lthough RN“i naturally exists, synthetic artificial siRN“ exerts similar

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effects as natural endogenous microRN“ miRN“ . ”oth sense and antisense strands of siRN“ can be synthesized separately and annealed to form double stranded siRN“ duplexes in vitro. “fter the siRN“ is delivered into the cytoplasm, the artificial siRN“ silences the target gene using similar biological processes as endogenous miRN“. Since the introduction of nucleotide artificial siRN“s that triggered gene silencing in mammalian cells [ ], synthetic siRN“ has generated much interest in biomedical research, in which the kidney is one of important key players. siRN“ as a strategic molecule has been highly expected in the field of innovative therapy. ”ecause siRN“ is highly efficient at gene silencing, it is possible to develop specific siRN“-based drugs that could target any genes, including those that have unknown pharmacological antagonists or inhibitors. Different types of synthetic siRN“ have been tested for their efficacy in various disease models, including cancer [ ], autoimmune disorders [ ], cardiovascular injuries [ ], and organ transplantation [ ], including native and transplanted kidney injuries [ ]. “s siRN“ is a posttranscriptional regulator, it must first be absorbed into the target cells. Therefore, the kidney could be an excellent target organ for siRN“ therapy because it benefits from rapid and vast blood flow physically, subsequent glomerular filtration, and tubular absorption. In fact, the systemic administration of siRN“ leads to rapid uptake by the kidney, yielding a significant decrease of target protein expression [ ]. Consequently, RN“i by siRN“ has advantages for the treatment of renal diseases due to the unique urological system [ ]. In addition, the preservation of donor kidneys before transplantation also provides a suitable time window for the intervention of siRN“. In this chapter, we highlighted the design and delivery of siRN“ and its therapeutic effects with a focus on kidney diseases. We also discussed future challenges of siRN“ therapy, targeting single/double/multiple genes, cell/allele specific delivery, time-controlled silence, and siRN“-modified stem cell therapy.

. Current principle of siRNA design The design of potent siRN“s has been greatly improved over the past decade. The basic criteria for choosing siRN“s include the consideration of thermodynamic stability, internal repeats, immunostimulatory motifs, such as GC content, secondary structure, base preference at specific positions in the sense strand, and appropriate length [ ]. Chemical modifications significantly enhance the stability and uptake of naked siRN“s. Importantly, siRN“s can be directly modified without crippling the silencing ability. Chemical modifications have been rigorously investigated for virtually every part of siRN“ molecules, from the termini and backbone to the sugars and bases, with the goal of engineering siRN“ to prolong half-life and increase cellular uptake. The most common chemical modification involves modifying the sugar moiety. For example, the incorporation of ′-fluoro ′-F , -O methyl, -halogen, -amine, or -deoxy can significantly increase the stability of siRN“ in serum. Locked nucleic acid LN“ has been also applied to modify siRN“. The commonly used LN“ contains a methylene bridge connecting the ′-oxygen with the ′-carbon of the ribose ring.

siRNA-Induced RNAi Therapy in Acute Kidney Injury http://dx.doi.org/10.5772/61838

This bridge locks the ribose ring in the ′-endo conformation characteristic of RN“ [ ]. “dditionally, recent studies, including ours [ ], have proven the efficacy of LN“-modified siRN“ in terms of prolonged half-life in serum, but without detectable adverse effects, suggesting that the natural RN“i machinery could accommodate a certain degree of alterations in the chemical structure of siRN“s [ ].

. siRNA delivery The biggest obstacle faced by siRN“ therapies is the in vivo delivery of genetic materials. The systemic delivery of synthetic siRN“ has the most medical and commercial potential. This type of delivery, however, remains a major challenge for translating siRN“ from the research to the clinic. Overcoming the delivery challenge requires effective siRN“ delivery vehicles. The virus-based delivery system, while efficient, may be fatally flawed due to raised safety concerns, such as inducing mutations and triggering immunogenic and inflammatory responses [ ]. Therefore, extensive research had been performed to develop efficacious nonviral delivery systems, including direct chemical modification of siRN“ as described above and/or optimization of delivery materials, such as liposome formulation, nanoparticle conjugation and antibodies that target cellular moieties [ ]. To date, studies on synthetic siRN“ therapy have been performed in a variety of cell culture and rodent models [ ] that produced exciting results and were cost effective but failed to faithfully mimic human diseases. Therefore, large animal models, such as porcine models, are indispensable to compensate for the limitations of rodent models due to their greater similarity to human beings. The investigations on siRN“ conducted in our laboratory have reflected this trend in the field [ , , ]. . . Direct delivery of synthetic siRNA in vitro/ex vivo The siRN“ could be easily transduced into various cells for scientific research. For example, we transfected synthetic caspase- siRN“ in porcine proximal tubular cells LLC-PK using cationic lipid-based transfection reagent. The caspase- siRN“ inhibited apoptosis and inflammation in LLC-PK cells that were subjected to hydrogen peroxide stimulation [ ]. In addition to in vitro delivery of siRN“, ex vivo/in vivo siRN“ delivery to target organs is an indispensable step before its clinical application. If it was directly delivered into the kidneys, siRN“ could obtain higher local concentrations, which would result in improved gene silencing efficacy. During kidney transplantation, ex vivo local delivery of siRN“ into the donor kidney is feasible because it could be facilitated by the unique structure of the kidney and the characteristics of kidney transplantation. We utilized an ex vivo isolated porcine kidney reperfusion system to assess the efficacy of naked caspase- siRN“. The caspase- siRN“ was directly infused into the renal artery locally and autologous blood perfusate mimic systemic delivery before -h cold storage CS , followed by a further reperfusion for h. The results demonstrated that the caspase- siRN“ improved ischemia reperfusion IR injury with reduced caspase- expression and apoptosis, better renal oxygenation, and acid–base homeo‐

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stasis [ ]. These promising proof-of-principle observations provide valuable guidance for further development before siRN“ used in clinical practice. . . Local or systemic delivery of siRNA in vivo Delivery of siRN“ via ex vivo route can be applied in donor kidneys, but most renal diseases need in vivo delivery. ”ased on the anatomical and physiological characteristics of the kidney, local delivery can be achieved through several routes: renal artery, first targeting the glomeruli or tubules [ , ] renal vein, predominately targeting tubulointerstitium [ ] intraureteral, administered into the renal pelvis and interstitium [ ] and subcapsular administration, achieves intraparenchymal silencing [ ]. Due to the rich blood flow through the glomeruli, siRN“ injection via the renal artery followed by electroporation could silence specific genes in the glomeruli, such as TGF-β , which subsequently ameliorates matrix expansion in an experimental glomerulonephritis model [ ]. We then used naked caspase- siRN“ in a porcine kidney autotransplant model for the first time. The left kidney was retrieved from mini pigs and was infused with University of Wisconsin solution, with or without . mg of naked caspase- siRN“, via the renal artery, which was followed by renal artery and renal vein clamping for -h cold storage CS, mimicking donor kidney preservation before transportation in clinic . “fter right nephrecto‐ my, the left kidney was autotransplanted into the right nephridial pit for h without systemic siRN“ treatment Figure . The expression of caspase- mRN“ and active caspase- protein, as well as its precursor, was downregulated by siRN“ in the post-CS kidney. In the siRN“ preserved posttransplant kidney, however, caspase- precursor was further decreased while caspase- mRN“, and its activated subunits were upregulated, which resulted in increased apoptosis and inflammation. This study indicated that the naked caspase- siRN“ was effective for cold preservation but was not effective at protecting posttransplant kidneys, which may be due to systemic compensative responses overcoming local effects. Therefore, to overcome the systemic response and to prolong the therapeutic time window, we subsequently utilized a novel, serum-stable caspase- siRN“, both locally as before and systemically via a pretransplantation intravenous injection, and observed the animals for up to weeks post‐ transplantation. The effectiveness of the novel caspase- siRN“ was confirmed by downre‐ gulated caspase- mRN“ and protein in the post-CS and/or posttransplant kidneys, as well as reduced apoptosis and inflammation. More importantly, renal function, associated with active caspase- , HMG” , apoptosis, inflammation, and tubulointerstitial damage, was improved by this novel, serum-stable caspase- siRN“ [ ]. It has also been revealed that an injection of a single-dose Fas siRN“ through the renal vein post ischemia provided a survival advantage in a murine IR model, which was due to the antiapoptosis and antiinflammation effects of the Fas siRN“ [ ]. Unilateral ureteral obstruc‐ tion UUO is a well-established model for tubulointerstitial fibrosis. Xia et al. injected the siRN“ of heat shock protein once via the ureter at the time of UUO preparation, leading to significantly reduced fibrosis-related protein expression and a remarkable alleviation of the accompanying interstitial fibrosis [ ]. Subcapsular administration is still used in some experiments due to its unique advantages, although it requires an invasive procedure and has

siRNA-Induced RNAi Therapy in Acute Kidney Injury http://dx.doi.org/10.5772/61838

Figure . Schematic drawing showed a series of our studies using caspase- siRN“. The caspase- siRN“ was first used to protect porcine renal tubular epithelia cells against hydrogen peroxide-induced injury [ ]. The renoprotection of naked caspase- siRN“ with the same sequences was further validated in a porcine ex vivo isolated reperfusion model and showed that the siRN“ was effective for cold preservation [ ], but not in autotransplanted kidneys with‐ out systematic siRN“ treatment [ , , ]. Finally, the chemically modified siRN“ of caspase- via locked nucleic acid stabilized the siRN“ in serum and significantly protected autotransplanted kidneys [ , , ].

limitations in clinical practice. Cuevas et al. reported that an infusion of DJ- an antioxidant specific siRN“ into the subcapsule silenced DJ- expression in the renal cortex and increased ROS production [ ]. Systemic delivery is a common and convenient clinical practice, although current clinical trials using siRN“s are almost directly administered to the target site, such as the nostril, eye, and lung, thereby avoiding the complexity of systemic delivery [ ]. The most common method of systemic siRN“ delivery is a hydrodynamic intravenous injection with hydraulic pressure to assist siRN“ cell entry. However, the pharmacokinetic metabolism of siRN“ is more compli‐ cated during systemic delivery because siRN“s can be rapidly degraded by nucleases in the serum and cleared by the kidney and liver. To enhance the in vivo efficacy of siRN“ treatment, a variety of approaches have been attempted for both siRN“ itself and delivery techniques [ – ], as mentioned above. Due to its anatomical and physiological characteristics, the kidney is the most preferable target organ of systemic siRN“ administration. siRN“ access to the kidney is thought to be depend‐ ent on the filtration and reabsorption functions of the kidney. Proximal tubule cells PTCs are

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the primary site for rapid and extensive endocytic uptake of siRN“ within the kidney following glomerular filtration. In an “KI model, naked synthetic siRN“ targeting p that was intravenously injected h after renal ischemic injury significantly reduced upregulated p expression and protected both the PTCs and kidneys [ ]. In another study performed by Zheng et al., siRN“ was systematically injected to target complement C and caspase- in a murine renal IR injury model. The results showed that the level of serum creatinine and blood urea nitrogen was significantly decreased in the siRN“-treated mice [ ]. “s most of “KI may be not associated with renal surgery, the systemic siRN“ delivery might be a desirable approach. However, for kidney transplantation, in which IR injury is inevitable, local siRN“ delivery via any above-mentioned method is feasible and more effective. . . Cell-specific delivery “s proposed by precision medicine, individual person should receive customized healthcare including diagnosis and intervention. The dysfunctional cells are the true targets for siRN“ delivery. For instance, it is known that p in PTCs promotes “KI, whereas p in other tubular cells does not [ ]. It is also expected that apoptosis-inducing siRN“ should be directly delivered into tumor cells rather than the surrounding normal cells. Therefore, the cell-specific delivery method is our key point in the next generation of siRN“ development. Recently, antibody conjugation technology has made tumor-targeting drug delivery systems available. The conjugate can be regarded as a guided molecular missile that specifically targets unique antigens [ ]. Inspired by cancer therapy strategies, siRN“s have also been packed to be delivered to target organs, even cells. Recently, a type of asymmetric liposome particle “LP has been developed, which highly efficiently encapsulates siRN“ without nonspecific cell penetration. The “LPs protected siRN“ from ribonuclease degradation. “LPs without any surface modification elicited almost no uptake into cells, while the polyarginine peptide surface-modified “LPs induced nonspecific cell penetration [ ]. Leus et al. delivered siRN“ targeting vascular cell adhesion molecule- VC“M- into inflammation-activated endothelium using anti-VC“M- -S“INTPEGarg formulated with additional mol% DOPEPEG in vivo. The antibody recognizes V“CM- , which can create specificity for inflamma‐ tion activated endothelial cells. The siRN“ homed to VC“M- protein expressing vasculature in TNF-α-treated mice without any kidney and liver toxicity [ ]. These results represent great progress in siRN“ delivery system development. “ntibody-mediated specific recognition rather than virus-mediated recognition may be a mainstream in the future. . . Allele-specific RNAi RN“i, in addition, discriminates between two sequences only differing by one nucleotide conferring a high specificity of RN“i for its target mRN“. This property was used to develop a particular therapeutic strategy called allele-specific RN“i devoted to silence the mutated allele of genes causing dominant inherited diseases without affecting the normal allele. Therapeutic benefit was now demonstrated in cells from patients and animal models, and promising results of the first phase Ib clinical trial using siRN“-based allele-specific therapy were reported in pachyonychia congenita, an inherited skin disorder due to dominant

siRNA-Induced RNAi Therapy in Acute Kidney Injury http://dx.doi.org/10.5772/61838

mutations in the keratin 6 gene [ ]. The allele-specific siRN“ silencing of the mutant keratin 12 allele was also applied in corneal limbal epithelial cells grown from patients with Mees‐ mann’s epithelial corneal dystrophy [ , ]. It has also been shown that modified siRN“s conferring allele-specific silencing against disease-causing “LK mutants found in fibrodys‐ plasia ossificans progressiva, without affecting normal “LK allele [ ]. . . Delivery of siRNA using a cargo system “lthough lentivirus vectors as vehicles together with liposome reagents are widely applied in the transduction of siRN“, nanoparticle systems have emerged in last few years as an alternative carrier for advanced diagnostic and therapeutic applications. The nanotechnology offers many merits and overcomes the range of challenges/barriers summarized in the previous section, such as the bioavailability and biodistribution of therapeutic agents. Recent reports have demonstrated that the kidney, the glomerulus especially, is a readily accessible site for nanoparticles. Zuckerman at al. intravenously administered nanoparticles containing polycationic cyclodextrin and siRN“/CDP-NPs, most of which deposited in the glomerular mesangial areas. Furthermore, the cultured mouse and human mesangial cells could rapidly internalize siRN“/CDP-NPs. This process could be accelerated by attaching targeting ligand mannose or transferrin to the nanoparticle surface [ ]. Complex nanoparticles, especially cationic polyplexes/lipoplexes and liposomes, dominat‐ ed the scene in the early days of RN“i therapeutic development. Their main advantage lies in their endosomal release activity and their ability to concentrate multiple RN“i triggers in one particle [ ]. Forbes and Peppas cross-linked polycationic nanoparticle formula‐ tions using “RGET “TRP or UV-initiated polymerization. The advantage of this method is the one-step, one-pot, and surfactant-stabilized monomer-in-water synthesis, which is simpler and faster compared with traditional complicated multistep techniques involving toxic organic solvents [ ]. Regardless of how much each mechanism plays in the transport of the drug, cell entry remains a focus for drug design and discovery. “n exciting and relatively new approach to transporting pharmaceutical agents into cells is making use of cell-penetrating peptides CPPs . CPPs are relatively short peptides, typically less than amino acids, and could be vectors for the delivery of genetic and biologic products. CPPs provide a safe, efficient, and noninvasive mode of transport for various cargos into cells [ ]. Recently, van “sbeck et al. discovered that CPP/ siRN“ complexes with the most negative zeta-potentials in serum were the most resistant to siRN“ release over a -h incubation period compared to less negatively charged complexes [ ]. They also found that the zeta-potential of CPP/siRN“ complexes in serum did not correlate with improved cellular association, which might demonstrate the importance of serum proteins or CPP conformation on the ability of CPP/siRN“ complexes to associate with the cell membrane. Huang et al. designed a bifunctional peptide named RGD - R, by which siRN“ was delivered in vitro and in vivo. ”ecause of their electrostatic interactions with polyarginine R , negatively charged siRN“s were readily complexed with RGD - R peptides, forming spherical RGD - R/siRN“ nanoparticles. This is also a novel siRN“ delivery tool [ ].

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Gemini or dimeric lipids GCLs are a recent type of amphiphilic molecules that contain two polar headgroups linked by a rigid or flexible spacer that may be hydrophobic or hydrophilic. “s each headgroup has a hydrophobic moiety, GCLs may be considered as two conventional monomeric surfactants connected by a spacer group [ ]. GCLs have been proved as promising candidates to transfect nucleic acids in gene therapy. The molecular structure of the GCLs offers a high number of alternatives to develop and to improve their capability as transfecting agents.

. siRNA therapy in AKI To date, siRN“ therapy has been successfully applied in a variety of acute kidney injuries. IR injury is the primary cause of “KI, particularly during kidney transplantation, in which the kidney is exposed to hypoxia and experiences a series of oxidative, inflammatory and apoptotic responses [ , ]. Consequently, specific siRN“s targeting critical molecules that are involved in the processes of oxidation, inflammation, and apoptosis have been developed. Caspase- , which mediates apoptosis and inflammation, is upregulated by IR injury. Multiple pharmacological interventions against caspase- , including enzyme inhibitors and genetic modification, have been investigated. In recent years, our group studied the delivery and efficacy of caspase- siRN“ in in vitro, ex vivo, and in vivo kidney injury models. The synthetic caspase- siRN“ was initially tested in porcine PTCs LLC-PK , with or without hydrogen peroxide H O stimulation. “poptotic cells and activated IL- β protein expression were significantly reduced by the caspase- siRN“, with improved cell viability [ ]. This outcome led to siRN“ application in an isolated organ perfusion system, as described above, and the efficacy of caspase- siRN“ was further proven, in terms of silenced caspase- mRN“ and protein expression, attenuated inflammation and apoptosis, and improved renal function and histology [ ]. The porcine kidney preserved by caspase- siRN“ was then autotransplanted in a -day model. However, the transplanted kidney was not protected without systemic treatment of the recipient. Moreover, new serum-stabilized caspase- siRN“s were applied locally in kidney preservation and intravenously in recipient in a -week autotransplant model. The transplanted kidneys were protected without significant off-target effects. These serials of stepby-step studies provided promising evidence to support siRN“ treatment to be further applied in clinic. p , another pivotal protein in the apoptotic pathway, has been identified as a mediator of transcriptional responses to IR injury [ ]. Molitoris et al. revealed that intravenously injected p siRN“ attenuated ischemic and cisplatin-induced “KI [ ]. Fujino et al. also tested the efficacy siRN“ targeting p via transarterial administration siRN“ injected into the left renal artery immediately after ischemia improved tubular injury and downregulated GSK- β expression [ ]. In a diabetic mouse model, p inhibition by siRN“ also reduced ischemic “KI [ ].

siRNA-Induced RNAi Therapy in Acute Kidney Injury http://dx.doi.org/10.5772/61838

Silencing of other important transcription factors or immunity related receptors using siRN“s have also been studied. Renal IR injury and inflammation are related to postsurgical healing and both processes can be influenced by toll-like receptor TLR signals. Effective TLR silencing by siRN“ decreases renal cell apoptosis, mitigates “KI severity, and increases the mice survival [ ]. NF-κ”, a pro-inflammatory transcription factor induced by TLR and other signals, plays a key role in “KI. NF-κ” activation depends on the activation of the inhibitor of κ” kinase β IKKβ . Wan et al. demonstrated that silencing IKKβ using siRN“ diminished inflammation and protected the kidneys against IR injury [ ]. These studies clearly demon‐ strate the therapeutic potential of siRN“-induced silencing of key “KI mediators, which are activated and involved in the pathways of apoptosis, inflammation, immunity, etc.

. Off-target side effects and toxicities of siRNA The siRN“ has been likened to a magic bullet due to this potency and specificity, but offtarget side effects and toxicities create additional challenges for researchers. The induction of various side effects may be caused by unexpected perturbations between RN“i molecules and cellular components. The off-target effects of siRN“ were first reported by Jackson and colleagues in [ ]. ”roadly speaking, off-target effects can be siRN“ specific or nonspe‐ cific. The former are caused by limited siRN“ complementarity to nontargeted mRN“s. The latter, resulting in immune- and toxicity-related responses, are due to the construction of the siRN“ sequence, its modification, or the delivery vehicle. The off-target effects associated with siRN“ delivery fall into three broad categories: miRN“-like off-target effects, referring to siRN“-induced sequence-dependent regulation of unintended transcripts through partial sequence complementarity to their ′UTRs inflammatory responses through the activation of TLR triggered by siRN“s and/or delivery vehicles such as cationic lipids and viruses and widespread effects on miRN“ processing and function through the saturation of the endogenous RN“i machinery by exogenous siRN“s [ , ]. . . miRNA-like off-target effects The siRN“s and miRN“s share similar machinery downstream of their initial processing. Using several different siRN“s targeting the same gene, microarray profiling showed that each siRN“ produced a unique, sequence-dependent signature. Sequence analysis of off-target transcripts revealed that the ′ UTR regions of these transcripts were complementary to the ′ end of the transfected siRN“ guide strand [ ]. It is now understood that for the off-targeting effects to occur, a perfect complementarity between the seed region of the antisense strand such as nucleotide positions – or – and the ′ UTR of the transcript is necessary [ , ]. Silencing the set of original off-target transcripts could be induced by base mismatches in the ′ end of siRN“ guide strands. However, a new set of off-target transcripts within ′ UTRs that were complementary to the mismatched guide strand could be generated [ ].

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RN“i regulation by miRN“s involves partial complementarity between the targeting RN“ and miRN“. ”ecause miRN“s cause gene silencing through mRN“ degradation and trans‐ lation inhibition, the siRN“-mediated off-target effects may also be acting at two levels. For this reason, there should be greater emphasis on improving siRN“ design as well as moni‐ toring gene and protein levels following RN“i therapy to account for any off-target effects. . . Recognition and stimulation of the innate immune system The recognition and stimulation of the immune system arenonspecific off-target effects of siRN“ therapy. The RN“-sensing pattern recognition receptors PRRs , localized in endo‐ somes, are the most important components of the innate immune system. The responses of PRRs to siRN“s are either TLR-mediated or non-TLR-mediated. The PRR responses are also associated with siRN“ sequence-specific side effects and have recently attracted many attentions from researchers [ ]. RN“-sensing TLRs TLR and TLR are predominantly located intracellularly and recognize nucleic acids released from invading pathogens. The nonTLR-mediated innate immune responses triggered by siRN“ binding are linked to RN“regulated expression of protein kinase PKR and retinoic acid inducible gene RIG , which further induce caspase- and NF-κ” expression, respectively. The activation of PRRs generates excessive cytokine release and subsequent inflammation [ ]. ”ased on this second type of off-target RN“i effects, our group further investigated the mechanism of how short-acting caspase- siRN“ impaired posttransplanted kidneys. The results suggested that the amplified inflammatory responses in caspase- siRN“ preserved autotransplant kidneys were associated with TLR , TLR , and PKR activation, which may be due to systemic compensative responses, although persistent actions initiated by short-acting caspase- siRN“ cannot be completely excluded [ ]. Other studies have also indicated that the horseshoe-like structure of TLR facilitates dsRN“ recognition [ , ]. Interactions between TLR and dsRN“ were originally reported in when TLR -deficient mice exhibited reduced immune responses to dsRN“ viruses [ ]. Several studies have demonstrated that the immune response to siRN“s is cell type-dependent due to the selective expression of TLRs. siRN“s stimulate monocytes and myeloid dendritic cells through TLR to produce proinflammatory cytokines, or activate plasmacytoid dendritic cells through TLR to produce type I interferons [ – ]. In addition, the volume of hydrody‐ namic naked siRN“ delivery influences immune activation. Rácz et al. compared the immune responses induced by µg siRN“ dissolved in either low-volume mL/mouse or highvolume % of body weight, . mL/mouse in average physiological salt solution delivered in vivo. Low-volume hydrodynamic injection induced slight alanine aminotransferase “LT elevation and mild hepatocyte injury, whereas high-volume hydrodynamic injection resulted in higher “LT levels and extensive hepatocyte necrosis. High-volume hydrodynamic injection also led to a time-dependent slight increase in IFN-related gene expression [ ]. Collectively, these studies suggest that there is a need for improving siRN“ design, establishing experi‐ mental controls and carefully interpreting results.

siRNA-Induced RNAi Therapy in Acute Kidney Injury http://dx.doi.org/10.5772/61838

. From bench to bedside: Clinical trials The numbers of RN“i-based preclinical studies and clinical trials have grown over the past several years. To date, there have been registered clinical trials using siRN“ worldwide. These studies include retinal degeneration, dominantly inherited brain and skin diseases, viral infections, respiratory disorders, metabolic diseases, and of particular note, kidney diseases. In , Quark Pharmaceuticals completed a phase I, randomized, double-blind, dose escalation, safety, and pharmacokinetic study NCT on QPI, also designated I NP, which was a synthetic siRN“ that temporarily inhibits p expression that is in early development for acute kidney failure therapy. I NP is the first siRN“ to be systemically administered in humans. ”ased on the preclinical data obtained from animal models, the siRN“ was intravenously injected within h to bypass surgery patients. Pharmacokinetic data were collected during the first h, and safety and dose-limiting toxicities were monitored until hospital discharge and – months after surgery. Recently, Quark initiated a subsequent clinical trial to determine whether a single administration of I NP can prevent delayed graft function in kidney transplant recipients. Data from this study will be used to identify I NP doses for follow-on efficacy studies NCT . “nother ongoing phase I trial investigat‐ ing solid tumors, including Renal cell carcinoma RCC , was conducted by Calando Pharma‐ ceuticals. The investigators used C“L““- , whose active ingredient is a type of siRN“, to inhibit tumor growth and/or reduce tumor size. This siRN“ inhibits the expression of the M subunit of ribonucleotide reductase and resists nuclease degradation by using a stabilized nanoparticle that targets tumor cells NCT .”esides, there is an ongoing study, in which patients with melanoma, kidney cancer, pancreatic cancer, or other solid tumors that are metastatic or cannot be removed by surgeryare treated by “PN , siRN“-transfected autologousperipheral blood mononuclear cells.These cells were modified by siRN“ targeting factors inhibiting the killing ability of immune cells in vitro and transfused back into the body, in order to kill more cancer cells NCT , the above clinical trials can be found at ClinicalTrials.gov, Table . Study

Target/siRNA drug

Status

Disease

NCT

I NP

Phase I, completed

Kidney injury acute renal failure

NCT

I NP

Completed

Delayed graft function in kidney transplantation

NCT

C“L““-

Phase I, terminated

Solid tumor cancers including RCC

NCT

“PN

Phase I, recruiting

Melanoma, kidney cancer, pancreatic cancer, or other solid

Table . Clinical trials of siRN“ therapy in kidney diseases.

. Perspectives and challenges Despite the enormous potential advantages of siRN“ therapy, additional research must be performed before its large-scale clinical application.

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. . Target gene selection Genome-wide or pathway-specific siRN“ libraries have become available using highthroughput screening approaches. Establishing in vitro prescreening leads to signaling pathway prediction and target validation in in vivo renal disease. However, choosing one or a set of reasonable target genes is the key for designing specific siRN“ treatments. The patho‐ physiological changes during kidney disease, like any other disease, refer to a complex gene and protein regulation network. For example, the network that exists during kidney trans‐ plantation involves the original conditions of the donors and the interactions between the donor kidneys and the recipients, which could direct the progression, as well as the recovery, of the injury. Fortunately, transcriptome measurements of the transplanted kidney may provide a comprehensive understanding of gene regulation and would be beneficial for target gene selection. Mueller et al. analyzed the transcriptome of postreperfusion implant biopsies in living donors LD and deceased donors DD . Hundreds of mRN“s were identified that predicted delayed graft function [ ]. In a recent prospective study using human posttransplant kidney biopsies, mRN“s and two miRN“s were identified as molecular signatures of “KI. Elevated secretory leukocyte peptidase inhibitor in “KI allografts was validated and miR- p was identified as a molecular regulator [ ]. These genes could be used as potential targets of siRN“ therapy. We recently identified times more differentially expressed genes in renal allograft biopsies between living donors and cadaveric donors at min than months posttransplan‐ tation. The majority of these differentially expressed genes are responsible for acute responses at min, but also involved in inflammation, nephrotoxicity, and proliferation at months. These divergent transcriptome signatures between two types of donors might be linked with not only the initial injury of the donors, but also the immune responses of the recipients. “nother method for selecting target genes is by identifying their translation product proteins. To find a single or a set of crucial proteins involved in kidney allograft rejection, Wu et al. explored potential transcriptional factors and regulation networks in kidney transplant recipients, of which suffered from acute rejection “R . The results demonstrated that the dominant processes and responses were associated with inflammation and complement activation in “R. “ number of transcription factors were identified in “R patients, including NF-κ”, signal transducer, and activator of transcription ST“T and ST“T [ ]. Their recent study further revealed inflammation-derived kidney allograft injury, such as “R, chronic rejection, and impaired renal function without rejection. Wu et al. common proteins and level-specific proteins from the phenotype-related protein–protein interaction networks [ ]. These potential biomarkers also provide valuable targets for siRN“ design relating to the treatment of transplant-related injury. . . Timely application Compared with shRN“, an advantage of siRN“ for “KI therapy is time-controlled, transient treatment. Silencing the target gene for a short time or a long time should be assessed before RN“i application. The silenced genes may be multifunctional according to the surrounding milieu. For example, caspase- , generally considered an executor in cellular apoptosis, should

siRNA-Induced RNAi Therapy in Acute Kidney Injury http://dx.doi.org/10.5772/61838

be inhibited in injured tissues. However, it is also a loyal scavenger in malignantly transformed cells, which could be an unavoidable side effect in any caspase- -targeting siRN“ therapy. For “KI, siRN“ ineffectiveness is needed after the therapeutic time window. “dditionally, siRN“ application avoids intracellular traffic. In certain circumstances, shRN“ delivery could be harmful to the organ or even fatal. “ study from Grimm et al. investigated the long-term effects of sustained high-level shRN“ expression in the livers of adult mice. “n evaluation of distinct adeno-associated virus/ shRN“ vectors, with unique lengths and sequences that were directed against six targets, showed that vectors resulted in dose-dependent liver injury, with ultimately causing death. The observed morbidity was associated with the downregulation of liver-derived miRN“s, indicating possible competition of the latter with shRN“s through saturation of the endogenous RN“i machinery by the exogenous siRN“s for the limited cellular factors required for the processing of various small RN“s [ ]. Therefore, controlling intracellular shRN“ expression levels will be imperative, but siRN“ would not influence the endogenous process of RN“ degradation mediated by miRN“s. . . siRNA targeting single, double or multiple genes The knockdown of two or more genes simultaneously using siRN“ cocktail has been recently reported. Many applications of siRN“ cocktail have demonstrated significant benefits compared with siRN“ targeted to a single gene, particularly in anticancer and antiviral therapy [ , ]. “ high concentration of individual siRN“s may represent the key off-target effect in terms of competition for endogenous miRN“ biogenesis machinery. Therefore, the other advantage of siRN“ cocktail is the relatively low concentration of each siRN“, which may also reduce off-target signatures without sacrificing silencing potency [ ]. . . Cell-specific siRNA targeting We have showed that the apoptosis of different types of cells leads to different outcomes. For instance, the apoptosis of inflammatory cells is associated with inflammation clearance and tissue remodeling, whereas the apoptosis of renal parenchymal cells is link to tubular atrophy and renal fibrosis. Therefore, using the genetic material such as siRN“ targeting specific cell at particular time frame is crucial to achieve high efficacy of treatment in “KI and also avoid site-effects [ , ]. It is still challenging to administrate siRN“ cell specifically, but it is feasible as there were a few studies showed delivering siRN“ into liver cells and antigen-presenting cells [ – ] using carbon nanotubes and mannose-conjugated liposomes. In addition, surface pegylation and cell-specific targeting ligands incorporation in the carriers may improve the pharmacokinetics, biodistribution, and siRN“ selectivity. Choosing appropriate siRN“ carriers has to consider the safety, effectiveness, ease of manufacturing, off-target effects [ ], and innate immune responses. Of course, the efficacy of siRN“ is still a most important factor dominating the selection of its carriers [ ].

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. . siRNA-modified stem cell therapy Mesenchymal stem cell MSC transplantation has attracted much attention in cell therapy in different organ systems such as myocardial infarction. One of the limitations is the poor survival of grafted cells in the ischemic microenvironment. To tackle this issue, a novel siRN“mediated prolyl hydroxylase domain protein PHD silencing system has been developed based on arginine-terminated generation poly amidoamine nanoparticles. This system, for the first time, exhibited effective and biocompatible siRN“ delivery and PHD silencing in MSCs in vitro. “fter transplant PHD siRN“-modified MSC in myocardial infarction models, MSC survival and paracrine function of IGF- were enhanced significantly in vivo, with decreased cardiomyocyte apoptosis, scar size, and interstitial fibrosis, and increased angio‐ genesis in the diseased myocardium, which ultimately attenuated ventricular remodeling and improved heart function. This study demonstrated that a great potential of siRN“-modified stem cells in therapeutic applications, which, of course, might be used in “KI [ ].

. Conclusions The kidney is a comparatively easy target organ for siRN“ therapy due to its unique structural and functional characteristics. siRN“ intervention is effective, feasible, and has great potential for fighting against kidney diseases. For the next-generation siRN“ development, cell-specific precise delivery should be pursued. “lthough the safety of siRN“ therapy has been proven by rapidly emerging clinical studies, off-target and compensative responses still need be overcome via various modification strategies. The time for realizing the therapeutic potential of RN“i has come because optimized siRN“ therapy, in conjunction with advanced genetic screening technologies, could facilitate timely and specific treatment of kidney as well as other organ diseases in the near future.

Author details Cheng Yang

,

and ”in Yang ,

*

*“ddress all correspondence to: by @le.ac.uk Department of Plastic Surgery, Zhongshan Hospital, Fudan University, Shanghai, China Shanghai Key Laboratory of Organ Transplantation, Shanghai, China Medical Research Centre, Medical School of Nantong University Department of Nephrology, “ffiliated Hospital of Nantong University, Nantong, China, United Kingdom Renal Group, Department of Infection, Immunity and Inflammation, University of Leicester,University Hospitals of Leicester, United Kingdom

siRNA-Induced RNAi Therapy in Acute Kidney Injury http://dx.doi.org/10.5772/61838

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cal siRN“ delivery into melanoma. Biomaterials j.biomaterials. . .

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.

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] Chen D, Koropatnick J, Jiang N, Zheng X, Zhang X, Wang H, Yuan K, Siu KS, Shun‐ nar “, Way C, Min WP: Targeted siRN“ silencing of indoleamine , -dioxygenase in antigen-presenting cells using mannose-conjugated liposomes: a novel strategy for treatment of melanoma. J Immunother : – . DOI: . /CJI.

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Preclinical Development of RNAi-Inducing Oligonucleotide Therapeutics for Eye Diseases Tamara Martínez, Maria Victoria González, Beatriz Vargas, Ana Isabel Jiménez and Covadonga Pañeda Additional information is available at the end of the chapter http://dx.doi.org/10.5772/61803

Abstract RN“ interference RN“i is a posttranscriptional mechanism of gene regulation present in eukaryotic cells. Inducers of RN“i are small molecules of RN“ that act in the cyto‐ plasm where they are able to impair translation of a specific mRN“ to protein, hence modifying gene expression. The discovery of this mechanism in mammals led to the de‐ velopment of a new class of therapeutics with the aim of exploiting this endogenous mechanism of action. In the last decade, great efforts have been put into understanding RN“i and translating this accumulated knowledge into the design of modern therapeu‐ tics. With several compounds in phase III clinical development, the field is getting closer to its first market authorization. Here we make a thorough overview of the field of RN“i therapeutics in ophthalmology, one of the fields in which RN“i has been most successful. Keywords: RN“i, eye diseases, ophthalmology, drug development

. Introduction Short interference RN“s siRN“ are small molecules of double-stranded RN“ of around base pair long that specifically downregulate the expression of a target gene [ ]. This mecha‐ nism of endogenous gene expression regulation, present in most eukaryotic cells, has been thoroughly used to study gene function [ ]. SiRN“s exert their function in the cytoplasm of the cell, where they assemble with a several proteins to yield the RN“-induced silencing complex RISC , a multimeric RN“–protein complex that recognizes complementary messen‐ ger RN“s mRN“ and promotes their degradation, thus blocking the synthesis of specific proteins. RN“i may be activated by endogenous siRN“s synthesized in the nucleus of the cell and generated by subsequent processing within the cell cytoplasm to yield siRN“s. “lterna‐

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tively, siRN“s can be exogenously introduced to mimic the action of endogenous RN“i triggers [ ]. “mong the benefits of RN“i are the potential of transiently silencing any given gene at any stage of development and to affect gene expression in specific anatomical regions without affecting nontargeted regions. These benefits are being used as a basis to develop a new class of innovative drugs that may reach the market in the next five years the present report highlights the advances made in RN“i therapeutics on the field of ophthalmology.

. The special environment of the eye: advantages and disadvantages The eye has traditionally been considered a good organ for proof-of-concept studies to assess the efficacy of innovative therapies. It has a very particular anatomy that allows the transfor‐ mation of sensory information into electrical signals that can be thereafter interpreted by the brain. Transformation and partial processing of sensory information takes place in the retina, located at the back of the eye. The correct function of the eye requires light to travel through several anatomical structures in order to reach the photosensitive retina hence, these struc‐ tures have to be transparent or semi-transparent to allow passage of light. The environment of the eye is extremely specialized to ensure that transparency is maintained, and there are several mechanisms in place to ensure that this specialized environment is preserved. One of the anatomical characteristics of the eye to allow light to travel through its structures is restricting the blood flow to regions where transparency is not strictly required. In addition, access to the innermost regions of the eye is controlled by several barriers these barriers isolate the eye from external aggressions and pathogens to but also limit the access of therapeutics. The influence of the particular anatomical features of the eye on drug delivery is further explained in Section of this review. The barriers of the eye do not only isolate this organ from external aggressions or substances but also limit the access of internal elements as such, the immune system has only limited access to the eye making the eye a partially immune-privileged region. Finally, the aqueous humor, the clear liquid that fills the eye and maintains its shape and pressure, has a very low content in proteins compared to serum. “mong the proteins that are significantly reduced compared to other tissues are RNases and elements of the complement cascade. The reduced concentration of RNases increases the halflife of RN“s used as therapeutics, and reduction in the elements of the complement cascade further decreases the likelihood of an unwanted immune reactions to drugs. In order to preserve its integrity, the eye has efficacious barriers to block the entrance of pathogens and substances that could potentially affect its sensory function. The eye has developed specific features that ensure that light travels through its tissues this specialization of tissues to preserve visual function is also observed by the immune system [ ]. Immune responses change the local environment of tissues and are frequently associated to tissue inflammation. In order to avoid these potentially harmful changes, the eye has a relatively immune privileged status. This immune privilege status is maintained by several mechanisms. “bsence of lymphatic and blood vessels in certain areas and abundance of immunosuppressive factors in the aqueous humor are among these mechanisms [ ]. On the other hand, the eye

Preclinical Development of RNAi-Inducing Oligonucleotide Therapeutics for Eye Diseases http://dx.doi.org/10.5772/61803

needs to be able to respond to situations in which its integrity can be compromised such as viral or bacterial infections. The innate immune response is the first system activated in response to aggressions it acts like a watchdog mechanism recognizing pathogen-associated molecular patterns P“MP . Depending on the molecular characteristics and location of the P“MP, different effectors of the innate response are activated these responses can be mediated by toll-like receptors TLR or independent of these receptors. Toll-like receptors discriminate self-motifs from non-self-motifs [ ]. There are ten known TLRs, and they differ in their subcellular localization and in the type of non-self-pattern they recognize. TLR , TLR , TLR , TLR , and TLR recognize components of bacterial walls and are located in the cell surface, whereas TLR , TLR , TLR , and TLR recognize oligonucleotides and are sequestered in intracellular compartment. TLR binds to single- and double-stranded RN“, TLR binds to single-stranded RN“, and TLR binds to unmethylated CpG motifs, usually found in bacterial DN“. In the eye, the expression pattern of each TLRs varies depending on the anatomical structure all TLRs are present in the corneal and retinal pigment epithelia TLR is the predominant TLR in the rest of the eye structures where it localizes in resident antigen presenting cells [ , ]. TLR-independent response mechanisms to cytoplasmic oligonucleotides include dsRN“-binding protein kinase, the RN“ helicase RIG-I , and oligoadenylate synthase enzyme. These proteins are cytoplasmic dsRN“ sensors belonging to the antiviral innate immune system, which plays an important role in antiviral defense in response to viral infection and replication [ ]. The first proof-of-concept studies to demonstrate the viability of silencing genes in the eye showed that the injection of siRN“s into the subretinal space or vitreous cavity could indeed downregulate specific genes. In these experiments, the downregulation of genes of the vascular endothelial growth factor VEGF family with siRN“s correlated with the inhibition of ocular neovascularization [ , ]. The first set of these experiments used an adenoviral vector that codified for an siRN“ designed against VEGF . The subretinal injection of this vector h after the induction of coroidal neovascularization CNV by laser reduced the areas of neovascula‐ rization compared to areas of mice injected with vector codifying for a scrambled siRN“ [ ]. In a subsequent study, Campochiaro and coworkers [ ] demonstrated that the inhibition of ocular neovascularization could also be achieved by injecting a VEGFR siRN“ directly, without an expression vector, into the vitreous cavity. The siRN“ used in this study had the so-called canonical design, which comprises a -nucleotide guide strand and a complemen‐ tary passenger strand annealed to form an siRN“ duplex with a -bp dsRN“ stem and nucleotide ′ overhangs at both ends [ ]. In , Kleinman and coworkers published a study in Nature demonstrating that the effect of siRN“s on CNV was mediated by activation of TLR rather than an effect on target [ ]. The results of these studies showed that the effect of the siRN“s on CNV was dosedependent but not dependent on sequence. In addition, the authors demonstrated that a minimum length of the siRN“ was required in order for the molecules to have an effect on CNV this length was show to be at least nucleotides. The study also showed that the internalization of the siRN“s was not required for the inhibition of CNV as cells of the retinal pigment epithelium RPE abundantly express TLR on the cytoplasmic surface. The authors used several sequences to point out that the inhibition of CNV was mediated

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through an off-target effect. Using docking models, the authors showed that TLR and siRN“s were indeed able to interact with each other but the interaction was unstable when siRN“s were shorter than nucleotides. “ subsequent study by the same group showed that activation of TLR by IVT siRN“s led to caspase- -mediated degeneration of the RPE questioning the safety these compounds as therapeutics for back of the eye diseases [ ]. The findings of Kleinman and coworkers boosted research on alternative designs that could successfully block immune recognition among the most commonly used strategies are incorporation of delivery systems and ’-ribose modifications. Finally, exogenous RN“s are quickly degraded by RNases present in tissues and body fluids [ ]. These enzymes cut RN“ into smaller components that are subsequently incorporated into the route of degradation of nucleotides. RN“ses are present at high concentrations both in tissues, such as in spleen, liver, and pancreas, and in biological fluids, such as in serum [ ]. In the eye, the tear fluid is rich in nucleases but the presence of these enzymes is considerably lower in most eye tissues, thus allowing for increased half-life of the siRN“s used for thera‐ peutic purposes.

. Efficacy studies: Animal models to study the eye Proof-of-concept studies are required in order to demonstrate that a particular drug has the proposed activity. siRN“s are designed using bioinformatics tools to target specific regions of the human genome. Therefore, assessing the activity of these molecules in animal models requires that the siRN“ has biological activity in the species chosen to perform the proof-ofconcept study. With the sequencing of the genome of most animal models used in biomedical research, evaluating the homology of a given sequence between two species is nowadays common practice, but this does not warrant finding a fully homologous siRN“ for all targeted genes. In cases where homologous sequences cannot be found, a surrogate compound can be used to perform animal efficacy studies this entails designing a compound that targets the exact same region as the human version but with the sequence of the gene of the animal species to be used. Several animal models can be used to assess the activity of a compound. Here we highlighted the animal models used in the developmental programs of products included in the ophthalmic siRN“ pipeline. . . Ischemic optic neuropathy Ischemic optic neuropathy is a sudden loss of vision caused by interruption or decreased blood flow in the optic nerve. There is a disagreement as to its pathogenesis, clinical features, and management because ischemic optic neuropathy is not a unique disease, but a spectrum of different types [ ]. Ischemic optic neuropathy can be primarily of two types: anterior “ION caused by the interruption of blood flow in the optic nerve head and posterior PION involving the posterior part the optic nerve. ”oth types can be further classified into different subtypes. “ION comprises arteritic “-“ION caused by giant cell arteritis and nonarteritic N“-“ION caused by other than giant cell arteritis. N“-“ION is by far the most common

Preclinical Development of RNAi-Inducing Oligonucleotide Therapeutics for Eye Diseases http://dx.doi.org/10.5772/61803

type and typically affects individuals between and years of age. The incidence of “ION has only been thoroughly studied in the US“ where there are . – . cases per , inhabitants for the nonarteritic type, the numbers are lower: . per , inhabitants. N““ION is characterized by visual loss, optic disc swelling, sometimes with flame hemorrhages on the swollen disc or nearby neuroretinal layer, and sometimes with nearby cotton wool exudates. Visual loss is usually sudden and may progress over several hours to days and even weeks [ ]. “nimal modes of this disease are used to assess efficacy of pharmaceuticals in development for these conditions and include the optic nerve crush model and the photoembolic stroke model. The optic nerve crush model is a general model in which surviving of the ganglionar cells can be studied in response to a physical damage to the optic nerve [ ]. In the photoem‐ bolic stroke model, a photosensitive dye, such as rose bengal, is injected intravenously, and laser is specifically applied to the optic nerve head to activate the dye. The activation of the dye results in damage to the endothelial cells of the optic nerve vascular system that ultimately leads to thrombosis of vessels and edema of the optic nerve head [ ]. . . Glaucoma Glaucoma is a group of progressive optic neuropathies characterized by vision impair‐ ment and degeneration of retinal ganglion cells that if left untreated can lead to blind‐ ness. Glaucoma is the second leading cause of global irreversible blindness, and it has been estimated that . million people were affected by primary open-angle glaucoma PO“G and primary angle–closure glaucoma P“CG globally in , a number expected to increase to nearly million by [ ]. The degeneration of the optic nerve is thought to be produced as a result of changes in intraocular pressure IOP , but specific molecu‐ lar mediators of these changes have yet to be identified. ”ecause glaucoma may be asymptomatic until a relatively late stage where the nerve damage has already occurred, early diagnosis and treatment are crucial for halting the progression of the condition [ ]. Reduction of IOP is the only proven strategy to treat the disease thus, first-line treat‐ ments are aimed toward achieving this goal. There are several compounds currently used to lower IOP, and although most of them are efficacious in lowering IOP, they all come with their own set of side effects and tolerance to the drug is a frequent phenomenon. Tolerance or reduced response of the drug requires change of drug regimen, frequently increasing the dose or combining the prescribed pharmaceutical with another drug [ , ]. Depending on the specific phase of the disease one wants to model, several animal models can be used [ ]. If studying the degeneration of the retina is the main objective, the models mentioned in the previous section can be used. For assessing the IOP lowering efficacy of pharmaceuticals, models with increased IOP are generally used. The increased IOP model induced by oral water overloading in rabbits is a very easy and physiologic model to screen compounds. The basis behind this model is to give the animal an oral overload of water that will result in a transient increase in IOP [ ]. “lthough the specific mechanism behind the increase in IOP following water overloading is uncertain, the model has been extensively used to perform rapid screens of IOP-lowering compounds. The main advantage of this model over other existing models of increased IOP is that the anatomical structure and physiology of eye

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structures are preserved allowing a normal response to hypotensive drugs. Other models of increased IOP include laser photocoagulation, intracameral injection of latex microspheres, topical application of prednisolone, light-induced reduced outflow facility, subconjunctival injection of betamethasone, or episcleral vein occlusion [ ]. . . Dry eye disease Dry eye disease DED is a multifactorial disease of the tear-fluid and ocular surface that results in symptoms of discomfort, visual disturbance, and tear film instability. It is accompanied by increased osmolarity of the tear film and inflammation of the ocular surface [ ]. Common symptoms of this condition include blurry vision, tearing, and ocular pain. There are several factors that contribute to the etiology of the disease, among them are insufficient tear secretion, excessive evaporation, and alteration in the composition of the tear film [ ]. Temporary changes of the composition of the tear film can cause an acute form of DED if changes persist, the condition can turn into chronic DED. Damage to the ocular surface is usually more severe in the chronic forms than in the acute types. DED is frequently associated to other conditions such as Sjögren’s disease or lachrymal gland dysfunction, but it can also be caused by vitamin deficiency, contact lens wear, and use of several prescription drugs. “cute DED is handled with lubricants and avoiding preservatives in concomitant eye drops. If DED persists, treatment options include procedures that favor tear retention such as punctal occlusion, moisture chamber spectacles, contact lenses, or pharmacologic agents that stimulate tear secretion. More severe forms may require the use of anti-inflammatory therapy [ ]. “lthough some advances have been made toward alleviating some of the symptoms of DED, pain associated to this condition is not usually addressed. Pain in the eye results from stimulation of sensory axons of the trigeminal ganglion neurons innervating the cornea [ ]. “nimal models to assess the efficacy of ocular analgesics are extremely complex in terms of interpreting efficacy outcomes [ ]. One of the commonly used models to study pain is the capsaicininduced ocular pain model developed by Gertrudis and colleagues [ ]. This model is based on the evaluation of animal behavior after topical ocular administration of capsaicin, a selective agonist of transient receptor protein vaniloid type TRPV . Capsaicin applied locally to the eye activates TRPV inducing palpebral closure. Latency to open the eye and time required for complete palpebral opening can be used as measurements of the discomfort caused by capsaicin. Reference products used in this model include analgesics, in particular, capsazepine, the antagonist of TRPV channels. . . Ocular allergy Ocular allergies constitute a heterogenic group of diseases with a broad spectrum of clinical manifestations and include mild forms such as seasonal allergic conjunctivitis S“C and perennial allergic conjunctivitis P“C , and more severe manifestations such as vernal keratoconjunctivitis VKC , atopic keratoconjunctivitis “KC , and giant papillary conjuncti‐ vitis GPC . The severe forms can be associated to complications such as corneal damage and may cause vision loss. S“C and P“C are commonly IgE-mast cell-mediated hypersensitivity reaction to external allergens, whereas “KC and VKC are characterized by chronic inflamma‐

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tion involving several immune cell types. In S“C and P“C, allergens, with the help of antigen presenting cells, trigger a Th -predominant immune response that induces ” cells to release IgE. In S“C and P“C, allergen-induced local release of IgE prompts infiltration and degranu‐ lation of mast cells in Ca +-dependent mechanism. Mast cells liberate preformed inflammatory mediators such as histamine and leukotriene that subsequently attract eosinophils amplify the allergic response [ ]. The prevalence of ocular allergies in the general population is estimated to be around % in the United States [ ] and up to % in Europe and the Middle East [ ], but it is probably underestimated in most epidemiologic studies [ ]. The primary treatment for ocular allergies includes avoidance of allergens, cold compresses, and lubrica‐ tion. In persisting cases, symptoms can be treated using topical and oral decongestants, antihistamines, mast-cell stabilizers, or anti-inflammatory agents [ ]. “llergic conjunctivitis can be modeled in animals by exposing them to allergens in the presence of an adjuvant [ ]. The model developed by Magone and coworkers uses Female ”alb/C mice that are sensitized with short ragweed and alum and several days later animals receive a topical dose of short ragweed pollen in the eye. “ prescreening of mice can be performed in order to select only those animals that respond to allergens. . . Age-related macular degeneration with choroidal neovascularization “ge-related macular degeneration “MD is the leading cause of severe vision loss in indi‐ viduals over years of age [ , ]. “MD is caused by a combination of genetic and environ‐ mental factors. Risk factors include hypertension, cardiovascular disease, smoking, and high ”MI. “mong the genetic factors that confer susceptibility to developing “MD are variants in genes encoding complement pathway proteins [ , ]. The underlying cause for “MD is accumulation of drusen or residual material produced by the renewal process of the external part of the photoreceptors of the retina in the retinal pigment epithelium RPE . The accumulation of this material in the RPE leads to the production of inflammatory mediators that cause photoreceptor degeneration in the macula and severe vision loss [ ]. In the early stages of the disease, accumulated drusen are small the size and amount of this material increase as the disease progresses and central vision deteriorates. There are two types of “MD: dry or wet. Dry “MD is characterized by the degeneration of the RPE and photoreceptors along with changes in pigmentation of the RPE. In the wet form, or choroidal neovascularization CNV , fragile blood vessels of the choriocapillaris grow into the RPE and frequently leak blood and fluid that accumulate between RPE and choriocapillaris. “s a result of these abnormal growths, dense scars are formed in the macula, and the RPE can detach. The wet form is more severe than the dry form and sometimes dry “MD can develop into wet “MD [ ]. The characteristic invasion of leaky blood to the RPE in wet “MD is mediated by VEGF. The discovery of the relationship between VEGF and changes in vasculature in “MD led to the development of different approaches aimed to decrease the levels of this growth factor. “ntibodies targeting VEGF are currently the first-line treatment for wet “MD [ ]. The hallmark of wet “MD is CNV thus, this is the lesion most extensively modeled in animals to assess efficacy of compounds targeting this disease. The laser-induced CNV model is by far

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the most used animal model. This model, initially developed for nonhuman primates NHP , was later adapted into rodents. The basis for this model is to induce a break in ”ruch’s membrane using a high-energy laser. The experimental CNV can be analyzed in vivo using fluorescein angiography or optical coherence tomography or postmortem studying the retina explants. The model has been successfully transferred and validated to rat and mouse, and in both species, the chain of events taking place after lesion induction resembles the events that tale place in humans with the disease. Other models include the injection of subretinal materials such as Matrigel, angiogenic substances, macrophages, lipid peroxides, or polyethy‐ lene glycol. “lthough these models are promising, they have yet to be appropriately validated in order to be used as a proof of concept tools [ ]. . . Diabetic retinopathy Diabetic retinopathy DR is an ocular complication of diabetes mellitus characterized by microaneurysms in the retinal vasculature that eventually lead to ischemia and macular edema. Changes in the retina can cause rapid vision loss, and this complication is the main cause of visual loss in working-age individuals [ , ]. The initial phase of DR, known as nonproliferative DR, is characterized by the thickening of the capillary basement membrane and apoptosis and migration of pericytes. These microchanges cause microaneurysms and small leakages in the vessels that irrigate the retina. “s the disease progresses, interaction between endothelial cells and pericytes weakens and the capillaries become permeable subsequent accumulation of fluids in the macula leads to edema. The microaneurysms in the retinal capillaries cause occlusions that compromise blood flow through the retina and cause ischemia. Local hypoxia upregulates angiogenic factors that cause capillaries to grow into the retina, preretinal space, and vitreous cavity stage known as proliferative DR [ ]. “mong the upregulated angiogenic factors, one of the most critical is VEGF the newly formed vessels are structurally deficient and very responsive to this growth factor. “s such, antibodies used to treat “MD are also used for the treatment of diabetic retinopathy. DR is usually treated with laser photocoagulation, a procedure that does not cure the disease but mitigates the damage. IVT steroids can also be used to reduce accumulation of fluids within the retina. If accumulation of blood in the vitreous humor physically impedes laser photocoagulation, a vitrectomy has to be performed in order to remove the blood accumulat‐ ed in the vitreous prior to laser photocoagulation. There are several animal models of diabetic retinopathy, each of them comes with its own set of advantages and disadvantages. One of the most extensively used is the streptozotocin STZ induced diabetes model. Intravenous or intraperitoneal injection of STZ causes a rapid and selective destruction of β-pancreatic cells leading to hyperglycemia and development of type I diabetes. The model has been used successfully in several animal species including rat, mouse, rabbit, dog, and monkey. Nonproliferative DR develops in this model, but microaneurysms and neovascularization are seldom observed hence, this model can be complemented with the laser-induced CNV model explained in the “MD section. Larger animal models can be generated by surgically removing the pancreas, but this model is significantly more complicated to generate than the STZ model and has the same drawbacks. “lternatively, animals can be fed a high-galactose diet, but the induction of diabetes is considerably slower [ ].

Preclinical Development of RNAi-Inducing Oligonucleotide Therapeutics for Eye Diseases http://dx.doi.org/10.5772/61803

. Biodistribution studies Despite the extraordinary potential that RN“i technology displays in the treatment of ocular conditions, the transition of siRN“s programs to the clinical setting still presents challenges. The in vivo efficacy of therapies based on siRN“s depends on the ability of a given siRN“ to reach the cytoplasm of its target cell in sufficient quantities to achieve its desired biological effect. The intrinsic characteristics of siRN“s such as their sensitivity to degradation by endogenous enzymes, their relative large size, and its negative charge limit their ability to cross biological barriers and reach the cytoplasm. “pproaches used to overcome the hurdles associated to the use of siRN“s range from delivery strategies to chemical modifications aimed towards improving the pharmacological properties of the therapeutic siRN“s. Drug delivery into the eye is challenging due to the presence of static and dynamics barriers that protect the internal tissues. The eye consists of two anatomically differentiated regions: the anterior and posterior segments. The anterior segment includes the cornea, conjunctiva, iris, ciliary body, lens, and anterior and posterior chambers this segment occupies approxi‐ mately the anterior third of the eyeball. The posterior segment is of greater size and comprises the sclera, choroid, retina, and vitreous cavity. There are significant anatomical, molecular, and immune differences between the two segments thus, strategies to deliver molecules to the eye will be very different depending on the targeted segment [ ]. The anterior region of the eye is protected from exterior aggressions by the cornea and tear film. The former is a specialized tissue composed of five layers that constitutes the main physical barrier to external molecules the latter is an enzyme-rich fluid that degrades many biological molecules, lubricates the eye surface, and washes away materials from the cornea. In addition, many components of the tear film impede adhesion of molecules to the eye surface further restricting the access of external molecules to the inside of the eye. Topical ocular administration of drugs is a patient-friendly administration route typically used for the treatment of pathologies affecting the anterior segment of the eye. However, molecules applied as eye drops are quickly cleared from the ocular surface being the bioavailability of a compound administered via this route less than % of the initially applied dose. The standard volume of a commercial eye drop is approximately µL whereas the normal volume of the tear fluid in the ocular surface is - µl. Once an eye drop is instilled in the inferior conjunctival sac, there is a transient increase of volume that activates the blinking reflex and increases the turnover of the tear film. Most of the content of the eye drop is spilled out by the blinking process or drained via the nasolacrimal duct, drastically reducing the amount of compound available to the eye. The cornea is a specialized tissue covering the anterior part of the eye whose main functions are protecting against harmful agents and provide the eye with a refractive surface that allows the entrance of light. The human cornea is approximately . – . mm thick, and it is comprised of three layers: the outer five cell layer-epithelium, a thick stroma rich in type I collagen fibrils and glycosaminoglycans, and the innermost endothelium consisting in a single layer of cuboidal cells. The corneal epithelium is separated from the stroma by the ”owman´s mem‐ brane, while the stroma and the corneal endothelium are separated by the Descemet´s

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membrane. There are no blood vessels irrigating the cornea this provides the required transparency for the transmission and refraction of light. Drugs can take two paths to penetrate the corneal epithelium: the intracellular path crossing through the cells or the paracellular path bypassing between cells. The cells of the corneal epithelium are tightly attached to each other with gap and tight junctions that restrict the diffusion of large molecules between them [ – ]. The cross-cellular pathway requires molecules to be able to cross cell membranes thus, lipophilic molecules have an easier access through this route. The stroma is an aqueous matrix composed mainly of hydrated collagen and proteoglycans with few keratinocytes interspersed [ ]. The hydophilicity/lipophilicity index determines the diffusion of molecules through this layer [ ]. The remaining layers of the cornea do not significantly hamper the diffusion of molecules. Contrary to the cornea, the conjunctiva is a highly vasculated tissue that covers the sclera and lines the inner surface of the eyelids. Its main functions are producing mucus and tears to lubricate the eye surface and preventing the entrance of pathogens. The human conjunctiva is composed of three layers: the outer epithelium, the substantia propia, containing nerves and blood vessels, and the submucosa layer, which provides a lightweight attachment to the underlying sclera [ ]. The histology of the stratified outer epithelium varies among the different regions of the conjunctiva, but it is always its apical portion that controls the permeability of the conjunctiva. The conjunctiva offers an attractive route for drug delivery when compared to the cornea as it presents an extended exchange surface as well as a superior rate of permea‐ tion to large hydrophilic molecules. The sclera is structurally continuous with the cornea and extends posteriorly from the limbus. The composition of the sclera is similar to that of the corneal stroma, mainly collagen and mucopolysaccharides leaving numerous channels through which drugs can freely diffuse [ ]. The sclera is poorly vascularized and significantly more permea‐ ble than the cornea but less permeable than conjunctiva. There are contradictory reports on the ability of charged molecules to cross the sclera. Some authors suggest that this layer is more permeable to negatively charged molecules [ , ], whereas other studies suggest that positively charged molecules cross the sclera more easily [ , ]. Ranta and colleagues suggested that the negative charge of mucopolysaccharides in the sclera prevented the diffusion of negatively charged molecules as a consequence of charge repulsion. Other studies have shown that negatively charged molecules are indeed able to cross the sclera, pointing out that size is the limiting factor in drug diffusion through this layer [ ]. It should be noted, however, that scleral drug binding does not necessarily impair drug delivery to inner structures of the eye it can also act as a drug-depot if the molecules are subsequently released [ ]. Ophthalmic drugs topically administered to de eye can thus be absorbed through two pathways: crossing the cornea to reach the aqueous humor or through the conjunctival-scleral pathway reaching the uvea. The relative quantity that enters through each of the abovementioned routes varies significantly depending on the size and hydrophilic/lipophilic ratio of the molecule. Generally, the conjunctival route is favored for large hydrophilic molecules, whereas small lipophilic drugs are mainly absorbed through the cornea. The ability of siRN“s to penetrate the cornea has been thoroughly demonstrated as well as the ability of these compounds to enter the cytoplasm of cells within the cornea. However, the capacity of siRN“s to cross the cornea is limited, as shown by the limited amount of siRN“s detected in the aqueous humor following eye drop instillation [ ].

Preclinical Development of RNAi-Inducing Oligonucleotide Therapeutics for Eye Diseases http://dx.doi.org/10.5772/61803

Increasing the amount of compound in the anterior part may be of interest for treating specific conditions. For this purpose, several strategies can be used in order to improve delivery: a increasing the residence time of the compound within the eye surface, b directing the molecule to a specific region to increase the concentration locally, and c increasing absorption by using physical methods. Increasing the contact time of the molecule with the eye surface can be achieved by the use of formulations or depots. Formulations that increase viscosity and/ or mucoadhesion of ophthalmic solutions are generally believed to increase absorption into the eye. Polymers such as methylcellulose or polyvinyl alcohol can be added to solutions to increase viscosity and consequently increase residence and reduce clearance time. Mucoad‐ hesion may be increased by formulating the oligonucleotides in polymers such as chitosans. These polymers have been used to deliver DN“ vectors into the eye [ ]. Encapsulation in liposomes and in thermosensitive gels has also been attempted as a means to increase the absorption and residence time of oligonucleotides in the eye [ ]. In these studies, a -mer was formulated in liposomes, a thermosensitve % poloxamer gel, and HEPES the results of these studies showed that the amount of compound reaching external tissues such as the conjunctiva or the cornea was higher when the compound was prepared in HEPES. ”y contrast, access to deeper regions of the anterior chamber such as the sclera or the iris benefited from the increased viscosity of the gel formulation [ ]. One of the main drawbacks of biodistribu‐ tion studies to assess the fate of a given siRN“ in a formulation is that most of these studies focus on the fate of nanocarrier rather than on that of the oligonucleotide and the relative distribution of the molecule among the tissues of the eye. Therefore, thorough biodistribution, studies are required to address the specific characteristics required for improving delivery for specific conditions. Targeting has scarcely been used to deliver oligonucleotides into the eye there are a few reports using dendrimers with the goal of increasing the intracellular concen‐ tration of therapeutic oligonucleotides in specific regions of the eye, but advances toward this goal are as of today very limited [ ]. Physical methods such as iontophoresis have also been studied aiming to increasing the amount of molecule that crosses the cornea and/or the sclera. “lthough iontophoresis certainly increases the amount of transcorneal and transcleral delivery of oligonucleotides mainly to the anterior chamber but also to some degree to the posterior chamber, the use of this method has not been extensively used most likely because the equipment required to apply the required current would entail in-office administration, which would significantly complicate repeated administrations [ ]. Drugs administered systemically enter the eye from the bloodstream crossing the capillaries of the choroid. The choroid is a vascular layer composed of capillaries and supported by ”ruch’s membrane, a connective membrane of – µm thickness. ”ruch’s membrane separates the choroid from the retina, forming the main barrier to permeation across the choroid-”ruch’s bilayer [ ]. The permeability of ”ruch’s membrane is relatively high charge and size do not generally affect drug diffusion through this membrane unless molecules are very big in this particular case, size can reduce the rate of permeation [ ]. The choroid is a thin and permeable membrane that is rich in melanin. Melanin has the ability to bind and retain many drugs hampering their entrance to the retina and inner tissues. Other drug-binding proteins, depending on the kinetic of binding/unbinding retention of drugs by melanin, can completely block the entrance or act as a reservoir for slow release [ ]. Studies to assess the binding of oligonucleotides to melanin have yielded different results suggesting that at least some

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oligonucleotides bind to melanin reducing the rate of entrance to the retina this is however not the case for all oligonucleotides [ , ]. The main restriction to free permeation of molecules from systemic circulation to the eye is the blood–retinal barrier ”R” . The ”R” is composed of the inner ”R” and the outer ”R”. The former includes the vessels of the retina, whereas the latter is constituted by the retinal pigment epithelium RPE . ”oth barriers possess cells with well-developed intercellular junctions that control the permeation of substances through them. Larger molecules, such as proteins and nucleic acids, are mostly able to permeate through the choroid but have limited ability to cross the inner ”R” thus, drugs need to exit the choroid and penetrate the eye crossing the outer ”R”. Crossing through the outer ”R” usually requires high systemic doses increasing the likelihood of systemic side effects [ ]. Delivery to the posterior segment of systemic or topically applied drugs requires thus crossing several biological barriers. Therefore, invasive administration procedures are frequently used to deliver drugs to the posterior segment. In addition, the outflow mechanisms of the eye rapidly remove drugs from the posterior chamber thus, reaching clinically meaningful concentrations is challenging. Most of the programs developing siRN“s for eye conditions target the back of the eye consequently, the route of administration is IVT injection. The concentration of siRN“s administered IVT is highest in the vitreous body, but they are also found in the RPE, choroid, and retina. Depending on the stability of the siRN“s, the compound can also be found in systemic circulation. There are numerous reports describing strategies that can be of benefit for increasing the concentration of drugs in the back of the eye. Several nonbiodegradable Retisert™, Illuvien™, and Vitrasert® and one biodegradable Ozurdex™ intravitreal ocular inserts are currently used in the clinical practice for delivering small molecules to the back of the eye. It is expected that these advances be soon incorporated into the pipelines of larger molecules such as proteins and oligonucleotides.

. Toxicology siRN“s are chemically synthesized oligonucleotides and are considered New Chemical Entities NCEs by the US and European Regulatory “uthorities since when the European Commission excluded siRN“s from the definition of advanced medicinal products [ ]. Toxicology assessment of RN“i-based drugs should be carried out following guidelines for NCEs, and the complete toxicology battery is usually performed following the recommenda‐ tions of the ICH M R guideline [ ]. The guideline recommends the assessment of toxicol‐ ogy in two species, a rodent and a nonrodent, at three dose levels and for a duration that should be similar or superior to the clinical trial to be carried out. This assessment should include acute or maximum tolerated toxicology studies and repeated-dose toxicity studies. “ddition‐ ally, pharmacokinetics, safety pharmacology, genotoxicity, carcinogenicity, and specific toxicology studies should be carried out depending of the nature, indication, and route of administration of the product. On the other hand, some aspects of RN“i-based products are closer to new biological entities N”Es rather than NCEs therefore, some of the requirements

Preclinical Development of RNAi-Inducing Oligonucleotide Therapeutics for Eye Diseases http://dx.doi.org/10.5772/61803

of the ICH S guideline also apply to the design of their developmental programs [ ]. “s such, a tailored toxicology assessment program should be designed combining the recommenda‐ tions outlined in the above-mentioned guidelines and the accumulated experience of numer‐ ous compounds tested in preclinical and clinical development. Toxicology of ocular products depends on their biodistribution and on their biological activity. Moreover, the disease process, age, sex, or eye pigmentation are other potential factors affecting the toxicity profile of the ocular drug assessed. “dditionally, the bioavailability of the RN“i compound will depend mainly on the route of ocular administration topical versus injected and on the physicochemical characteristics of the drug. Toxicities arising from oligonucleotides, including siRN“s, can be classified into hybridiza‐ tion-dependent toxicities and hybridization-independent toxicities. Hybridization-dependent toxicities can be caused by a exaggerated pharmacology: excessive activity on the intended target or by b off-target effect: modulating gene expression of an unintended target by an RN“i-mediated mechanism. Hybridization-independent toxicities are often associated to the chemistry of the siRN“. Identified hybridization-independent toxicities include prolongation of activating partial thromboplastin time aPTT , complement activation, and immunostimu‐ lation [ , , ]. . . General toxicology Up to date, numerous siRN“s indicated for different eye conditions have entered clinical trials Table the administration route of four of these compounds is by IVT injection, whereas the remaining two compounds are topically administered in eye drops. The toxicology assessment of these products follows the traditional schedule for NCEs this schedule entails general toxicology studies in two species, a rodent and a nonrodent species of variable length. Most programs up to date have used NHP as the nonrodent species this is because siRN“s are species specific, and it is likely that the assessment of toxicology was performed in the only species in which the compound was pharmacologically active. The rabbit is very frequently used to assess toxicology of compounds under development for eye conditions. Many sponsors of programs using NHP or dog as nonrodent species chose to use the rabbit as second species, although this animal is not a rodent per se. Reasons behind this choice include the similarity of the volume of the eye to that of humans and the difficulty of administering controlled doses to smaller animals. This is particularly relevant when the compound is administered by IVT injection. This rationale has also been followed for developing siRN“s for eye conditions only in one case, PF, rats were used as the rodent species, and the rest of the programs developing siRN“s for eye indications used the rabbit New Zealand White rabbits or Dutch ”elted rabbit as second species for toxicology assessment. Most programs developing siRN“s for eye conditions include acute/maximum tolerated dose and repeat-dose toxicology studies. The length of these studies is determined by the indication, stage of development, and envisioned duration of treatment. In addition, most programs do not only perform toxicology studies using the envisaged route of administration but also include studies using intravenous route to challenge the systemic exposure to the drug and assess potential dose limitations and target organs.

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. . Genotoxicity “s mentioned in the previous section, both the ICH M R and the ICH S guidelines apply to programs developing siRN“s [ ]. The ICH S states that the range and type of genotoxicity studies routinely conducted for NCEs are usually not applicable to N”Es pointing out that performance of these studies is only required when there is a cause of concern. The European Medicines “gency EM“ issued a reflection paper on the assessment of the genotoxic potential of antisense oligodeoxynucleotides in January . This paper recommends addressing at least two issues in regards to oligonucleotides which may indicate a cause of genotoxic concern: a analyzing the potential of incorporation of phosphorothioated PS oligonucleotides into the DN“ and b addressing the potential of triplex formation of oligonucleotides with the DN“ fiber [ ]. Several years of experience with siRN“s indicate that full-length molecules are very unlikely to interact with the DN“. Thus, the potential cause of concern may arise from the genotoxic potential of metabolites or chemical contaminants. The metabolism of nonmo‐ dified oligonucleotides yields naturally occurring nucleotides that are subsequently incorpo‐ rated to the natural degradation pathways of endogenous nucleic acids thus, toxicities derived from these degradation products are not expected. Modified oligonucleotides, on the other hand, incorporate very frequently backbone modifications to reduce nuclease activity and improve other pharmaceutical properties of the molecule. The most commonly used backbone modification is the replacement of a nonbridging oxygen on the backbone between two ribonucleotides with a sulfur to create a PS linkage [ ]. Extensive genotoxicity studies performed with Vitravene, a PS antisense oligonucleotide administered by IVT injection, indicate that oligonucleotides with a PS backbone do not pose genotoxic potential [ , ]. These results are in line with those obtained in the analysis of over compounds studied in the standard battery, all of which have yielded negative results. Other modified nucleotides could potentially be incorporated into nucleotide pools and be thereafter used to synthesize DN“. The standard battery of tests would detect eventual damaging potential of theses degradation products. The EM“ reflection paper also recommends assessing the potential of triplex formation with the DN“ fiber. For this to happen, siRN“ molecules would have to enter the nucleus of the cell and their structure should include an uninterrupted homopurine stretch of at least – base pairs that should be homologous a given region of the DN“. In silico design of siRN“s usually addresses these issues and candidates with the ability of forming triplex are avoided prior to lead selection. . . Carcinogenicity Standard carcinogenicity studies are generally not required for N”Es, but these studies may be required for siRN“s depending on their chemical structure, clinical dosing, patient population, or biological activity. If the in vitro test genotoxic studies indicate that there is cause of concern for carcinogenic potential, further studies should be required in relevant models.

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For RN“i products under development for eye conditions, the systemic bioavailability of these products is usually very low, and a waiver to perform these studies may be justified. Strategies should be discussed in a case-by-case base with the competent health authorities. . . Reproductive and developmental toxicity The assessment of reproductive and developmental toxicity is required to support the use of a given pharmaceutical in pregnant women, women of childbearing potential, or children. These studies are regulated by the ICH S guideline [ ], which recommends assessing the effect of drugs on all phases of the reproductive cycle. These recommendations apply to siRN“based products. Nevertheless, due to the unique features of these compounds, a case-by-case approach should be followed for each product, and the requirements for these studies should be discussed with the competent authorities. The target, indication, chemical modifications, and systemic bioavailability of the RN“i-based drugs are features that may influence in the nature of the required studies. ”ecause the toxicity of siRN“s can be caused by exaggerated pharmacology whenever reproductive toxicity studies are required, they should be performed in a pharmacologically active species. Standard reproductive toxicity species in rodents or rabbits can give information on toxicity related to chemical structure. However, if the compound is not active in these species or if the biological activity is not deemed to be equivalent to the foreseen activity in humans, the assessment of reproductive risk may be conducted using an active analog or in a nonrodent species in which the compound has biological activity. If the former strategy is chosen, the toxicity and toxicokinetic profile of the surrogate should be taken into account when interpreting the results. If the compound is only active in NHPs, studies should only be performed in cases where there is cause for concern. In these particular cases, the number of animals should be optimized, and a combined enhanced pre-and postnatal developmental study can be performed as recommended for N”Es. In NHP studies, the assessment of reproductive toxicology is usually studied by histopathologic examination of the reproductive organs as part of the general toxicology studies of at least three months. The timing of reproductive and developmental studies depends on when women of childbearing potential are to be included in clinical trials. If NHPs are required for the assessment, the timing is more flexible due to the length and complexity of the studies [ ]. . . Local tolerance Local tolerance studies are required for all topically administered drugs. In most cases, the potential adverse events caused by local tolerance issues are evaluated in the single or repeated-dose toxicology studies, reducing the number of animals required for the program. . . Safety pharmacology “ccording to ICH S “, safety pharmacology studies can be reduced or eliminated for locally applied products as well as for N”Es that achieve highly specific receptor targeting [ ]. For siRN“s under development for ophthalmology indications, separate safety studies are not

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usually required instead, functional safety end points are incorporated into the repeated-dose toxicity studies. If the results of the toxicology studies indicate that there is cause of concern, separate safety pharmacology studies should be performed.

. Programs in development and future ahead Table summarizes the status of siRN“-based therapies under development for ocular conditions. “s mentioned in Section , the eye offers multiple advantages for developing innovative therapies therefore, studies in the eye pioneered the field of siRN“ therapeutics. The first siRN“ to enter clinical development for an ophthalmology indication was bevasiranib in shortly followed by sirna. ”evasiranib targeted VEGF“, whereas sirnatargeted VEGFR . These compounds were being developed for the treatment of “MD as both showed a dose-dependent inhibition of experimental CNV in animal models that correlated with knockdown of their respective target genes [ , ]. “s mentioned in Section , in Kleinman and coworkers published a study demonstrating that the effect of siRN“s targeting VEGF and VEGFR on CNV was not mediated by an on-target effect but by activation of TLR [ ]. The results of these studies indicated that the effect of the compounds on CNV was sequence-independent and mediated by siRN“s of base pairs or longer. The study also showed that the internalization of the siRN“s was not required for the inhibition of CNV as cells of the RPE abundantly express TLR on the cytoplasmic surface. The authors used several sequences, including those of the siRN“s undergoing clinical trials at the time, to point out that the inhibition of CNV by both bevasiranib and sirnawas mediated through an offtarget effect. “ subsequent study by the same group showed that activation of TLR by IVT siRN“s led to caspase- -mediated degeneration of the retinal pigment epithelium RPE questioning the safety of these compounds as therapeutics for back of the eye diseases [ ]. The clinical development of bevasiranib was halted in and of sirnain both as a result of not reaching or being unlikely to reach their respective efficacy end points in phase III trials. The findings of Kleinman and coworkers boosted research on alternative designs that were not able to activate TLR , and as result, a new generation of compounds is currently under‐ going clinical trials. Currently, the most advanced siRN“s-based programs for ocular indica‐ tions are Quark’s QPIand Sylentis’ bamosiran SYL . QPIis a -nt modified siRN“-targeting caspase currently in phase II/III for the treatment of nonarteritic anterior ischemic optic neuropathy N“ION [ ]. QPIhas shown to be safe when IVT injected to animal models and humans. The ongoing phase II/III trial for this compound analyzes the potential of multiple IVT doses to improve visual acuity in patients suffering N“ION [ , ]. ”amosiran is a canonical-designed naked siRN“-targeting β -adrenergic receptor “DR” under development for the treatment of increased IOP associated to glaucoma [ , – ]. Glaucoma is a degenerative, chronic disease of the optic nerve that can lead to blindness if left untreated [ ]. The mechanistic details of optic nerve degeneration observed in glaucoma are yet to be fully detailed, but it is well established that reduction of intraocular pressure avoids

Preclinical Development of RNAi-Inducing Oligonucleotide Therapeutics for Eye Diseases http://dx.doi.org/10.5772/61803

development of the disease. “DR” controls the production and release of aqueous humor. The aqueous humor is responsible for maintaining optimal IOP. Treatment with topic betablockers has shown to efficiently reduce intraocular pressure, but currently approved betablockers are small molecules and are thus able to reach systemic circulation and systemic organs were they cause unwanted effects. The rationale behind bamosiran is developing a locally active compound that efficiently knocks down “DR” in the eye but that is not able to reach systemic tissues reducing the likelihood of side effects. The compound is administered in eye drops and has been shown to be well tolerated in animal models and humans [ , ]. Three different doses of bamosiran are currently being studied in an active controlled phase IIb trial. Previous clinical trials with this compound have shown promising results in healthy individuals and patients with ocular hypertension [ , ]. SYL is a naked -bp siRN“-targeting transient receptor potential vanilloid- TRPV for the treatment of ocular pain. TRPV is a cation channel permeable to calcium activated by heat, low pH, and capsaicin among other signals. This receptor is present in several structures of the eye where it has been related, among other roles, to nociception [ ]. SYL has shown to be safe when administered in eye drops to animals and humans and to have analgesic effect in the capsaicin-induced eye pain model. The compound is currently undergoing a phase I/II for the treatment of ocular pain associated to dry eye disease, a condition for which no specific treatment currently exists [ ]. PFis a chemically stabilized siRN“-targeting RTP , a stress-induced adaptor protein that inhibits mTOR function upstream to TSC /TSC complex in response to a variety of stresses. Expression of RTP is upregulated in response to ischemia, hypoxia, and/or oxidative stress. Intravitreal injection of PFin preclinical animal models of laser-induced CNV leads to silencing of RTP via a RN“i mechanism without TLR activation and reduction of CNV volume, vessel leakage, and infiltration of inflammatory cells into the choroid [ – ]. This compound has undergone phase II clinical trials for the treatment of diabetic macular edema and wet “MD. Treatment with PFof patients with diabetic macular edema over a period of months caused a dose-dependent improvement in visual acuity compared to the visual acuity observed in patients treated with laser photocoagulation [ ]. “ subsequent phase IIb trial was conducted with a new set of doses but terminated because the primary end point was not likely to be achieved. The compound was thereafter tested in combination with ranibizumab, a monoclonal antibody fragment that targets VEGF and is the current gold standards for treatment of the disease. The results of this study have not yet been disclosed. PFhas also been studied in patients suffering wet “MD. In this indication, the compound did not show improvement as a single agent or in combination with ranibizumab in mean visual acuity after months of dosing. Self-delivery rxRN“s sd-rxRN“s incorporate ’-F and -’O-Me modifications and a sterol conjugate on the sense strand with the goal of improving stability and cellular uptake. These compounds have a -nt antisense strand and a sense strand usually shorter than nt resulting in an asymmetric duplex with a phosphorothioated single-stranded tail on the antisense strand [ ]. These compounds have been tested in vitro where they have shown to be able to induce target knockdown in different cell lines. In vivo analysis of their activity

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showed these compounds are readily taken-up by retinal cells and that the compound is evenly distributed throughout the mouse retina. Several of these compounds are under development for different eye conditions and are expected to enter clinical development shortly. SYL is a naked -bp siRN“ targeting the calcium release-activated calcium modulator OR“I . Store-operated Ca + entry SOCE is activated in response to depletion of endo‐ plasmic reticulum Ca + pools. “ctivation of SOCE induces Ca + entry from extracellular compartments, and this is mediated by store-operated CR“C channels. CR“C channels are composed of calcium sensing proteins called STIM stromal interaction molecule and poreforming subunits named OR“I [ ]. Mammalian cells have three OR“I isoforms: OR“I , OR“I , and OR“I although OR“I and fulfill the same role as OR“I , the Ca + currents generated by these proteins are around two- to threefold smaller than the ones generated by OR“I [ ]. There is growing evidence that indicates that short-term and long-term activation of immune cells in allergic responses is mediated by influx of Ca + to immune cells from the extracellular compartment. Short-term responses include the degranulation of mast cells and the activation of effector cytolitic T cells. Indeed, mast cells lacking either STIM or OR“I show a considerable defect in degranulation [ , ]. Long-term responses involve the modulation of gene expression that controls ” and T cell proliferation and differentiation. SYL is being developed for the treatment of ocular allergies and has shown to reduce immediate clinical signs in a mouse model of ragweed pollen-induced ocular allergy. The decrease in clinical signs was accompanied by a reduction in the number of infiltrating eosinophils in the conjunctiva and reduction of allergy biomarkers. TTis an ““V‐encapsidated construct that expresses a single shRN“ modeled into a miRN“ backbone that inhibits the expression of VEGF‐“ for the treatment of wet “MD. VEGFa protein is responsible initiating a signaling cascade that stimulates the growth of new blood vessels, a hallmark of wet “MD. TTis a second-generation candidate designed to express three shRN“s, which target three different genes, VEGF receptor , PDGF-β, and human complement factor ”, proteins that play a major role in the progression of wet “MD. ”oth these compounds are yet in a preclinical phase IND filing is planned for . STP is a multitargeted siRN“ cocktail nanoparticle formulation administered by IVT injection under development for treatment of wet “MD, proliferative diabetic retinopathy, and herpetic stromal keratitis. The cocktail includes three -mer siRN“ duplexes targeting VEGF, VEGFR , and VEGFR . Inhibiting this clinically validated pathway at the endothelial cells lining the interior of the growing blood vessels is thought to halt the progression of “MD. This product is currently in preclinical stage. “Q“ is a single-stranded long chain nonmodified ribonucleotide connected by a prolinederived linker that self-anneals to form a shot-hairpin structure within the molecule. The compound targeting periostin acts through an RN“i mechanism and is being developed for the treatment of diabetic retinopathy. The compound has shown positive result in a proof-ofconcept study of CNV [ ].

Preclinical Development of RNAi-Inducing Oligonucleotide Therapeutics for Eye Diseases http://dx.doi.org/10.5772/61803

Name ”evasiranib

Indication

Target

Route

Sponsor

Status

“MD with choroidal

VEGF“

IVT

Opko Health

Halted in Phase III

VEGFR

IVT

“llergan

Halted in Phase III

Caspase

IVT

Quark

“ctive, Phase II/III

neovascularization Sirna-

“MD with choroidal neovascularization

QPI-

N“ION Primary “ngle Closure

“ctive, Phase IIa

Glaucoma PF-

“MD with choroidal

RTP

IVT

Quark/Pfizer

Completed, Phase

neovascularization

II

Diabetic macular oedema

Completed, Phase IIb

SYL

Glaucoma

β “DR

Eye Drop

Sylentis

Completed, Phase IIb

SYL

Ocular pain associated to TRPV

Eye Drop

Sylentis

“ctive, Phase IIa

dry eye disease Undisclosed

Retinal scaring

Undisclosed

Intraocular

RXi

Preclinical

Undisclosed

Corneal scaring

Undisclosed

Eye Drop

RXi

Preclinical

Undisclosed

Macular degeneration

Undisclosed

Intraocular

RXi

Preclinical

SYL

Ocular allergy

OR“I

Eye Drop

Sylentis

Preclinical

TT-

“MD

VEGF-“

IVT

”enitec

Preclinical

TT-

“MD

VEGF-“, PDGFβ IVT

”enitec

Preclinical

IVT

Sirnaomics

Preclinical

IVT

“qua TherapeuticsPreclinical

and CF” STP

“MD and retinopathy

VEGF-VFGFR VEGF

“Q“

Diabetic macular oedema Periostin

Table . siRN“s in development for ocular indication.

. Conclusion RN“ interference is on the verge of becoming a new class of therapeutics [ ]. The field of ophthalmology has played a major role in advancing siRN“s from laboratory tools to the clinic. In the last few years, significant advances have been made in the understanding of how these molecules enter and exert its action in the eye and in the identification of the main hurdles that still need to be addressed. The introduction of chemical modifications as well as the under‐ standing of the immune activation in the eye has significantly improved the pharmaceutical properties of compounds for eye conditions. However, the following years will tell whether

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improvements on these molecules are enough to be of therapeutic value in the field of ophthalmology or not.

Author details Tamara Martínez#, Maria Victoria González#, ”eatriz Vargas, “na Isabel Jiménez and Covadonga Pañeda* *“ddress all correspondence to: [email protected] Sylentis S“U, Tres Cantos, Madrid, Spain These two authors contributed equally to the review.

#

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Chapter 13

RNAi-Induced Immunity Wenyi Gu Additional information is available at the end of the chapter http://dx.doi.org/10.5772/61632

Abstract RN“ interference has a close relationship with the host defense system including adap‐ tive immunity. It is not only involved in regulating immune cells at different stages of the immune response but also directly induces or enhances antigen presentation and subse‐ quent immune responses. We have previously reported that a small hairpin RN“ shRN“ targeted the downstream site of a dominant cytotoxic T lymphocyte CTL epit‐ ope of human papillomavirus HPV type oncogene E can stimulate an immune re‐ sponse against E expressing tumors in C ”L/ mice. This results in the elimination of tumor growth in vivo, whereas an shRN“ that targets the upstream site does not. Our re‐ cent data further confirm the long half-life of the '-mRN“ fragment after shRN“ degra‐ dation and its involvement in protein synthesis. This chapter summarizes these findings and provides some updated explanations for the findings. Keywords: RN“i, shRN“, immune response, HPV E , miRN“

. Introduction RN“ interference RN“i is a conserved gene-regulation mechanism in all eukaryotic cells, where small RN“s including small interfering RN“ siRN“ , small hairpin RN“ shRN“ , and micro-RN“ miRN“, miR interact with message RN“s mRN“s in a sequence-specif‐ ic manner and cause the cleavage or translational blockage of a gene [ , ]. ”ecause of its specificity and efficiency, it has been widely utilized as a routine tool for gene functional studies in biology laboratories worldwide. In addition, since RN“i blockage is very specific and at the transcriptional level, RN“i-based gene therapy RN“i therapy is thought to hold a great potential for treating many diseases, especially viral infections and genetic disorders. Synthesized siRN“ is thus regarded as a specialized drug for gene therapies. So far, promis‐ ing results have been obtained with RN“i therapy in various diseases and many are being tested in clinical trials, including viral infections, cancers, and genetic or inflammatory dis‐

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orders [ - ]. “s cancers and other emerging diseases such as dementia and super-bug infec‐ tions become major public health issues, RN“i therapy can offer a new solution and has the additional ability to overcome drug resistances. ”eside the fundamental gene-silencing and gene-regulating roles of RN“i, which will also regulate the gene functions of immune cells and thus the immune responses, RN“i pathway itself has an additional function and involvement in inducing adaptive immunity. This func‐ tion has not been well-studied, and the mechanism is not clear. Its relationship with the im‐ mune system in terms of antigen reactivation and antigen presentation is still a new area to be investigated. Indeed, in plants and primitive species, RN“i is a part of the defense sys‐ tem against viral infections. However, in mammals, RN“i seems not directly involved in the immune system, probably due to the development of an advanced and sophisticated im‐ mune system. This chapter summarizes the evidence of RN“i-induced immunity against tu‐ mors and provides some updated possible explanations for the findings. The possible link between miRN“ and its degraded products with the immune system has been also dis‐ cussed. Exploring the relationship between RN“i and the immune system may lead to new discoveries in RN“i biology and approaches for more effective cancer immunotherapy or treatment for viral and intracellular pathogen infections.

. The discovery of RNAi-induced adaptive immunity against tumors In , we reported a discovery about RN“i-induced immunity [ ]. We investigated two shRN“s encoded by lentiviral vectors on their ability to suppress tumor cell growth and stimulate antitumor immunity in vivo. One shRN“ targeted the downstream site of a domi‐ nant cytotoxic T lymphocyte CTL epitope of the oncogene E of human papillomavirus HPV type termed downstream shRN“ , while another shRN“ targeted the upstream site of this epitope termed upstream shRN“ . ”oth shRN“s were equally effective at silenc‐ ing E gene expression in mRN“ and protein levels and led to the inhibition of tumor cells growth in vitro and in vivo [ ]. In spite of this, TC tumor cells expressing HPV E and E treated with downstream shRN“ stimulated an immune response against E in C ”L mice and resulted in elimination of tumor growth in vivo, whereas cells treated with the up‐ stream shRN“ did not. When untreated TC tumor cells were injected to the same mice challenging tumors , the group of downstream shRN“ exhibited a total inhibition of chal‐ lenging tumor growth, whereas no inhibition was observed in the upstream shRN“ group. This ability of downstream shRN“ was absent in Rag-/- mice lack of T- and ”-cells , suggest‐ ing adaptive immune response or T-cell response was required. To prove that the immune response was antigen-specific, we carried out a same animal experiment by immunizing C ”L/ mice with TC- cells treated with these shRN“s but challenged with another tumor cell line C , which has the E expression and H- b genetic background as C ”L/ mice. “gain, we observed that only mice immunized with downstream shRN“ treated cells had a loss of tumor formation, indicating tumor clearance was specific to E . Our data indicate that a more effective treatment can be developed for cervical cancer by combining RN“i

RNAi-Induced Immunity http://dx.doi.org/10.5772/61632

treatment with immunotherapy. Our results also reveal that RN“i may be widely used as an antitumor immunity stimulator or enhancer Fig .

Figure . Schematic diagram shows RNAi targeting site and ' mRNA fragment. It is known that in cervical cancer HPV E and mouse lymphoma EG /OV“ ovalbumin models, the shRN“ targets the downstream site of the CTL epitope that produce the ' fragment of mRN“. This makes immature proteins and further induces an immune re‐ sponse.

To prove the applicability of immune response to general tumor antigens, we tested a model antigen ovalbumin OV“ expressed in EG cells that are a mouse thymoma cell line with C ”L genetic background. We chose the major CTL epitope of OV“, SIINFEKL, as the tar‐ get site and designed two upstream shRN“s OV“- and - and a downstream shRN“ OV“- . The shRN“-treated EG cells were used to immunize the mice, which were subse‐ quently challenged with untreated EG cells. We observed that only mice inoculated with OV“- -shRN“ treated cells had significantly reduced tumor formation but not with OV“and - shRN“s.

. Confirmation of mRNA fragments and truncated proteins in treated cells To determine if the RN“i-induced immune response was actually from degraded products of RN“i, we designed a series of primers that would amplify RN“ fragments inside and

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outside the targeting sites of upstream shRN“ E - and downstream shRN“ E - , Fig “ . If an inside fragment was present while the outside fragment was absent, it would indi‐ cate that the mRN“ had been cleaved by shRN“ at the target site. The cells were treated with E - and E - shRN“s and incubated for – days before real-time RT-PCR was car‐ ried out. “s expected, the shortest PCR fragment, R , was observed in all samples Fig ” . The full-length R PCR fragment was only observed in untreated TC cells or cells infected with the lentiviral vector control PLL, Fig ” . In cells treated with both E - and E - , R was not found, indicating that shRN“-mediated cleavage was occurring. Of most interest was the R fragment which was found in all samples except cells treated with E - . These results suggest that R or R short fragments of E - and E - existed in the cells, at least temporarily at the time we isolated RN“. These short-form mRN“s may act as templates for short-form proteins truncated proteins and trigger antigen presentation to CD + T-cells and a CTL immune response to E . “part from our data, a previous study reported that degradation of the ' mRN“ fragment resulting from siRN“-mediated cleavage was blocked for some mRN“s, leaving an mRN“ fragment that could act as a template for cDN“ synthesis. They suggested that this could give rise to false negative results and that this phenomenon may be avoided by the careful design of RT-qPCR primers for each individual siRN“ experiment [ ]. This report further confirms that mRN“ fragments from RN“i do sometimes exist in the cells. In addition, it was noticed by researchers that un-degraded fragments of an siRN“-targeted mRN“ may cause false positive effects of microarray analysis [ ]. To avoid this, they developed a qRTPCR protocol, which allowed for the determination of the optimal time point for mRN“ analyses, indicating mRN“ fragments after RN“i can be present in cell for a certain time. What is the functional role of these mRN“ fragments after RN“i? Our data demonstrated that they can be involved in translational machinery and produce truncated proteins. To ex‐ perimentally prove this, we utilized the OV“-expressing EG cell model again. The cells were treated with downstream and upstream shRN“s and further treated with the protease inhibitor MG to reduce protein degradation before immunoblotting was performed. The blots were probed with an antibody against the N terminus of OV“ protein. The predicted size of a truncated protein produced by the cleavage of OV“- shRN“ was . kDa. We observed a protein band about -kDa in cells treated with the OV“- shRN“ but not in un‐ treated and OV“- or OV“- shRN“ treated cells. It proves that truncated proteins can be produced in cells by the translation of mRN“ fragment cleaved by shRN“. The predicted truncated product by shRN“-OV“ was not observed due to cross-reacting proteins on the blot [ ]. Our recent data unpublished showed that the cleaved ’ and ’ fragments of human papil‐ lomavirus type HPVE / mRN“ after shRN“ treatment were unevenly degraded. The ’ mRN“ fragment was more abundant and displayed a greater stability than the corre‐ sponding ’ fragment in the treated cells. Further analysis revealed that the ’ fragment was polysome-associated, indicating its active translation, and this was further confirmed by us‐ ing tagged E protein to show that C-terminally truncated proteins were produced in treat‐ ed cells Singhania et al. submitted .

RNAi-Induced Immunity http://dx.doi.org/10.5772/61632

Figure . The shRNA targeting sites and primer design for HPV E /E mRNA “dapted from Gu et al . “ The shRN“ and primer sites on E /E mRN“. ” The PCR products on an agarose gel. Notes: TC , the untreated cell con‐ trol. PLL, lentiviral vector control. *: indicates the site of CTL epitope. F: forward primer, R: reverse primer.

. Possible models for explaining RNAi-induced immunity It is well established that miRN“s play an important role in regulating innate and adaptive immune responses as a part of their gene-regulating roles. Evidence has accumulated that miRN“s are involved in the adaptive immune responses by regulating T-cells, ”-cells, and antigen-presenting cells “PCs . For example, miRwas reported to target phosphatase and tensin homolog PTEN and increase proliferation and the activation of T-cells [ ], while miR,, and let- have been shown to be involved in the development of T-cells into memory cells [ ]. In addition, miRwas shown to inhibit nuclear factor of activated T-cells- NF“T- in the activation of CD T-cell in the early stage of adaptive immune re‐ sponses [ ] and miR-a could promote CD and CD double positive T-cell develop‐ ment [ ]. For ”-cells, miRis required for their normal function such as production of isotype-switched, high-affinity antibodies and for memory responses [ ]. It has also been demonstrated that miRis induced by ”-cell receptor ”CR [ ]. However, overexpres‐ sion of miRcan immortalize ”-cells and lead to transformation, for instance, E”V was shown to have induced miRexpression and transformed ”-cells [ , ]. In addition, miRis important in ”-cell development [ ] and so is miR- [ , ]. For dendritic cells DCs , a recent review summarized the need of miRN“s in their lineage commitment from bone marrow progenitors and for the development of subsets such as

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plasmacytoid DCs and conventional DCs [ ]. Liu et al. used software to predict and then conducted experiments to confirm that three members of the miRfamily, miRa, miRb, and miRare negative regulators of the innate immune response and antigen-presenting capacity of DCs. They showed that miR/ expression was up‐ regulated in DCs on maturation and activation induced by TLR , TLR , and TLR agonists. These miRN“s in turn inhibited the production of cytokines including IL- , IL- , TNF-al‐ pha, and IFN-beta and upregulation of MHC class II expression and DC-initiated antigenspecific T-cell proliferation by targeting Calcium/calmodulin-dependent protein kinase II alpha CaMKIIα [ ]. In addition, miRand miRhas been shown to play an impor‐ tant regulatory role in Langerhans cells LCs by cross-presenting a soluble antigen to anti‐ gen-specific CD + T-cells [ , ]. ”eside “PCs such as DC and LC, miRN“ is shown to be directly involved in antigen presentation. For example, ”artoszewski et al. demon‐ strated that the mRN“ of human endoplasmic reticulum ER antigen peptide transporter T“P is a direct target of miR. They showed that the '-UTR un-translational region of T“P contains a -mer seeding region for miRand the ER stress-associated reduction of T“P mRN“ and protein levels could be reversed by inhibitory miRN“ of miR[ ]. “s T“P plays an important role in MHC class I-associated antigen presentation, their data provide an insight for miRN“-regulating MHC class-I-associated antigen presentation dur‐ ing ER stress. The above-highlighted results clearly indicate miRN“’s regulatory role in many aspects of adaptive immunity. However, is it possible that miRN“ also takes part in host immunity through mRN“ fragments produced after RN“i just like shRN“ described in section and ? Normally, miRN“s target the ' UTR and lead to the translation block or degradation of the targeted mRN“s. So when they degrade mRN“s, it is supposed to produce long ' mRN“ fragments and may also produce truncated proteins. If this is true, the translated de‐ fective proteins could be treated as truncated protein and be processed by proteasome. If there is a CTL epitope in the defective structure, it could be coupled with the MHC class I molecule and presented to T-cells by DCs through antigen cross-presentation, as described above with shRN“. This could be a link between RN“i pathway and antigen presentation or adaptive immune response. In the shRN“ case discussed above, the target should be at the downstream of a CTL epit‐ ope to induce immunity. When miRN“s target ' UTR, a site certainly at the downstream site of any possible CTL epitopes, it is assumed to have the ability to produce truncated pro‐ teins and so to induce immune responses. Therefore, an important question for miRN“ biol‐ ogy is whether miRN“s can routinely induce immune responses by degrading mRN“ at ' UTR and generating ' mRN“ fragments or truncated proteins that contain CTL epitopes? So far, there is no answer for this question. “nother critical question is: what is the differ‐ ence between blocking and degrading mRN“ by miRN“s at ' UTR? Does this relate to an‐ tigen presentation of different proteins? ”ecause it has been shown that miRN“ can act as siRN“ and shRN“ can be produced in the same pathway as miRN“ [ ], it is important and interesting to investigate if miRN“ can induce the same immune response as shRN“. The systems of HPV E /TC and

RNAi-Induced Immunity http://dx.doi.org/10.5772/61632

OV“/EG can be used as good models to investigate this. “s miRN“s are routinely transcri‐ bed and involved in interacting with mRN“, this mechanism can be considered as a routine way in cells to generate CTL containing truncated proteins. However, because most mRN“s in cells are for self-proteins, their CTL epitopes will not be presented to T-cells. This leaves the question of whether this is a mechanism just for cells that express viral genes such as HPV E in TC and C as above or for cells expressing foreign genes/antigens such as OV“ in EG ? The next question is: can this be generalized to any tumor antigens including self-antigens? This is an interesting subject to investigate and will facilitate our understand‐ ing of how RN“i pathways interact with and are involved in adaptive immune responses antigen presentation to utilize them for cancer immunotherapy. “lthough some miRN“ are highly conserved between lower animals and higher animals, mammals have far more miRN“s compared to nonmammals. This suggests that during evo‐ lution, as gene regulation became so complex and important in higher animals, miRN“ or RN“i pathway gradually specialized into gene regulation. “t the same time, as the adaptive immune system became well developed and highly specialized, these two systems got sepa‐ rated, but as described above, they still have some links. Future investigations leading to in‐ sight into these links will provide answers to the above questions.

. Conclusion In summary, RN“i-induced immunity opens a new perspective in which to explore the rela‐ tionship between RN“i pathways and the immune system, especially its involvement in an‐ tigen presentation in the adaptive immune response. For RN“i biology, it will provide an insight into the understanding of function roles of RN“i including miRN“ and siRN“ in host defense. In the field of gene therapy for cancers, RN“i can be used as an approach to silence oncogenes as well as a strategy to enhance immunity against cancer antigens at least viral infection related cancers and further explored as a novel cancer immunotherapy. Fi‐ nally, for intracellar pathogens, it can be used as a strategy for developing new vaccine through RN“i reactivating their antigens to the immune system.

Author details Wenyi Gu* “ddress all correspondence to: [email protected] “ustralian Institute of ”ioengineering and Nanotechnology, University of Queensland, QLD, “ustralia

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Chapter 14

Perspectives on RNA Interference in Immunopharmacology and Immunotherapy Zhaohua Hou, Qiuju Han, Cai Zhang and Jian Zhang Additional information is available at the end of the chapter http://dx.doi.org/10.5772/61575

Abstract RN“ interference RN“i , mediated by short interfering RN“ siRN“ , vector-derived short hairpin RN“ shRN“ and microRN“ miRN“ , brings about revolutionary fea‐ tures to basic biomedical research and clinical application. New drugs based on RN“i have been developed for therapeutic applications. The family of RN“i molecules are effi‐ cient agents to modulate mammalian immune system, and many studies reported that these molecules could manipulate immune defence, surveillance and homeostasis. ”oth perfect match of siRN“/shRN“ and non-perfect match of miRN“ could be beneficial for designing RN“i-based drugs for treatment of tumour and viral infection. This chapter provides a view to control or utilize the immune regulation of various small RN“s that should help researchers to understand the successful clinical application of RN“i. Keywords: RN“ interference, siRN“, miRN“, immunopharmacology, immunotherapy

. Introduction RN“ interference RN“i is a conserved mechanism against exogenous nucleic acid and transposon transcripts in plants and lower animals. No matter of transfected siRN“, vectordelivered shRN“ or pre-miRN“ transcribed mainly by Pol II , Dicer DCLs and “gronaute “GO family proteins efficiently process small RN“s into short double-stranded RN“ dsRN“ . Further, dsRN“s assemble into the RN“i-induced silencing protein complex RISC to guide and cleave target mRN“, promote mRN“ degradation or inhibit mRN“ translation. The great potential of RN“i is to specifically repress the expression of diseasecausing genes while avoiding undesirable effects. It is well accepted that siRN“ can be recognized by endosomal pathways, Toll-like receptor TLR , TLR , TLR and cytoplasmic pathways, retinoic acid-inducible gene I RIG-I ,

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melanoma differentiation-associated antigen MD“- and RN“-activated protein kinase PKR , resulting in immune activation [ – ]. For example, it has been demonstrated that siRN“ can cause activation of at least three key transcription factors, including NF-κ”, interferon regulatory factor IRF- and IRF- , and stimulate interferon IFN secretion. This activates T cells and dendritic cells DCs in the spleen in a TLR -dependent manner [ , ]. Furthermore, ′-triphosphate siRN“ was demonstrated to activate RIG-I signal pathway, and then natural killer NK cells and DCs were activated [ , ]. In most circumstances, immune system stimulation is regarded as an unwanted side effect therefore, siRN“-induced immune response should be controlled by using proper delivery system or chemical modification, although immune stimulation has been proved to be essential in cancer treatment and viral infection. miRN“s are critical in regulating the development, differentiation, function and destiny of immune cells, including DCs, granulocytes, monocytes/macrophages, NK and natural killer T NKT cells and ” and T lymphocytes. miRN“s influence both innate and adaptive immune defence and individual miRN“s may contribute their implications to various immunemediated diseases. Furthermore, pattern recognition receptors PRRs , kinases, adaptors, inflammatory factors and IFN could all be targets of miRN“s. Extra effort has been made to develop miRN“-based oligos or vectors for anti-infection purpose by manipulating corre‐ sponding immune genes. In addition to silencing of targeted genes in a sequence-specific manner, components of RN“i technology often induce immune response. Several strategies were reported to design RN“i molecules with gene silencing and immune regulatory properties. ”ifunctional molecules rely on the activation of PRRs such as TLR / , TLR or RIG-I, or just rely on down-regulation of target gene. This chapter summarizes RN“i-involved immune responses in the past years and discusses the anticipated therapeutic application.

. Chemically synthesized siRNA and vector-derived shRNA . . RNAi drugs based on targeting specific immune genes Immune disorders, both autoimmune diseases and immune defective or deficiency, are always caused by high-level overexpression of certain immune genes. “ variety of immune inhibitory genes can serve as targets for RN“i-mediated gene silencing. Targeting specific immune suppressor could re-balance immune network and subsets. Elevated activity of signal transducer and activator of transcription ST“T has been found in several kinds of human tumours. Use of RN“i to knockdown ST“T expression and inhibit its activation would reduce the tumour cell growth such as pancreatic cancer, colorectal cancer, melanoma and hepatocarcinoma cells. ST“T knockdown could induce bystanders immune response in vitro and in vivo, where CD +, CD + and NKT cells were activated as well as the secretion of interferon- IFN- , interleukinILand tumour necrosis factor alpha TNF-α was increased significantly [ – ]. siRN“-ST“T , synthetical‐

Perspectives on RNA Interference in Immunopharmacology and Immunotherapy http://dx.doi.org/10.5772/61575

ly linked to CpG agonist of TLR , was demonstrated to silence immune suppressor ST“T gene in TLR + myeloid cells and ” cells. This strategy of therapy leads to activation of various populations of immune cells, including DCs and macrophages, that ultimately induce potent anti-tumour immune responses [ , ]. Hossain DM et al. recently report‐ ed that CpG-siRN“-ST“T conjugates could efficiently silence the target expression, and abrogate inhibition of CD + T cells in patients who received myeloid-derived suppressor cells MDSCs [ ]. Researchers proved that immune-stimulation-inducing CpG “ -ST“T siRN“ was non-toxic for normal human leukocytes [ ]. In another experiment, Luo Z et al. [ ] generated a nano-vaccine loaded with poly I:C a TLR agonist and ST“T siRN“. Researchers found this kind of siRN“ could promote the maturation of DC and reverse immunosuppression in the tumour micro-environment the function of inhibitory cells in tumour-draining lymph nodes were inhibited thus, anti-tumour immune responses were potently induced and the survival were prolonged [ ]. Therefore, ST“T siRN“s are expected to be a promising immunomodulatory drugs to improve the treatment efficacy of cancer vaccines by abrogating tumour immunosuppression. Suppressor of cytokine signalling SOCS is a negative regulator of antigen-presenting cell “PC -based immune response. Silencing of SOCS gene expression by RN“i is essential for DCs to enhance “g-specific anti-tumour immunity [ ]. SOCS -silenced bone marrow dendritic cells ”MDCs were more potent in suppressing tumour growth [ ]. When SOCS was silenced, maturation of DCs i.e. expressions of CD , CD , CD and major histocom‐ patibility complex II [MHC II] was significantly accelerated. “s a result, SOCS inhibition upregulated the expression of IFN- and IL- , and decreased IL- secretions, which induced Th cell differentiation and thereby affected the development of Th cell. The combined nanoparticle NP delivery, which can render both tumour antigen and siRN“-SOCS to ”MDCs, simultaneously could enhance immunotherapeutic effects in ”MDC-based cancer therapy [ , ]. DC-targeted delivery of SOCS siRN“ has been shown to enhance antifungal immunity in response to Candida albicans in vitro and HIV-specific cytotoxic T cell in mice [ , ]. This evidence suggests the use of SOCS –siRN“, as a potent adjuvant to improve immune response. “ is usually regarded as an attractive target for siRN“-mediated gene knockdown in DCs because it is a negative feedback regulator of multiple pro-inflammatory signal transduction. Several reports demonstrated that RN“i-mediated “ silencing in DCs enhanced expression of co-stimulatory molecules CD , CD , CD and MHC class II and pro-inflammatory cytokines IL- and TNF-α . Tumour-infiltrating cytotoxic T lympho‐ cytes CTLs , T helper cells that produced IL- and TNF-α were also activated by si“ DC. “ silencing in DCs can enhance the immune response against self-tumourassociated antigens [ , ]. Furthermore, “ -silenced DCs were proved to overcome CD +CD +regulatory T Treg cell suppression [ , ]. “ -silenced DCs could skew naive CD + T cells towards Th cell, but not Treg, Th or Th cells. ”ecause a high amount of IL- was produced in “ -silenced DCs, simultaneous down-regulation of IL- and “ resulted in enhanced T cell stimulatory capacity in DCs. “ down-regulation resulted in enhanced CTLs immune response by the NF-κ” and “P- pathways [ , ]. RN“i of “

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has enabled DCs to gain a potent ability to activate CTLs and Th cells, and inhibit Treg, providing a novel strategy to promote a tumour immune response. Programmed death ligand PD-L is another exciting target on the surface of antigenpresenting cells “PCs PD-L/PD- interactions were related to functional impairment and exhaustion of tumour antigen-specific CD + T cells. “lthough PD-L antibody exerts a potent anti-tumour effect, previous reports [ ] have demonstrated that PD-L -siRN“-PEI were preferentially and avidly engulfed by tumour-associated CD c+PD-L + tolerogenic DCs at ovarian cancer locations. This kind of nanoparticle uptake stimulated multiple TLRs signalling, mainly via myeloid differentiation factor MyD . Then, regulatory DCs activated into potent stimulators of CTLs that led to significant anti-tumour immunity in mouse models of ovarian cancer. Most importantly, PD-L knockdown DCs showed superior potential to expand minor histocompatibility antigen MiH“ -specific CD + effector and memory T cells from leukaemia patients early after donor lymphocyte infusion and later during relapse. Combined PD-L and PD-L knockdown resulted in improved prolifera‐ tion of CD + T cells and enhanced cytokine production [ , ]. In addition, another report demonstrated the improved effector functions of tumour-specific CD + and CD + human T cells by siRN“-mediated silencing of PD- ligands, PD-L or PD-L [ ]. These results suggest that siRN“-mediated knockdown of PD-L is a fascinating strategy to inhibit a negative regulatory mechanism of tumour-specific T cells. siRN“-CD , delivered by a novel delivery system with a poly-d“ extension at the '-end of the siRN“ sense strand that was stably incorporated into , -β-glucan, was captured and incorporated into DCs through its receptor, Dectin [ ]. This strategy could induce antigenspecific Tregs, resulting in the permanent acceptance of mouse cardiac allografts. CD knockdown significantly suppressed Th -type cytokines and induced Th -type cytokines in rats with myocarditis. Knockdown of CD in experimental autoimmune myocarditis E“M rats promoted Foxp gene expression and increased Treg cells [ ]. In addition, when silencing of CD or CD /CD , DCs exhibited suppressed allostimulatory activity with impaired “PC function. In the well-established collagen-induced arthritis CI“ model, multigene-silenced DCs were capable of delaying onset of joint pathology. Therapeutic effects of gene-silenced DCs were mediated by the inhibition of collagen II-specific “b production and suppression of T cell recall responses. “lso, multigene-silenced DCs inhibited Th and Th response, demonstrating IFN- and IL- inhibition [ ]. Thus, inhibition of specific co-stimulatory molecules of DCs reveals a promising approach of suppressing immune responses in autoimmunity. These findings highlight the potential of immunomo‐ dulation of siRN“-CD , and have important implications for developing RN“i-based clinical therapy in the transplantation field. It is well documented that tumours could secrete immunosuppressive molecules, including the cytokines transforming growth factor β TGF-β and IL- . This creates an immunosup‐ pressive environment, which inhibits anti-tumour immunity. The suppression of Treg cell, induced by targeting TGF-β using siRN“, can enhance the efficacy of a DC vaccine against a poorly immunogenic tumour in mice [ ]. Nanoparticle-delivered TGF-β siRN“ enhances

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vaccination against advanced melanoma, and the tumour micro-environment was modified with increased levels of tumour-infiltrating CD + T cells and decreased level of regulatory T cells [ ]. siRN“ targeting IL- receptor α siIL- R“ initiated the significant antigen-specific CD + T cell immune responses. Concordantly, the combination of knockdown of IL- R“ and TGF-βR in DCs showed significant up-regulation of MHC I, enhancing co-stimulatory molecules CD , CD , CD and chemokine CCR after lipopolysaccharide LPS stimula‐ tion. It induced the strongest anti-tumour effects in the TC- P a cervical cancer model expressing the human papillomavirus [HPV]- E antigen tumour model, and even in the immune-resistant TC- P ones [ ]. These data revealed that siRN“ co-targeting immuno‐ suppressive molecules enhance the potency of DC-based immunotherapeutics. High-mobility group box HMG” is highly expressed in tumour cells and increased levels of HMG” in tumour cells are usually associated with a greater tumour angiogenesis, growth, invasion and metastasis. Knockdown of tumour cell-derived HMG” by shRN“ did not affect tumour cell growth, while naturally acquired long-lasting tumour-specific IFN- - or TNF-αproducing CD + T cell responses were induced, and ability to induce Treg was attenuated. This led to naturally acquired CD T cell-dependent anti-tumour immunity [ ]. Foxp , a master gene that controls the development and function of Treg cells, contributes to pathogenesis of several different tumours. Owing to the intracellular localization of Foxp , RN“i technology was employed to knockdown its activation to suppress Treg activity in vivo. Tsai et al. [ ] performed a study targeting silencing Foxp gene expression by shRN“smediated RN“i using a lentivirus vector in a murine model of leukaemia. The lentiviral vector was used to overcome poor transfection efficiency. Lentiviral-mediated Foxp RN“i showed suppressive effects on tumour growth and prolonged the survival of tumour-transplanted mice. Furthermore, Foxp knockdown mediated by siRN“ increased the ratio of Th /Th in chronic hepatitis ” patients transcription factors T-bet and G“T“- may be partly involved in this progress [ ]. This strategy provides a novel view about how to decrease the number of Treg cells and weaken its function. Selective knockdown of CCL and CCL expression in monocyte-derived DC MoDC by siRN“ decreased the ratios of CD + to CD + as well as lowered the frequency of Tregs recruited by MoDC. Furthermore, intratumoural injection of MoDC, which was transfected with siCCL and siCCL , significantly reduced the number of Tregs while inducing CD + T cells infiltration in thymic nude mice with human tumour xenografts [ ]. Using siRN“ to selectively silence chemokines may lead to a new strategy for DC vaccine development to improve cancer biotherapy. High expression of indoleamine , -dioxygenase IDO in DCs leads to the suppression of T cell responses. Gene silencing by siRN“ or shRN“ of IDO in DC would up-regulate IL- and IFN- and inhibit apoptosis in CD and CD T cells as well as Treg cells IL- expression was significantly down-regulated, thus finally restraining tumour growth. DC-based vaccine with IDO silence was demonstrated to augment and enhance the anti-tumour response against breast cancer, melanoma, bladder tumour and liver cancer [ – ]. “ novel “PC-targeted siRN“ delivery system using mannosed liposomes Man-lipo with encapsulated siRN“-IDO

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Man-lipo-siIDO was demonstrated to preferentially silence IDO in “PCs and efficiently enhance anti-tumour immune response [ – ]. It was reported that natural killer group , member D NKG D activation was involved in NK cell and CD + cell-mediated liver inflammation, and blockade of NKG D by silencing of multiple NKG D ligands on hepatocytes was considered efficient in liver disease intervention. Huang et al. [ ] constructed a plasmid containing the three shRN“ sequences shRae shMult -shH . “fter hydrodynamic injection into mice, they found that the expression of all three NKG D ligands on hepatocytes was down-regulated, and fulminant hepatitis mediated through NKG D in NK cell was attenuated. Furthermore, simultaneous knockdown of multiple human NKG D ligands MHC class I polypeptide-related sequence ”/“ MIC“/” , UL -binding protein [UL”P ] and UL”P also significantly attenuated NK cell cytolysis. Simultaneous knockdown of multiple ligands of NKG D is a potential therapeutic approach to treat liver diseases induced by NKG D-expressing NK cells and CD + cells. Furthermore, inhibition of human leukocyte antigen-G HL“-G by siRN“ boosted NK cell lytic function [ , ]. “mong several molecules involved in immune response, the choice of targets should be carefully reviewed and validated comprehensively according to the emerging knowledge about their function. . . Advantage of non-target immune effect of siRNA/shRNA drugs siRN“/shRN“ have the potential to recruit immune receptors specialized in RN“ sensor, such as TLR , TLR / [ , ]. ′-triphosphate siRN“ p-siRN“ was demonstrated to be detected by RN“ sensors RIG-I. These immunostimulatory siRN“ or shRN“ can non-specifically induce innate immune response, the so-called off-target’ effects that have considerable implications for clinical application to cure cancer and infection disease. IFN response is a common side effect of siRN“s and siRN“s with GU-rich sequences, which are very potent in inducing IFN-α response. “ newly published report demonstrated that siRN“ could induce IFN-α responses, and then induced the analgesic effects in the spinal cord. This off-target analgesia is dose- and sequence-dependent while non-GU-rich sequences also produced off-target analgesia at high doses, where pain relief by a designed siRN“ may not be attributable to target gene knockdown but IFN response [ ]. Early in , Karikó et al. [ ] demonstrated that siRN“s and shRN“s induce immune activation by signalling through TLR and activate sequence-independent inhibition of gene expression. Kleinman et al. [ ] showed that non-targeted against non-mammalian genes and targeted against vascular endothelial growth factor [VEGF] or VEGFR siRN“s suppressed choroidal neovascularization CNV via cell-surface TLR and its adaptor TIR-domaincontaining adaptor-inducing interferon-β TRIF , leading to the induction of IFN-α and IL- . The effect of non-targeted siRN“ to suppress dermal neovascularization in mice was as effective as vascular endothelial growth factor VEGF siRN“. This finding showed that two investigational siRN“s in clinical trials owe their anti-angiogenic effect in mice, which was not due to target knockdown but due to TLR activation. The efficiency of RN“i by siRN“ is

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believed to be comparable with anti-VEGF antibodies. Kleinman’s group then concluded that a -nucleotide nt non-targeted siRN“ suppresses both hemangiogenesis and lymphangio‐ genesis in mouse models of neovascularization, induced by corneal sutures or hindlimb ischemia, as efficiently as a -nt siRN“ targeting VEGF-“ [ ]. “mong siRN“s, Khairuddin et al. [ ] identified an extremely immunostimulatory siRN“s, targeting the HPV, which exerted potent anti-tumoural function. This bifunctional siRN“ could reduce growth of established TC- tumours in C ”L/ mice, and its effect was TLR dependent, where ablation of TLR recruitment via ′O-methyl modification of the oligo backbone reduced these anti-tumour effects. Flatekval et al. [ ] designed either monofunc‐ tional siRN“s devoid of immunostimulation or bifunctional siRN“s with IDO silencing and immunostimulatory activities. They showed that bifunctional siRN“s were able to knock‐ down IDO expression and induce cytokine production through either endosomal TLR / or RIG-I. In the past years, several studies reported that bifunctional p-siRN“ Exp:targeting ”cl / TGF-β/Survivin/Glutaminase/IDO with target silencing and an innate immunity stimulation via RIG-I activation could confer potent anti-tumour efficacy. This is illustrated for the first time by the work of Poeck et al. [ ], who reported that bifunctional siRN“s, with ′-triphos‐ phate targeting ”cl p-siRN“ , led to better melanoma tumour reduction than OH-siRN“ or ′-triphosphate siRN“s containing target mismatches. Poeck and his colleagues revealed that siRN“ with ′-triphosphate ends could be recognized by RIG-I and activate an innate immune cells such as DC then, expression of IFNs was directly induced, leading to apoptosis in tumour cells. These bifunctional p-siRN“s with RIG-I activation and RN“i-mediated ”cl silencing could provoke massive apoptosis of tumour cells in lung metastases in vivo. This was the first report demonstrating that p-siRN“ represents a single molecule-based approach in which RIG-I function activates immune cell and gene silencing, leading to a key molecular event. Researchers subsequently found that p-TGFβ -siRN“ combining RIG-I activation with gene silencing of TGF-β induced profound tumour cell apoptosis and revealed potent antitumour efficacy in pancreatic cancer. This kind of p-siRN“ induces a Th cytokine profile, demonstrating IFN- induction and IL- inhibition. High level of IFN and CXCL recruited more activated CD + T cells to the tumour. Frequency of immunosuppressive CD b+ Gr- + myeloid cells was reduced after p-TGFβ -siRN“ treatment [ ]. In addition, p-siRN“ against survivin gene was designed and generated. This finding demonstrated that p-survivin-siRN“ inhibited lung cancer cell proliferation and induced a RIG-I-dependent type-I interferon response [ ]. Recently, ′-triphosphate siRN“ combining glutaminase GLS silencing with RIG-I activation was demonstrated to induce more promi‐ nent anti-tumour responses than RIG-I ligand or GLS silencing capability alone. p-siRN“GLS effectively induced intrinsic proapoptotic signalling, and GLS silence sensitized malignant cells to apoptosis induced by RIG-I activation. Moreover, cytotoxicity was en‐ hanced, resulting from disturbed glutaminolysis induced by GLS silencing. Finally, RIG-I activation by p-siRN“-GLS blocked autophagic degradation, leading to dysfunction of mitochondria, whereas GLS silencing severely impaired reactive oxygen species ROS scavenging systems, leading to a vicious circle of ROS-mediated cytotoxicity [ ]. Immature

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monocyte-derived DCs had been transfected with siRN“-bearing ′-triphosphate-activated T cells [ ]. In addition, p-siRN“ can inhibit hepatitis ” virus H”V , Influenza “ Virus and Coxsackie‐ virus, by gene silencing and RIG-I activation. RN“i provides a promising approach for the specific treatment of H”V infection. Our laboratory has previously demonstrated that p-H”xsiRN“ and shRN“-H”x not only directly inhibit H”V replication but also stimulate innate immunity against H”V, which are both beneficial for the inversion of H”V-induced immune tolerance [ ]. In H”V-positive hepatoma HepG . . cells, p-H”x-siRN“ combining RIG-I activation with H”x gene silencing induce stronger type I IFN response than non-target pscramble-siRN“, indicating that a potent immunostimulatory effect may partly contribute to the reversal of immune tolerance through decreasing H”V load p-H”x-siRN“ more strongly inhibited H”V replication and promoted IFN production than H”x-siRN“ in primary H”V + hepatocytes, and this effect was mediated by RIG-I activation [ ]. This was consistent of the other two reports [ , ]. Our dually functional vector containing both an immunostimulatory single-stranded RN“ ssRN“ and an H”x-silencing shRN“ could reverse H”V-induced hepatocyte-intrinsic immune tolerance TLR signalling pathway was attributed to this progress [ ]. Lin et al. [ ] designed and tested a p-mNP -siRN“ against influenza virus. They found that p-mNP -siRN“ could activate the RIG-I-mediated IFN-β pathway and significantly reduce virus load and virus-induced pathogenesis. The inhibition effect was in an siRN“- and RIG-I-dependent manner, demonstrating siRN“ playing dual antiviral roles: viral genespecific silencing and non-gene-specific RIG-I activation. This strategy was also proved to elicit potent antiviral effects in coxsackievirus myocarditis, and virus-specific p-siRN“ was superior to both conventional virus-specific siRN“ and non-target p-siRN“ in inhibiting viral replication and subsequent cytotoxicity [ ]. In the attempt to inhibit the expression of woodchuck hepatitis virus WHV , Meng et al. [ ] found that innate immune responses could be enhanced by RN“i through the PKR- and TLRdependent signalling pathways in primary hepatocytes. The immunostimulation by RN“i may contribute to the antiviral activity of siRN“s in vivo. Furthermore, siRN“ can also synergistically enhance DN“-mediated type III IFN a newly characterized antiviral interferon response in non-immune or primary immune cells. This enhancement is mediated by crosstalk signalling pathway between RIG-I RN“ sensor and IFI DN“ sensor [ ]. Designing with GU sequences, addition of triphosphate motifs to siRN“, co-treatment with CpG oligos are believed to activate innate immunity when siRN“ was applied in vitro and in vivo. “ccumulating evidence suggests these bifunctional siRN“s could activate NK cells and CD + T cells in different models. Thus, specific clinical applications of RN“i can benefit from a concurrent activation of the immune system.

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. miRNA It has been well discussed how miRN“s regulate signalling pathways, and the dynamics of the immune response, tolerance and homeostasis. Here we summarize and explore updated achievements of special miRN“s in immunopharmacology. . . miRNAs as intrinsic targets in antiviral immunity In addition to the conventional innate and adaptive immune responses, even in the earlier phase after virus invasion, the host cell suppresses viral replication by evolving the profile of special and constitutively expressed genes. These cell-intrinsic antiviral approaches based on host restriction factors may be no less important than in considerations of conventional immunity. “t the same time, viruses also gain some countermeasures or adapt the unique phenotype of their hosts substantially to survive. Moreover, miRN“s may also be involved in the inextricably intertwined relationship between viruses and their hosts. In , a liver-specific miRN“, miR , which is involved in cholesterol and lipid metabolism [ ], was illustrated to be necessary for hepatitis C virus HCV accumulation in cultured liver cells [ ]. Researchers found that miR directly binds to two close sites in the ′ non-coding region of the HCV genome and promote HCV translation [ – ]. This miRN“ kept conserved among all HCV subtypes [ , ]. Even in non-hepatic cell, miR could boost HCV replication [ ]. Moreover, miR was further proved to be significantly reduced after IFN-β treatment, and miR mimics neutralized IFN-induced anti-HCV effect [ ]. Epidemiological and genomic researches further suggested that the level of miR in individuals with HCV might be an ’indicator’ for IFN therapy, and only those patients with high levels of miR responded well to IFN therapy [ , ]. Therefore, miR antagonist would also be called as IFN ’sensi‐ tizer in HCV immune treatment. Santaris Pharma designed and synthesized an LN“-based miR inhibitor, named Miravirs‐ en or SPC , to eradicate HCV. The product was first evaluated in preclinical studies in mice [ ], cynomolgus monkeys [ ], green “frican monkeys and chimpanzees [ , ]. Here the key concern is that whether miR inhibitor can effectively lower the level of free miR and inhibit HCV replication without disturbing normal cholesterol and lipid metabolism or without any potential chemical toxicity. Interestingly, although there was a reduction of cholesterol levels in plasma by nearly %, Miravirsen caused a dose-dependent reduction of miR and maintained ∼ -week-long half-life in the liver of monkeys and chimpanzees [ , ]. Moreover, in the high-dose treatment group, Miravirsen decreased HCV subtype a or b more than orders of magnitude compared to control group. In all animal species, Miravirsen was reported to be safe, without serious adverse effects or dose-related toxicities in rats, monkeys and human [ , ]. In May , Miravirsen was put into human clinical trials as the first miRN“-based drug https://clinicaltrials.gov/ct /show/NCT . There was a significant, dose-dependent reduction and sustained decrease of HCV viremia after drug administration in human subjects, and several patients became even HCV undetectable during the study. “t the same time, only

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infrequent and moderately adverse effects were caused to some volunteers and did not influence the trial process [ ]. ”ecause miR is only liver enriched in physiological condi‐ tions and there is high amount of miR in adult human liver, it may be an ideal target to design highly specific anti-HCV drugs with good resistance to HCV infected person, particu‐ larly to those who have no tolerance to traditional treatments. In the following years, miR [ ], let- b family [ ] and some other miRN“s were then proved to influence HCV life cycle, providing new target to restrict hepatitis C infection and avoid chronic infection. ”esides HCV, some other kind of viruses also encode miRN“s or regulate the miRN“s expression in host cells to disturb the expression of many immune-associated genes directly and/or indirectly, so that they can be critical regulators for viral life cycle. For example, in HEK T cell, prototype foamy virus I PFV- encodes Tas protein to counteract cell-encoded miR , which could inhibit PFV- gene expression and accumulation [ ]. Kaposi’s sarcomaassociated herpesvirus KSHV -induced miR could silence p expression, which is critical for the transcription initialization of many antiviral genes, to help themselves maintain long-time latency [ ]. The hematopoietic-cell-specific miR - p potently restricts the replication of eastern equine encephalitis virus in myeloid-lineage cells by binding to the ′untranslated region UTR of viral genome [ ]. Even Drosha, the enzyme that processes miRN“ biogenesis and maturation, was an independent factor for limiting RN“ virus replication along with canonical type I IFN system in particular cell type [ ]. “bove of all, it is much likely that miRN“ mimics for viral inhibitory miRN“s or antagonists for viral beneficial miRN“s can be effective antiviral strategies as intrinsic immune drugs. . . miRNA regulation antimicrobial and anti-tumour immunity 3.2.1. miRNA in antimicrobial innate immunity Of the known PRRs, TLRs and RIG-like receptors RLRs have been well studied in mediating antimicrobial and inflammatory responses during infections, which may be targets of patho‐ gens or host-encoded miRN“s. The first PRR targeting miRN“ let i was reported in [ ], which targeted TLR mRN“ in a MyD /NF-κ”-dependent way during Cryptosporidium parvum infection, controlling the production of inflammatory factors. During Bacillus Calmette-Guérin ”CG infection, miR exerts its function by targeting multiple components of the TLR signalling pathway, including TLR , MyD , TNF receptor-associated factor TR“F and TNF-α in mouse lung cell [ ]. “fter HCV infection, miR was induced and negatively regulated the type I IFN signalling pathway by suppressing Janus kinase J“K and IRF in hepatocytes [ ]. Experimental evaluation using miR inhibitors or miR knockout up-regulated ”CG-induced pro-inflammatory factors or type I IFN and so as to inhibit ”CG or HCV more efficiently. ”esides using host miRN“s, human cytomegalovirus HCMV targeted TLR by encoding its own miRN“, miR-UL - p, and reduced the expression of IL- β, IL- and IL- upon stimulation with a TLR agonist [ ]. Neutralizing this miRN“ might recover normal cytokines production.

Perspectives on RNA Interference in Immunopharmacology and Immunotherapy http://dx.doi.org/10.5772/61575

”esides immune inhibitory miRN“s, dengue virus DENV -induced miR e* up-regulated IFN-β and the downstream IFN-stimulated genes ISGs by suppressing Ik”α and promoting NF-κ”-dependent IFN production [ ]. The transfection of miR e* would increase the expression of ′- ′-oligoadenylate synthetase O“S- , myxovirus resistance “ Mx“ and interferon-induced transmembrane protein IFITM . In , miR [ ] was proved to enhance RIG-I-induced viral replication by suppression of the expression of cylindromatosis CYLD , which suppresses RIG-I K- -linked polyubiquitin. Moreover, Enterovirus EV inhibited miR transcription in an IRF-dependent way and so as to attenuate virus-triggered type I interferon production. These studies suggested that recruitment or increase of miR e* or miR would stimulate type I IFN expression and inhibit virus more quickly. 3.2.2. ’Immune miRs as immunopharmaceutic agents With the general knowledge of immunologically relevant miRN“s established in the past years, many miRN“s have been intensively investigated using gain- and loss-of-function methods, showing how this novel class of small non-coding RN“ participates in mammalian immunity. “nd individual immune miRN“ might contribute its implications to various immune-mediated diseases. The role of miR b in immune signalling may be paradoxical. “fter stimulation with LPS, miR b was down-regulated and TNF-α, one of miR b targets, was overexpressed in R“W . , which is essential for antimicrobial activity. Moreover, during M. tuberculosis infection, the overexpression of miR a significantly attenuated the antimicrobial effects in macrophages through targeting UV radiation resistance–associated gene UVR“G [ ]. Nevertheless, in diffuse large ” cell lymphoma DL”CL , miR a and miR b directly target a negative NF-κ” regulator tumour necrosis factor alpha-induced protein TNF“IP and present a positive self-regulatory property to maintain prolonged NF-κ” activity. Taken together, whether overexpression or inhibition of miR b in an anti-infection therapeutic study depends on concrete circumstances. miR a also acts as a negative regulator in immune sensing. ”oth in mouse and human, miR a was always exploited by virus to attenuate innate and adaptive antiviral immunity mainly in DC [ ], lymphocyte [ ] and hepatocytes by inhibiting interleukin- receptorassociated kinase IR“K , TR“F [ ], son of sevenless homolog SOS [ ] and ST“T [ ]. Silencing of miR a via the delivery of sponge or antagomiR could restore the expression of inflammatory factors, augment type I IFN production and promote clearance of vesicular stomatitis virus VSV [ ], dengue virus [ ], enterovirus EV [ , ] and H”V [ ]. ”ecause miR a was also abnormally expressed in hepatocellular carcinoma HCC and exerted negative anti-tumour effects by up-regulation of immunoinhibitory cytokines such as TGF-β, IL- , VEGF, miR a may also be a novel immunotherapeutic target for HCC [ ]. Unlike miR a, miR always promotes immune signal transduction, enhances immune function or speeds lymphocyte proliferation. Mice lacking miR have impaired CTL cell responses to infections with lymphocytic choriomeningitis virus and the intracellular bacteria Listeria monocytogenes because of insufficient activation of “kt pathway after TCR cross-linking [ ]. miR knockout mice died soon after Erdman a variant from severe acute respiratory

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syndrome [S“RS] infection and held higher level of colony-forming units CFU in lungs than wild-type mice [ ]. During HIV infection, miR inhibited the HIV-activating effects of tripartite motif-containing protein TRIM , and therefore, it might promote a return to latency in CD + reservoir cells [ ]. In addition, in NK cells, miR might regulate T cell immunoglobulin- Tim- /T-bet/ST“T- -signalling axis, and following cytokine expression that balanced antiviral response and immune injury during chronic HCV infection [ ]. “ remarkably ectopic up-expression of miR can be observed by delivering hepatotropic adeno-associated virus ““V vectors to the liver of mice, and then high level of miR enhanced G“P’s protective capacity against parasite [ ]. These studies imply miR as an immune-augmenting adjuvant in improving the antigenicity of vaccination. miR was already proved to be of importance in myeloid progenitor cells proliferation and responsiveness to pathogenic stimuli in neutrophils by targeting myocyte-specific enhancer factor C MEF C , acting as a fine-tune regulator both in normal granulocytes generation and in preventing aberrant expansion and over-activated inflammatory responses. In recent years, miR was involved in inflammasome response by targeting NLR family pyrin domain containing NLRP in human [ ]. Moreover, Epstein–”arr virus E”V encoded a mimic of hsa-miR , called miR-”“RT , targeting the same site within the NLRP ’-UTR to repress inflammasome activation. Furthermore, this miRN“ can be secreted from E”V-infected ” cells into exosomes to rheostat NLRP inflammasome activity in non-infected cells [ ]. miR sponge would balance the amount of NLRP and absorb’ E”V-miR-”“RT in macrophages and DCs. It is noteworthy that two groups of miRN“s, which shaped NK-mediated cytotoxicity, have potent value for developing antiviral and anti-tumour biodrugs. First, NKG D–NKG D-L interaction plays a predominant role in NK cell–abnormal cell’ recognition. MIC”/“, UL”Ps targeting miRN“s, are not only encoded by human genome as stress regulators but also synthesized by some virus e.g. HCMV-miR-UL [ ], E”V-miR”“RT [ ], KSHV-miRK - [ ] and ”K virus ”KV -miR-” - p, JC virus JCV -miR-J - p[ ] , to escape from NK cell killing. Meanwhile, viral infected cell and tumour cell always express low MIC“/” because of up-regulated MIC”/MIC“, targeting miRN“s such as miR a, , , b [ ] to maintain a compromised micro-environment. Furthermore, non-classical human leukocyte antigen G HL“-G is known as an inhibitory ligand, which suppresses the cytotoxic activity of T and NK cells. Studies demonstrated a strong post-transcriptional gene regulation of the HL“-G by miR a, miR b and miR , and lower expression of these miRN“s in renal carcinoma [ ] and placental choriocarcinoma cells [ ]. Stable manipulation of these activating and inhibitory miRN“s may enhance NK and L“K cell-mediated cytotoxicity against infected and tumour cells. Therefore, it could be concluded that modulating the expression or inhibition of specific miRN“s could boost immune response during viral infections or against cancers. . . miRNA in maintaining immune homeostasis ”ecause several miRN“s participate in immune cell development and differentiation, abnormal expression of miRN“ may cause a disturbance of homeostasis by changing the ratio of helper and regulatory cell subsets, or perturb the functionality and survival of effect-

Perspectives on RNA Interference in Immunopharmacology and Immunotherapy http://dx.doi.org/10.5772/61575

memory cells that lead to lymphoproliferative disease. Utilization of miRN“ interference techniques may recover regular immune balance. 3.3.1. miR17-92, miR146a and miR155 in Systemic Lupus Erythematosus SLE In , a unique mouse strain, sanroque’, presented a pattern of lupus pathology, revealing the core role of T follicular helper Tfh in systemic autoimmunity [ ]. miR - was found to regulate Tfh cell differentiation, which is essential for maintenance of the germinal centre formation and sustained antibody responses. Overexpression of this miRN“ in T cells would enhance Tfh cell proliferation and survive an autoantibody production [ ]. Similarly, miR increased IL- -mediated ST“T signalling in T cell [ ], which might accelerate Tfh differ‐ entiation and maturation as well. Moreover, miR deficiency ameliorates autoimmune inflammation of SLE by targeting s pr in mice [ ]. Therefore, miR - and miR might be a new target to restrain aggressive autoimmune response in SLE. 3.3.2. miR29 and miR146 in type 1 diabetes Type diabetes T D is a chronic autoimmune disease that results from the persisting destruction of pancreatic β-cells by autoreactive CD + T cell and Th cytokines. Dicer deletion in β-cell would disrupt normal β-cell development and survival, lead to impairment of insulin secretion and diabetes development [ ], apparently suggesting that miRN“s network is necessary for normal glycometabolism. Recently, endogenous miR b released from pancre‐ atic β-cells within exosomes stimulated TNF-α secretion in spleen cells isolated from diabetesprone non-obese diabetic NOD mice. Delivery of miR b to mice activated myeloid cell and pDCs to induce IFN-α, TNF-α and IL- production [ ]. “bnormal expression of miR is associated with high serum titers of glutamic acid decarboxylase antibody in T D patients, indicating the involvement of miR in the sustained immune imbalance during T D progress [ ]. These findings raised the possibility of developing a new clue for T D immu‐ notherapy using miRN“-based agents. 3.3.3. miR146a and miR155 in rheumatoid arthritis The role of miR a in controlling Treg-mediated decrease of Th responses has been dem‐ onstrated [ ]. In contrast, miR promoted Th and Th differentiation and cell formation and lowered T cell sensitivity to IFN- -driven proliferation by targeting C-M“F and IFN Rα [ ]. Therefore, imbalance of miR a and miR may be an epigenetic phenotype for autoimmune response. In rheumatoid arthritis R“ , decreased expression of miR a contributes to an abnormal Treg phenotype and allows Th /Th skewing while low level of miR failed to support effective Th immunity [ ]. Systemic administration of miR a has potential therapeutic intervention for preventing bone destruction by inhibited Th and Th cells, as well as IL- β, IL- and TNF-α [ ]. 3.3.4. miR15 and miR326 in multiple sclerosis Multiple sclerosis MS is manifested by chronic and progressive inflammatory demyelination of the central nervous system and is one of the main causes of regressive neurological diseases.

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Study on MS animal model illustrated that mice with fewer Th cells were less susceptible to experimental autoimmune encephalomyelitis E“E [ ]. Therefore, Th -targeting biother‐ apeutic approaches may be a promising way to cure multiple sclerosis. Gang Pei’ s laboratory [ ] found that miR promoted Th differentiation by targeting Ets- a negative regulator of Th polarization and antagonizing miR by sponge vector that resulted in fewer Th cells and Th cytokines and remitting E“E symptom. Inversely, increased miR in primary human microglia up-regulated pro-inflammatory cytokine secretion and co-stimulatory surface marker expression suggested that miR inhibition in myeloid cell might be useful to suppress allogeneic T cell responses [ ]. In conclusion, reverse pathological expressed miRN“s and re-balance dysregulated immune genes are of consideration to treat multiple sclerosis.

. Conclusion RN“i technology holds promise for treating various human diseases. It is becoming apparent that clinical outcome of cancer immunotherapy and infectious diseases can be improved by targeted strategies to abrogate tumour-induced immunosuppression. “nti-tumour strategies using siRN“/shRN“/miRN“ for both silencing of oncogenes and recruiting of innate recep‐ tors were designed. The present researches highlighted the potential therapeutic applications of this new generation of siRN“s in immunotherapy. “dditionally, but importantly, siRN“/shRN“ or miRN“ drugs with regard to pharmacody‐ namic difficulties and unwanted side effects are even more complicated compared to low molecular weight drugs and hard to be delivered into immune cells. This requires more extensive procedure than any other traditional drugs. Considering clinical challenges for RN“based nucleic acid drugs, including barriers and RNases, the advanced tissue-directed delivery systems with safety, high efficiency and specificity, long-term function and controllability are required. “lthough the exploration of such tiny regulators causally bring pharmacists a considerable effort to draw up individualized and tailor-made strategies, we believe that immunoregulation triggered by siRN“/shRN“/miRN“ can be used to regulate the host immunity against cancers or viruses. The development of multifunctional RN“i molecules will greatly contribute to the future arsenal of tools to combat not only microbial pathogens but also hard-to-treat cancer.

Acknowledgements This work was supported by the Natural Science Foundation of China , , , National ”asic Research Program of China No. C” and National Mega Project on Major Infectious Diseases Prevention and Treatment ZX , The Special Foundation of Taishan Overseas Distinguished Experts and Scholars, The Priority Research Program of Shandong “cademy of Sciences, Natural Science Foundation of Shandong

Perspectives on RNA Interference in Immunopharmacology and Immunotherapy http://dx.doi.org/10.5772/61575

Province No.”S No.

QN

YY

, Natural Science Foundation of Shandong “cademy of Sciences

and Science and Technology Development Foundation of Shandong

“nalysis and Test Center.

Author details Zhaohua Hou , Qiuju Han *, Cai Zhang and Jian Zhang *“ddress all correspondence to: [email protected] Laboratory of Immunology for Environment and Health, Shandong “nalysis and Test Center, Shandong “cademy of Sciences, Jinan, China Institute of Immunopharmaceutical Sciences, School of Pharmaceutical Sciences, Shandong University, Jinan, China

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] Nachmani D, Stern-Ginossar N, Sarid R: Diverse herpesvirus microRN“s target the stress-induced immune ligand MIC” to escape recognition by natural killer cells. Cell Host Microbe. : – . DOI: . /j.chom. . .

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Chapter 15

RNA Interference as a Tool to Reduce the Risk of Rejection in Cell-Based Therapies Constanca Figueiredo and Rainer Blasczyk Additional information is available at the end of the chapter http://dx.doi.org/10.5772/61829

Abstract Remarkable progress in the experimental and clinical applications of cell-based therapies has identified stem cells and their derived products as potential candidates for regenera‐ tive therapies for many disorders. The use of autologous stem cells as source for regener‐ ative therapeutic products is strongly limited by their low availability. Therefore, the future applications of in vitro pharmed therapeutic cell products will most likely occur in an allogeneic manner. However, the high variability of the human leukocyte antigen HL“ represents a major obstacle to the application of off-the-shelf products. We have developed a strategy to decrease the immunogenicity of in vitro generated cell products by silencing HL“ expression using RN“i. HL“ expression was permanently silenced in CD + hematopoietic stem and progenitor cells and induced the pluripotent stem cells to generate HL“-universal cells sources, which were then used for the differentiation of low immunogenic cell products. In this chapter, we will provide an overview about an RN“ibased strategy to reduce the immunogenicity of cell-based therapies, and in particular in the generation of HL“-universal platelets and tissues. Keywords: HL“, immunogenicity, transplant rejection, blood pharming

. Introduction The high variability of the human leukocyte antigen HL“ constitutes a major hurdle in allogeneic transplantation and to the application of off-the-shelf cell products in regenerative medicine. Recently, remarkable progresses in the field of stem cell biology, cell pharming, and tissue engineering have made feasible the differentiation of cells and tissues that might serve as a bridging strategy or even an alternative to the very scarce donated tissues and organs. However, HL“ incompatibility may pose a threat to the applicability of such in vitro generated cell products by increasing the risk of immune rejection after the transplantation.[ ] To

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overcome this major hurdle in the fields of transplantation, cell and tissue engineering, we have developed an RN“ interference RN“i -based approach to reduce the immunogenicity of cells and tissues, allowing their application in an universal manner. “ lentiviral vector encoding for specific short hairpins RN“ shRN“ , targeting HL“ transcripts were used to achieve a permanent silencing of HL“ expression. “s HL“ residual expression is crucial to prevent the natural killer NK cell activity, RN“i appears as a superior tool in comparison to gene editing technologies that cause a complete gene deletion such as the transcription activator-like effector nuclease T“LEN or clustered regularly interspaced short palindromic repeats CRISPR /Cas systems.[ - ] ”y decreasing the immunogenicity of cells and tissues, the need for immunosuppressive regimens might be reduced after HL“-mismatched trans‐ plantation. Furthermore, as HL“ plays a pivotal role in the recognition of virus-infected cells and cancer cells, the combination of conditional promoter systems allows the re-expression of HL“ and constitutes a safety mechanism. The generation of low immunogenic cells and tissues would bring enormous benefits to the patients and open novel horizons in the field of transplantation and regenerative medicine.

. The HLA system Evolution conferred highly refined mechanisms to all animals from sponges to mammals to distinguish self from non-self, and thereby allowing an immune response against potential pathogens. In humans, a tight interplay between adaptive and innate immune systems allows their defense against virtually all pathogens and cancer cells.[ , ] Nevertheless, those sophisticated surveillance mechanisms pose a major hurdle to allogeneic transplantation. The alloimmune responses are mainly based on the recognition of mismatched major histocom‐ patibility complex MHC antigens by antibodies and T-cells. In humans, the MHC is known as HL“ and it comprises a group of linked genes. MHC class I and II regulate the immune response through the presentation of peptides to T-cells. “llogeneic MHC in the graft’s antigen-presenting cells “PCs is recognized after transplantation through a direct pathway. “fterwards, own patient “PCs process and present the allogeneic antigens to T-cells by an indirect alloantigen recognition pathway.[ , ] HL“ loci are encoded on the short arm of human chromosome . ”ased on their structure, HL“ molecules are grouped into class I and class II Figure . HL“ class I classical genes comprise the “, ”, and C loci, and are expressed in the majority of cells. HL“ class II genes include DR, DQ, and DP and are constitutively expressed only in professional “PCs.[ ] The non-self recognition mediated by the engagement of the T-cell receptor with the donor HL“ is the basis for the allogeneic immune response. . . HLA incompatibility increases the risk of rejection Despite the progresses in the field of transplantation, graft rejection remains the major concern regarding the application of off-the-shelf products. HL“ comprises the most polymorphic loci of the entire human genome. The probability to find a complete HL“-matched donor for a specific patient is very low and, therefore, in most of the cases, patients will be treated with partially HL“-mismatched tissues and organs. In addition, even in fully HL“ donor/recipient

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      class   I HL“-”     ‐and   ”   HL“   class   II   HL“-DQ   ‐ molecules.     struc‐   Figure .  Structure representation of   “  HL“ The tures were designed using the software http://www.mh-hannover.de/institute/transfusion/histocheck/.             ‐  

pairs, disparities at the minor histocompatibility antigens mH“g derived from other polymorphic proteins and presented at the HL“ complexes are capable of triggering antigen    cause  graft   rejection.       specific immune responses that Improved use of post-transplant immu‐ nosuppression to prevent acute and chronic rejection allowed allogeneic transplantation as a                                 widespread, successful therapy.[ - ] However, graft rejection remains a major concern in   ‐ ‐         the               Heart   and   Lung   the field of  transplantation. The registry of International Society for           ‐                               Transplantation ISHLT reported that within the first year after lung transplantation, up to % patients need only five       to   be treated     for acute   ‐rejection and       % are    alive after       years.[     ] “lso, the number of  HL“ mismatches between a donor/recipient pair  is associated with                     stronger immunosuppressive regimens. In particular, the number of HL“-DR mismatches and                         ‐     the number of HL“-“ and -” mismatches as well as rejection treatment showed significant                 ‐             associations with the dose of maintenance steroids. “lthough immunosuppression allows the                         acceptance of the allogeneic graft, it has severe side effects that may contribute to death even                                     with a functioning graft.[ ] The occurrence of post-transplant complications related to the             opportunistic       infection,          fractures     immunosuppressive therapy such as cancer, toxicity, and hip have indicated the therapeutical      the   necessity     to  develop   alternative       strategies       to allow             use  of off-the-shelf  HL“ and ] Furthermore, due to     mH“g-mismatched       cell-based    products.[       the   shortage of  organs and tissues for transplantation, there is a high demand regarding the       ‐             ‐    ‐             development of in vitro pharmed cell products, and engineered tissues or organs. Progresses                         in the fields of stem cell biology and tissue engineering have demonstrated the feasibility to                                         generate in vitro potential alternative cell-based products that might serve as an alternative or   need   for   using donated     ‐ tissues.   Nevertheless,       future     overcome the the use of such  products     in an  allogeneic  manner,  and therefore,       it will  be required       those products       will occur that will be able to escape an allogeneic immune response.                 ‐ ‐       ‐   ‐    

 

 

   

 

 

 

 

 

 

 

 

 

     

 

 

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. . RNAi-mediated HLA targeting as a strategy to decrease the cell immunogenicity Rejection of allogeneic grafts is based on the recognition of the HL“ complexes by the specific pre-formed complement-binding anti-HL“ antibodies or by the engagement of the T-cell receptor, which leads to T-cell activation and the initiation of the immune response.[ , ] RN“ interference is an invaluable technique in cell biology and regenerative strategies to silence the target gene expression. Our studies have focused on the downregulation of HL“ class I and class II expression on the graft cells. So far, several strategies have been developed to induce the acceptance of the allogeneic graft. Similar to the immunosuppression, those strategies involve the modulation of immune responses and aim the induction of tolerance to the graft. In our studies, we genetically modify the graft to silence its HL“ expression to prevent the recognition of the allogeneic graft as non-self by the recipient’s immune system. In this approach, we do not induce tolerance toward the allogeneic graft, but we generate a condition of immunological blindness in which the recipient’s immune system is not able to recognize the allogeneic cells due to the missing HL“ but is fully capable of defending the patient against common clinical conditions associated with the use of immunosuppressive drugs such as opportunistic infections and leukemia. To prevent the recognition of the grafted cells as off the shelf, we have downregulated the expression of HL“ class I and class II antigens using RN“i. We have constructed lentiviral vectors encoding for short-hairpin RN“ sequences targeting β -microglobulin shβ m or the alpha-chain of HL“-DR shDR“ to silence the expression of HL“ class I and class II antigens, respectively. Our studies demonstrated the feasibility to stably downregulate HL“ class I and II expression in several cell lines e.g., ”LCL, MonoMac- , HeLa as well as in primary cells e.g., endothelial cells, CD + progenitor, induced pluripotent stem cells . Cell transduction for the delivery of shRN“s targeting specific HL“ transcripts resulted in a decrease by up to % of β m or HL“-DR“ transcript levels and HL“ class I expression. In vitro assays have shown that HL“ class I-silenced cells were protected against the antibody-mediated complement-dependent cytotoxicity. Furthermore, in T-cell cytotoxicity assays, significantly lower cell lysis rates were observed when HL“silenced cells were used as targets in comparison to fully HL“-expressing cells. In addition, HL“-silenced cells demonstrated to induce significantly lower T-cell proliferation, proinflammatory cytokine secretion, and degranulation. The residual HL“ class I expression showed to be sufficient to prevent NK cell cytotoxicity. “ltogether, HL“-silenced cells showed a protective effect against the humoral and cellular allogeneic immune response.[ , , ] . . MHC-silenced cells survive after fully HLA-incompatible transplantation Despite the widespread use of immunosuppressive regimens to prevent graft rejection, their therapeutic window is very narrow. Immunosuppressive drugs frequently cause adverse effects including thrombocytopenia, leukopenia, hypercholesterolemia, stomatitis, nephro‐ toxicity, and diarrhea, and they lead to an increased risk for infections and cancer.[ , ] Silencing HL“ expression using RN“i may represent an alternative to immunosuppression hence it has the potential to offer many benefits for the patients. In addition, silencing HL“ expression may allow the future application of HL“-mismatched off-the-shelf products in a universal manner independently of the genetic background of the donor and recipient. In an

RNA Interference as a Tool to Reduce the Risk of Rejection in Cell-Based Therapies http://dx.doi.org/10.5772/61829

allogeneic transplantation rat model, we have confirmed the improved capacity of MHCsilenced cells to survive in allogeneic environment upon transplantation and even in the absence of immunosuppression. “ lentiviral vector encoding for a shRN“ sequence targeting rat MHC class I RT -“ and the sequence for firefly luciferase as a reporter gene was used to silence MHC class I Lewis rat-derived fibroblasts. In contrast to nonmodified fibroblasts, MHC class I-silenced fibroblasts were able to survive after subcutaneous transplantation in a complete MHC-mismatched setting. MHC class I-silenced fibroblasts were able to engraft and were detectable during the entire monitoring period weeks . Nonmodified cells were rejected in all animals.[ ] This study showed the superior performance of MHC-silenced cells after MHC-incompatible transplantation. . . Generation of HLA-silenced platelets Since the s, blood transfusion therapy has become routine clinical practice however, the concept of blood pharming – ex vivo production of mature blood cells – is quite new. In humans, platelet production is sustained by a well-regulated process known as thrombopoi‐ esis. In the bone marrow, CD + progenitor cells differentiate into polyploid megakaryocytes the precursor of platelets . Megakaryocytes lack the expression of CD , but express several glycoproteins essential for the platelet function.[ , ] In general, platelet numbers in blood range from x to x per liter, and an estimated x platelets are produced each day in the adult human. Thrombocytopenia and severe thrombocytopenia characterized as platelet counts less than x and x per liter, respectively, increase the risk of spontaneous bleeding and represent a threat for the patient’s life. Platelet transfusion has been widely used to prevent and treat life-threatening thrombocytopenia however, prepara‐ tion of a unit of concentrated platelets for transfusion requires at least – units of whole blood, thereby significantly increasing the risk of blood-borne infections and adverse immunologic reactions. Furthermore, platelet transfusion refractoriness – lack of adequate post-transfusion platelet counts – remains a major complication often observed in patients receiving multiple transfusions. This condition is frequently caused by the development of antibodies specific to HL“. Currently, platelet transfusion relies on volunteer blood donation however, the demand for blood products in particular of platelets often exceeds their availability.[ ] The potential of multipotent progenitor and stem cells in regenerative medicine has been recognized.[ , ] Platelet transfusion refractoriness due to the presence of anti-HL“ antibodies constitutes a life-threatening risk for many patients suffering from hematological disorders, and hence require multiple platelet transfusions. Thus, it would be highly desirable to produce HL“-deficient platelets to facilitate the management of severe alloimmunized thrombocytopenic patients. In our studies, we have combined the concept of blood pharming with RN“i as a strategy to downregulate HL“ gene expression. The ultimate goal of this approach is the large-scale production of platelets in vitro that may be used as an alternative to the conventional donated blood platelets. In addition, we aim for the production of genetically modified platelets with the capacity to survive even under platelet transfusion refractoriness.

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In our studies, CD + hematopoietic progenitor cells and induced pluripotent stem cells iPSCs were used to produce HL“-silenced platelets in vitro. CD + cells or iPSCs were transduced with a lentiviral vector encoding for the shRN“ sequence targeting β m which induces HL“ class I silencing. We have demonstrated the feasibility to generate HL“-universal CD + cells and iPSCs which might be used for the differentiation of HL“-silenced cellproducts. In our studies, we have differentiated HL“-silenced megakaryocytes and platelets from both cell sources. In our previous studies, we have demonstrated the possibility to generate HL“-silenced platelets with comparable function to blood-derived platelets. How‐ ever, in contrast to blood-derived platelets, in vitro generated HL“-silenced platelets were able to escape antibody-mediated complement-dependent cytotoxicity as well as cellular-depend‐ ent cytotoxicity. “lso, in a platelet transfusion refractoriness mouse model, HL“-silenced platelets showed the capacity to survive and were even detectable days after transfusion. [ ] The limited availability of CD + cells derived from G-CSF mobilized donors is a major obstacle to the large-scale production of in vitro pharmed platelets. The breakthrough Nobel Prize–winning research by Yamanaka and colleagues to induce pluripotency in somatic cells has reshaped the field of stem cell research. Human iPSCs can be used for studying embryo‐ genesis, disease modeling, drug testing, and regenerative medicine.[ ] In contrast to CD + cells, iPSCs represent an unlimited cell source for the in vitro production of a variety of cellbased products including platelets. Therefore, we have recently established an efficient protocol for the differentiation of megakaryocytes and platelets from iPSCs Figure . First, we have generated an HL“-universal iPSC line. Then, the lentiviral vector containing the shRN“ targeting β -microglobulin was used to silence HL“ expression on iPSCs. “ significant and durable reduction of HL“ expression was observed even after passaging. Nevertheless, the HL“-silenced iPSC line showed comparable expression of pluripotency markers such as SSE“- and Tra- as the original HL“ class I-expressing iPSC line Figure . The data indicate that silencing HL“ expression does not affect the pluripotency potential of iPSCs.

Lentiviral vector encoding the shRNA

Cell division

Platelets Endomitosis

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Megakaryocyte Proplatelet producing megakaryocyte

Figure . Schematic representation of the differentiation of HL“-silenced platelets from iPSCs. “ lentiviral vector en‐ coding for an HL“-specific shRN“ is used to transduce the iPSCs. “fterward, HL“-silenced iPSCs will be differentiat‐ ed using a cytokine cocktail containing thrombopoietin TPO until the release of platelets.

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Figure . Generation of an HL“-universal iPSC line. “ iPSCs were adapted to monolayer culture conditions and transduced with a lentiviral vector encoding for a nonspecific shRN“ shNS or a β -microglobulin-specific shRN“ to β silence the expression of HL“ class I ” expression of SSE“ and Tra- - on nonmodified and shβ m-expressing iPSCs C expression of HL“ class I of a HL“-silenced shNS-expressing iPSC lines. Cells were stained with an HL“ class I antibody W / and the expression of HL“ was measured by flow cytometry after different number of passag‐ β es. Mean fluorescence intensities MFI detected on HL“-silenced iPSCs were normalized to the MFI measured on shNS-expressing iPSCs at the same passage.

In addition, the future application of iPSCs will occur most likely in an allogeneic manner to facilitate their availability during the time of need and standardization of the protocols. Hence, the use of HL“-silenced iPSCs may facilitate  the application of HL“-mismatched iPSCderived cell products. For megakaryocyte differentiation, HL“-silenced iPSCs were cultured   in monolayer in the presence of vascular endothelial growth factor VEGF and ”MP- for mesoderm induction and afterward in the presence of TPO. During ontogeny, definitive hematopoietic cells are generated de novo from a specialized subset of endothelium, known as hemogenic endothelium. Endothelial-to-hematopoietic transition during embryogenesis provides the first long-term hematopoietic stem and progenitor cells in the embryo.[ ] In our differentiation cultures of iPSCs into megakaryocytes, structures resembling the hemogenic endothelium were observed Figure . In suspension, megakaryocytes could be detected by an increase in DN“ content higher than n and the expression of typical megakaryocytic markers such as CD and CD a. In addition, the megakaryocytes showed the capability to build pro-platelets. Moreover, the shRN“-mediated silencing effect was maintained during the entire differentiation. Importantly, iPSC-derived megakaryocytes and pro-platelets showed a significant reduction of β -microglobulin and HL“ class I antigens in comparison to those differentiated from iPSC expressing a nonspecific shRN“ control Figure . Differentiation rates of iPSC into megakaryocytes by up to % were observed Figure .

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Figure . Formation of hemogenic-like endothelium during the differentiation of iPSCs into megakaryocytes. The pho‐ tos display an island of “ x ”    x.       of   hemogenic-like ‐  endothelium at  a magnification          

 

 

   

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Figure . Differentiation of HL“-universal megakaryocytes from HL“-silenced iPSC. “ The histogram represents the flow cytometric analysis of HL“-silenced iPSC-derived megakaryocytes after staining with propidium iodide ” light microscopic and C fluorescence microscopic analysis of an iPSC-derived megakaryocyte after staining with ′, -Dia‐ midin- -phenylindol D“PI, blue D light microscopic analysis of pro-platelets indicated with white arrows E realtime PCR analysis of β -microglobulin levels in megakaryocytes derived from iPSCs transduced with a lentiviral vector encoding the shRN“ targeting β -microglobulin shβ m or a nonspecific shRN“ shNS as a control.

 

RNA Interference as a Tool to Reduce the Risk of Rejection in Cell-Based Therapies http://dx.doi.org/10.5772/61829

day 12

isotype

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0.2 %

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day 19

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82.1 %

CD41 Figure . Phenotypic analysis of HL“-universal megakaryocytes derived from iPSCs. The expression of the megakar‐ yocyte markers CD and CD a was measured by flow cytometry in the cells harvested from the iPSC differentiation cultures at different time points. The gates in the dotplots indicate megakaryocytes characterized by the double expres‐ sion of CD and CD a.

The complement-dependent cytotoxic CDC crossmatch is an informative assay that detects alloantibodies in pre- and post-transplant patients, which may guide the most appropriate clinical management of transplant patients.[ ] The capacity of in vitro generated HL“silenced megakaryocytes and platelets to escape antibody-mediated complement-dependent cytotoxicity was evaluated in CDC tests. HL“-silenced megakaryocytes incubated with specific HL“ antibodies and complement showed comparable cell lyses rates to the megakar‐ yocytes incubated with nonspecific HL“ antibodies. In contrast, significantly higher cell lysis rates were observed when HL“-expressing megakaryocytes were incubated with specific antiHL“ antibodies Figure . These data suggest that HL“-universal iPSC-derived megakaryo‐ cytes are protected from anti-HL“ antibody-mediated complement-dependent cytotoxicity and have the potential to survive under refractoriness conditions.

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Figure . HL“-silenced megakaryocytes are protected from antibody-mediated complement-dependent cytotoxicity. HL“-expressing shNS or HL“-silenced shβ m iPSC-derived megakaryocytes were incubated with a nonspecific antibody NC or an HL“-specific antibody and complement. Cell lysis was detected by flow cytometric analysis upon staining with propidium iodide. The bar graph represents meansβand standard deviations of three independent experi‐ ments. Statistical significance was calculated using Student’s t-test *p < . , **p < . ***p < . .

. . Generation of HLA-silenced tissues Due to the high variability of the HL“ loci and the shortage of organs and tissues for trans‐ plantation, it is very difficult to find a complete HL“-matched graft for a given recipient. The number of HL“ mismatches between the graft and the recipient are associated with a higher risk for graft rejection and even morbidity and mortality due to immunosuppression-related side effects. Thus, it would be desirable to engineer the grafts in order to decrease their immunogenicity by silencing HL“ expression. Worldwide, the demand for organs and tissues for transplantation is very high and it is not possible to satisfy all needs. This discrepancy is even accentuated in the Middle East and countries in the East such as India and China. “ccording to the World Health Organization WHO , there are over million people in the world who are blind in one or both eyes due to corneal injury or disease and up to million people could benefit from corneal transplants. However, according to data from eye banks and health agencies, less than , corneal transplants are done annually worldwide due to the shortage of human cadaver corneas. Furthermore, during the first five years after penetrating keratoplasty, rejection is responsible for – % of total corneal graft loss. High risk-corneal recipients even showed increased rejection rates – % .[ , ] The cornea presents a simple anatomical structure, in which the endothelium is easily accessible to the shRN“-encoding viral vector containing supernatant Figure . The integrity of the endothelial cell layer is crucial for the transparency of the cornea and it is the major target for rejection. Therefore, silencing HL“ expression on the corneal endothelium may   allogeneic keratoplasty. improve cornea survival in high-risk patients after  

RNA Interference as a Tool to Reduce the Risk of Rejection in Cell-Based Therapies http://dx.doi.org/10.5772/61829

Figure . “natomy of the cornea. Optical coherence tomography of a mouse cornea showing the three main layers.

We have silenced HL“ expression in human and mice corneas. The tissue was transduced for h with the lentiviral vector encoding for the MHC-specific shRN“s as described above. We were able to silence the MHC expression on the cornea endothelium, which is the major target during graft rejection. In this study, we demonstrated the feasibility to generate HL“ universal tissues in their original D structure Figure . Silencing HL“ expression on tissues is expected to significantly improve graft survival rates in high-risk keratoplasty patients.

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Figure . Generation of MHC-silenced corneas. “ mouse cornea was explanted and transduced with a lentiviral vector encoding the shβ m to cause a downregulation of MHC class I antigens and the expression of green fluorescence pro‐ tein GFP sequences. “ Light microscopic and ” fluorescence   microscopic analyses of a transduced mouse cornea.

 

. . Conclusion Recently, gene regulation or editing strategies have emerged as powerful tools to improve the design and efficacy of personalized cell-based therapies. In the field of histocompatibility and transplantation, RN“i seems to be a favorite approach to reduce the immunogenicity of

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allogeneic and in vitro generated cells and tissues. The lentiviral vector-mediated delivery of shRN“s targeting HL“ transcripts prevents the activation of cellular and humoral allogeneic immune responses that cause the rejection of the foreign cells. However, this RN“i-based strategy permits the residual expression of HL“ class I antigens which are crucial to inhibit NK cell cytotoxicity. With the establishment of iPSCs, the concept of cell pharming came one step closer to reality as iPSCs may serve as unlimited cell sources for different cell products such as platelets. The combination of RN“i-mediated HL“ silencing and the capacity to generate platelets in vitro may represent a novel therapeutic approach for the management of alloimmunized thrombocytopenic patients with an increased risk to develop refractoriness to platelet transfusion. Furthermore, our results also indicate the feasibility to reduce MHC expression in the D original structure of tissues. “brogating the histocompatibility barrier between donors and recipients may improve therapeutic efficacy, reduce the adverse events associated with strong immunosuppressive regimens, and improve transplant patient life’s quality. In conclusion, RN“i-mediated silencing of HL“ expression may open new avenues in tissue engineering and transplantation.

Author details Constanca Figueiredo* and Rainer ”lasczyk *“ddress all correspondence to: [email protected] Institute for Transfusion Medicine, Hannover Medical School, Hannover, Germany

References [ ] ”arry J, Hyllner J, Stacey G, Taylor CJ, Turner M. Setting up a Haplobank: issues and solutions. Curr Stem Cell Rep. : – . [ ] Figueiredo C, Seltsam “, ”lasczyk R. Class-, gene-, and group-specific HL“ silencing by lentiviral shRN“ delivery. J Mol Med Berl . : – . [ ] Jaimes Y, Seltsam “, Eiz-Vesper ”, ”lasczyk R, Figueiredo C. Regulation of HL“ class II expression prevents allogeneic T-cell responses. Tissue Antigens. : – . [ ] Wiegmann ”, Figueiredo C, Gras C, et al. Prevention of rejection of allogeneic endo‐ thelial cells in a biohybrid lung by silencing HL“-class I expression. Biomaterials. : – . [ ] Hirano M, Das S, Guo P, Cooper MD. The evolution of adaptive immunity in verte‐ brates. Adv Immunol. : – .

RNA Interference as a Tool to Reduce the Risk of Rejection in Cell-Based Therapies http://dx.doi.org/10.5772/61829

[ ] ”uchmann K. Evolution of innate immunity: clues from Invertebrates via fish to mammals. Front Immunol. : . [ ] “li JM, ”olton EM, ”radley J“, Pettigrew GJ. “llorecognition pathways in transplant rejection and tolerance. Transplantation. : – . [ ] Sagoo P, Lombardi G, Lechler RI. Relevance of regulatory T cell promotion of donorspecific tolerance in solid organ transplantation. Front Immunol. : . [ ] Shiina T, Hosomichi K, Inoko H, Kulski JK. The HL“ genomic loci map: expression, interaction, diversity, and disease. J Hum Genet. : – . [

] “lmoguera ”, Shaked “, Keating ”J. Transplantation genetics: current status and prospects. Am J Transplant. : – .

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] de Rham C, Villard J. Potential and limitation of HL“-based banking of human pluri‐ potent stem cells for cell therapy. J Immunol Res. : .

[

] Hess SM, Young EF, Miller KR, et al. Deletion of naive T cells recognizing the minor histocompatibility antigen HY with toxin-coupled peptide-MHC class I tetramers in‐ hibits cognate CTL responses and alters immunodominance. Transpl Immunol. : – .

[

] Kwun J, Malarkannan S, ”urlingham WJ, Knechtle SJ. Primary vascularization of the graft determines the immunodominance of murine minor H antigens during organ transplantation. J Immunol. : – .

[

] Trulock EP, Christie JD, Edwards L”, et al. Registry of the International Society for Heart and Lung Transplantation: twenty-fourth official adult lung and heart-lung transplantation report. J Heart Lung Transplant. : – .

[

] Opelz G, Dohler ”. “ssociation between steroid dosage and death with a functioning graft after kidney transplantation. Am J Transplant. : – .

[

] Robson R, Cecka JM, Opelz G, ”udde M, Sacks S. Prospective registry-based observa‐ tional cohort study of the long-term risk of malignancies in renal transplant patients treated with mycophenolate mofetil. Am J Transplant. : – .

[

] Wood S, Feng J, Chung J, et al. Transient blockade of delta-like Notch ligands pre‐ vents allograft rejection mediated by cellular and humoral mechanisms in a mouse model of heart transplantation. J Immunol. : – .

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] Wedel J, ”runeau S, Kochupurakkal N, ”oneschansker L, ”riscoe DM. Chronic allog‐ raft rejection: a fresh look. Curr Opin Organ Transplant. : – .

[

] Figueiredo C, Horn P“, ”lasczyk R, Seltsam “. Regulating MHC expression for cel‐ lular therapeutics. Transfusion. : – .

[

] Moes DJ, Guchelaar HJ, de Fijter JW. Sirolimus and everolimus in kidney transplan‐ tation. Drug Discov Today. : -

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[

] Khan S, Khan S, ”aboota S, “li J. Immunosuppressive drug therapy - biopharmaceut‐ ical challenges and remedies. Expert Opin Drug Deliv. : – .

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] Figueiredo C, Wedekind D, Muller T, et al. MHC universal cells survive in an alloge‐ neic environment after incompatible transplantation. Biomed Res Int. : .

[

] Thon JN, Italiano JE. Platelet formation. Semin Hematol.

[

] Patel-Hett S, Wang H, ”egonja “J, et al. The spectrin-based membrane skeleton sta‐ bilizes mouse megakaryocyte membrane systems and is essential for proplatelet and platelet formation. Blood. : – .

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] Stanworth SJ, Navarrete C, Estcourt L, Marsh J. Platelet refractoriness - practical ap‐ proaches and ongoing dilemmas in patient management. Br J Haematol. : -

[

] Crane “M, Kramer P, ”ui JH, et al. Targeted correction and restored function of the CFTR gene in cystic fibrosis induced pluripotent stem cells. Stem Cell Reports. : – .

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] Tong Z, Solanki “, Hamilos “, et al. “pplication of biomaterials to advance induced pluripotent stem cell research and therapy. EMBO J. : – .

[

] Gras C, Schulze K, Goudeva L, Guzman C“, ”lasczyk R, Figueiredo C. HL“-univer‐ sal platelet transfusions prevent platelet refractoriness in a mouse model. Hum Gene Ther. : – .

[

] Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryon‐ ic and adult fibroblast cultures by defined factors. Cell. : – .

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] Zape JP, Zovein “C. Hemogenic endothelium: origins, regulation, and implications for vascular biology. Semin Cell Dev Biol. : – .

[

] Lawrence C, Willicombe M, ”rookes P“, et al. Preformed complement-activating low-level donor-specific antibody predicts early antibody-mediated rejection in renal allografts. Transplantation. : – .

[

] Stuart PM, Yin X, Plambeck S, Pan F, Ferguson T“. The role of Fas ligand as an effec‐ tor molecule in corneal graft rejection. Eur J Immunol. : – .

[

] Yin XT, Zobell S, Jarosz JG, Stuart PM. “nti-IL- therapy restricts and reverses lateterm corneal allorejection. J Immunol. : – .

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Chapter 16

Utility of Potent Anti-viral MicroRNAs in Emerging Infectious Diseases Zhabiz Golkar, Donald G. Pace and Omar Bagasra Additional information is available at the end of the chapter http://dx.doi.org/10.5772/61687

Abstract MicroRN“s miRN“s are small, noncoding RN“ molecules that have emerged as im‐ portant posttranscriptional regulators of gene expression. miRN“ provides intracellular immune defense when the body is faced with challenges from transgenes, viruses, trans‐ posons, and aberrant mRN“s. miRN“ molecules trigger gene silencing in eukaryotic cells. To date, more than , different human miRN“s hsa-miRs have been identified, and it is generally agreed that cellular gene regulation is significantly impacted by the presence of miRN“s. “ single miRN“ has the complex capacity to target multiple genes simultaneously. In a viral infection context, miRN“s have been connected with the inter‐ play between host and pathogen, and occupy a major role in the host–parasite interaction and pathogenesis. While numerous viral miRN“s from DN“ viruses have been identi‐ fied, characterization of functional RN“ virus-encoded miRN“s and their potential tar‐ gets is still ongoing. Here, we describe an in silico approach to analyze the most recent Ebola virus E”OV genome sequences causing West “frican epidemics. We identified numerous candidate miRN“s that can be utilized to quell the Ebola virus. Future ap‐ proaches will focus on experimental validation of these miRN“s during quelling the Ebo‐ la target transcripts for further elucidating their biological functions in primates and other animal models. Keywords: Ebola virus, gene alignment, miRN“, prevention, vaccine

. Introduction . . Inhibition of Ebola virus by anti-Ebola miRNAs In silico Since the HIV- pandemic of the s and more recent outbreaks of bird flu, severe acute respiratory syndrome S“RS , and Middle East respiratory syndrome-Corona Virus MERCoV , it has been widely believed that there would be new pandemics of highly pathogenic

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viruses [ ]. Fortunately, until recently, many of the new emerging pathogenic agents, such as Ebola virus E”OV and Marburg virus M“RV , have failed to demonstrate the transmissi‐ bility or animal reservoirs required to become true pandemic threats [ , ]. Few, if any, antivirals can claim to be specific enough to halt the epidemic. Recently, a whole E”OV replication-defective vaccine E”OVdVP has been found to be very effective in nonhu‐ man primates, while two others are in Phase II trials [ ]. The testing of the recently developed replication-defective recombinant chimpanzee adeno‐ virus type –vectored ebolavirus c“d -E”O vaccine is based on a demonstration of efficacy in a nonhuman primate model [ ]. However, a curious finding has puzzled the investigators preexisting neutralizing antibodies against c“d and c“d in human serum samples were found in ~ % of Ugandans and in % of the US and European volunteers. The increased prevalence of neutralizing antibodies against chimpanzee adenoviruses in sub-Saharan “frica may indicate cross-species transmission of these viruses from chimpanzees to humans. The possibility of fairly high levels of neutralizing antibodies against c“d may complicate the evaluation of the effectiveness of Ebola vaccines currently underway. . . Virology of Ebola virus Filoviruses are taxonomically classified within the order Mononegavirales, a large group of enveloped viruses whose genomes are composed of a nonsegmented, single-stranded minus [ , ] RN“ molecule. Following their discovery, filoviruses were originally grouped with rhabdoviruses, as the appearance of these virus particles appeared similar [ ]. However, subsequent filamentous morphology and extensive genetic, physiochemical, and virologic studies of Marburg virus M“RV and Ebola virus E”OV revealed distinctive characteristics, and these viruses were placed into a separate family, the Filoviridae [ ]. Further characterization of these agents demonstrated that E”OV and M“RV represent divergent lineages of filovi‐ ruses, and that their variances were significant enough to warrant the formation of the two genera, M“RV and E”OV [ ]. Subsequent to the International Committee on Taxonomy of Viruses recommendation, the M“RV genus contains a single species, the Lake Victoria Marburg virus, as this strain exhibits only limited genetic variation. E”OV is the causative agent of Ebola virus disease EVD [ ]. The mortality rate can vary from % to % depending on the strains [ ]. The viral life cycle begins with host cell entry through a mechanism that is still poorly understood. The incubation period for EVD is – days, and typical early symptoms include fever, chills, malaise, and myalgia all of which could be misdiagnosed as malaria, which is highly prevalent in West “frican nations , followed by the onset of symptoms indicative of multi-organ stress and subsequent failure, sometimes followed by hemorrhagic episodes that can easily be misdiagnosed as Lassa fever. E”OV is a negative, single-stranded RN“ virus with an unusual, variable-length, filamentous, branched morphology whose helical capsid is enclosed inside a membrane. The mechanism of attachment and entry into the cell is still not completely defined see discussion below . Once inside, the viral RN“ polymerase L protein begins to copy the negative strand –ve RN“ to make the positive strand +ve transcripts that mimic the structure of mRN“ and are translated by host ribosomes. Replication is thought to occur in the cytoplasm. “n unusual

Utility of Potent Anti-viral MicroRNAs in Emerging Infectious Diseases http://dx.doi.org/10.5772/61687

feature of the transcription and translation of the Ebola genes is the fact that the glycoproteins GP are only expressed through transcriptional editing. The genome of the Zaire Ebola virus E”OV , the most pathogenic among all species of E”OV, is , nucleotides nts in length and contains seven transcriptional units that guide synthesis of at least nine distinct primary translation products: the nucleoprotein NP , virion protein VP , VP , glycoprotein GP , soluble glycoprotein sGP , small soluble glycoprotein ssGP , VP , VP , and the large L protein. L is the catalytic subunit of the viral polymerase complex Figure . Similar to other nonsegmented negative-sense NNS RN“ viruses, E”OVs encode a multiprotein complex to carry out replication and transcription. In the case of E”OV, viral RN“ synthesis requires the viral NP, VP , VP , and L proteins. Transcription of filovirus mRN“s is presumed to occur as in other NNS viruses, where there is a gradient of viral mRN“s with the abundance of each mRN“ transcript decreasing as the polymerase transcribes toward the ′ end of the template [ ]. Each E”OV mRN“ is presumed to be efficiently modified with a ′– ’-methylguanosine m G cap and a ′p “ tail [ ].

Figure . The genome of E”OV, kb in length, has the following gene order: ′ leader nucleoprotein NP , virion proteins VP VP -VP , membrane glycoprotein GP , viral polymerase VP VP -VP , viral polymerase L protein, and ′ trailer.

The Ebola virus genus possesses greater diversity, and four viral species have been recognized: Zaire Ebola, Sudan Ebola, Reston Ebola, and Ivory Coast Ebola E”OV-Z, E”OV-S, E”OV-R, and E”OV-IC, respectively . Each of the E”OV species has a different degree of pathogenicity and mortality rate [ ]. Therefore, E”OV-S and E”OV-Z, which are the predominant E”OVs associated with known outbreaks, are more pathogenic than E”OV-R and E”OV-IC [ ]. E”OV-IC has only caused a single nonfatal human infection, but E”OV-R has caused fatal infection in nonhuman primates [ ]. However, E”OV-S, E”OV-Z, and E”OV-” often cause severe hemorrhagic diseases with markedly high case fatality rates – % [ ]. The E”OV genome is . kb in length with the following gene order: ′ leader nucleoprotein NP , virion protein VP -VP , glycoprotein GP , VP , VP , polymerase L , and ′ trailer. The GP differences between any two species range from % to % at the nucleotide level and from

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% to % at the amino acid level [ ]. However, variations within E”OV-Z species are very low ∼ – % [ ]. Thus, GP nucleotides are usually used in the phylogenetic analysis of E”OV Figure .

Figure . The Ebola Pandemic Map depicts the history of Ebola in “frica. The sporadic cases of Ebola were common in the central “frican countries such as the DRC cases with fatalities , Uganda cases with fatalities , South Sudan cases with fatalities , Gabon cases with fatalities , the Republic of the Congo cases with fatalities , and South “frica cases with fatality .

”ecause of their high mortality rate, which can vary from % to %, and their potential for person-to-person transmission and lack of an approved vaccine or antiviral therapy, M“RV and E”OV are classified as biosafety level ”SL- viruses by World Health Organization [ , ]

Utility of Potent Anti-viral MicroRNAs in Emerging Infectious Diseases http://dx.doi.org/10.5772/61687

. . History of MARV and EBOV Viruses are obligate intracellular parasites and essentially rely on host cells for raw materials, replication, transcription, and translations of their genetic codes. Until a few years ago, we assumed that the major intracellular defenses against viral pathogens were interferons [ ]. Since the discovery of RN“ interference RN“i and miRN“s, we know that one of the fundamental functions of miRN“s is to prevent replication of foreign viruses by pre- and posttranscriptions and suppressions of viral expression [ ]. Therefore, besides endogenous gene regulation, miRN“s are the primary intracellular immune defense system [ ]. Viruses have also evolved to counter the antiviral effects of miRN“s by viral miRN“s vmiRN“s . Recently, Li and Chen [ ] have conducted molecular epidemiologic analyses of presently extant Ebola viral genomes to ascertain their evolutionary viral history. Of considerable potential importance are interpretations derived from a dataset that is between , and , years old and includes four Ebola species E”OV-Z, E”OV-S, E”OV-TF, E”OV-R [ ]. Logically, one could assume that over the past , years, humans have evolved counter‐ measures to the Ebola virus via innate, adaptive, and miRN“-based immunity. The identification in a human database of miRN“s capable of potentially quelling E”OV strongly suggests that Homo sapiens already have developed primary intracellular defens‐ es to quell E”OV infection [ ]. This raises a question: Why have E”OVs been circulating for about , years, and yet they seem to have emerged only recently? The earliest known cases of Ebola date to the s. One theory proposes that E”OV-Z experienced a recent genetic bottleneck [ ]. ”efore Ebola viral strains were introduced to primates, they had already been circulating among small mammals, including bats, rodents, marsupials, shrews, and so on [ ]. “lthough these bats and other animals were infected [ , ], no evidence demonstrated that such infections were fatal to them [ ]. This indicates that a natural balance had been achieved between the viruses’ pathogenicity and the host’s immune system, especially at the intracellular levels where miRN“s provide immunologi‐ cal protection [ ]. This homeostasis, this balance, apparently was broken in , when E”OV genetic diversity experienced a dramatic drop [ ]. “ccordingly, most lineages of the various E”OV species became extinct because of such influences as threatening human activities, climate change, and a steep decline in the number of animals to serve as a reservoir for viral replication. Probably due to altered patterns of positive selection in the glycoprotein GP , which diversified substantially and was found to be part of fusion and receptor binding within cellular membranes, infection patterns through direct exposure were changing. Therefore, by about , few lineages that possessed broader tropism and enhanced fitness had the capacity to infect primates via direct exposure [ ]. Similar examples can be seen in the emergence of HIV- , which appeared to have surfaced in the s through a zoonotic event that involved common infections among chimpanzees i.e., SIV and then accidentally jumped to humans [ – ]. Due to the paucity of significant differences in E”OV genetic diversity since , the decreased number of surviving viruses may have become the only circulating lineages in primates and viral reservoirs. E”OV-Z has the ability to traverse a long distance through bats, which serve as a migratory reservoir. Outbreaks with their epicenter in Congo have been caused by the E”OV-Z species [ – ].

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Through analysis of miRN“ numbers that demonstrate high homologies in seed sequences and that show high identity to E”OV species, we have deduced that the genetic variations at the GP may serve as a type of “chilles’ heel. “fter all, only one miRN“ showed identity to GP, while eight proved capable of blocking polymerase steps. This indicates that minor variations within the GP amino acid sequence could allow for viral entrance into host target cells in humans. The subsequent transcription of negative-stranded RN“ viruses into positive RN“ strands occurs amid a struggle to overcome the miRN“s with quelling potential that can halt this process. It is possible that at the time of exposure to E”OV, all of the protective miRN“s may not be present in the target cells, or may be present, but not in sufficient quantities to block early E”OV replication [ ].

Figure . The illustration depicts a simplified structure of Ebola virus. The functions of various viral proteins are de‐ scribed in the text. GP, glycoprotein NP, nucleoprotein VP , matrix protein VP , transcription factor and polymer‐ ase enzyme.

Figure shows the VP , VP , VP , VP , and L nucleoproteins that constitute the nucleo‐ capsid, which is crucial in both the transcription and viral replication processes [ , ]. The glycoprotein is located in the lipid membrane of the Ebola virus this is also the place in the host target cells where receptors that facilitate viral entry are embedded [ ]. Viral matrix proteins VP and VP are essential to viral budding, stability, and structure. VP is the primary matrix protein, and is the viral protein that is expressed most abundantly. It plays a central role in the process of Ebola budding from the plasma membrane. For example, in mammalian cells, the mere expression of VP is sufficient to create virus-like particles VLPs with morphological characteristics that are similar to those of the actual Ebola virus [ , ]. Given VP ’s absence, studies have found that the nucleocapsid was not transported effec‐ tively into the plasma membrane, and as this membrane is the site of assembly, budding, and

Utility of Potent Anti-viral MicroRNAs in Emerging Infectious Diseases http://dx.doi.org/10.5772/61687

incorporation into the virions, considerable attention should be given to the role of this matrix protein [ ]. The utilization of miRN“s that specifically target VP mRN“ degradation is important to our understanding of just how VP functions and what potential roles it might play in the regulation of VLP assembly in both in vitro and live cell settings. hsa-miR-4692 and hsa-miR-54 -az effectively target VP therefore, the overexpression of these particular miRN“s within host cells could totally disrupt the viral life cycle and may have a decisive impact in the categorization of therapeutic targets data unpublished . The tendency of Ebola VP to assemble virus-like particles VLPs presents an appealing model for analysis of the Ebola viral assembly at biosafety level made possible by the noninfectious nature of geneti‐ cally engineered VLPs [ ]. VP ’s association with the plasma membrane is of fundamental importance [ ] it is here that assembly is initiated as well as oligomerization [ ], and nucleoprotein recruitment. ”esides membrane association, VP also associates or otherwise interacts with host cell factors, including the endosomal sorting complex that supports transport ESCRT machinery [ , ], the vesicle coat II proteins COPII [ ], as well as the protein actin [ , ] these host cell factors, respectively, have been shown to enable VP budding, transport, and movement. Moreover, host cell protein kinases could contribute to Ebola infectivity as c-“bl can phos‐ phorylate Tyr in VP [ , ]. Still we have an inadequate understanding of how VP actually assembles on the plasma membrane before virion release occurs. Localization of VP in the plasma membrane is believed to be important as studies give evidence that hydrophobic residues located within the C-terminal domain, including Leu , are essential in the localiza‐ tion and budding processes [ ]. Detection of VP oligomers in VLPs and UV-inactivated virions has occurred [ , ] they have been detected mainly in filamentous structures stemming from the plasma membrane [ ]. Therefore, VP oligomerization apparently occurs on the same plasma membrane in which oligomers selectively have found to reside [ ]. In terms of structure, VP has predominantly been found to oligomerize into either hexamers or octamers [ , ]. These share a comparable monomer–monomer or intradimeric antipar‐ allel interface. However, the detection of oligomeric structures in live cells suggests that these structures, too, could exert a critical influence on both viral assembly and egress [ ]. We discovered that hsa-miR-4692 and hsa-miR-54 -az both target VP data unpublished . The formation of virus-like particles VLPs requires VP oligomers these are associated with membranes that are resistant to detergent [ ], which underscores the active part that the plasma membrane may play in VP oligomerization. Moreover, on the plasma membrane, matrix protein oligomerization may function as a scaffold in host protein recruitment, and also supply the force needed to effect the formation of virus particles and the deformation of membranes. “ comprehension of VP plasma membrane association thus becomes crucial to our understanding of how the formation of protein buds occurs on the plasma membrane. Gupta K [ ] recently investigated the role that the VP C-terminal domain plays in mem‐ brane association as well as in membrane penetration. These investigators utilized the monolayer penetration methodology to conduct in vitro research into the molecular basis of the penetration of the VP membrane. To study VP assembly and its associated egress in cells, they employed a multipronged methodology that blended cellular imaging, number and

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brightness N&” analysis, analysis of the egress of virus-like particles, site-directed muta‐ genesis, and total internal reflection TIRF microscopy. N&” analysis permitted them to ascertain the average number of molecules and also the brightness within each pixel within a fluorescence microscopy image. This permitted them to detect the oligomeric status of proteins that are labeled fluorescently. They concluded that within the VP C-terminal domain, a hydrophobic interface actually penetrates the plasma membrane, which plays a key role in the oligomerization of VP . The knocking out of plasma membrane penetration by hydrophobic mutants also substantially reduces the egress of VLPs [ , ]. Therefore, degradation of VP mRN“ by a two-pronged attack from hsa-miR-4692 and hsa-miR-54 -az can stop Ebola. “ distinguishing characteristic of filovirus genomes is their ′- and ′-UTRs that are long related to other RN“ viruses of the nonsegmented negative-strand NNS variety [ ]. Of particular note, Kochetov “V [ ] concentrated on the ′-UTRs in the mRN“ of seven E”OV viruses, due to the critical importance of the ′-UTRs in translation initiation. In four of these seven mRN“s, small alternate upstream open reading frames uORFs were identified, but their significance is yet to be fully characterized. In cellular mRN“s, uORFs are known to be a common feature they are critical in modulating translation of primary ORFs pORFs , which was accomplish by reducing the efficiency and quantity of the scanning ribosomes associated with the reinitiation that occurs at the start codon of pORFs [ ]. “t a u“UG, rather than a p“UG, translation initiation frequency is affected by a variety of factors, including the strength of the Kozak consensus sequence that surrounds the u“UG. Moreover, between the p“UG and the upstream open reading frame uORF is an intercistronic space that, combined with the phosphorylation status of and the eIF- α [ , ], controls whether translation takes place at the principal protein initiation site p“UG or at the termination codon u“UG . When eIF- α∼P is absent, cap-dependent translation has been found to be efficient, which permits higher ribosome initiation rates at the uORF [ ]. When eIF α∼P is enhanced, impairment of translation initiation occurs, which causes a ribosome to continue scanning beyond the u“UG in this case, initiation occurs at the p“UG. In short, when cell stress occurs, eIF α∼P facilitates translation initiation of select mRN“s that possess uORFs at the primary open ready frame pORF [ ]. They characterized how the E”OV ′-UTRs modulate translation. Mutating any of the four u“UGs present in the E”OV genome enhances translation at the corresponding pORF. The most dramatic effect was with the L gene where the L u“UG can potently suppress pORF translation however, in response to eIF α∼P, the L u“UG maintains L translation. Modulat‐ ing viral polymerase levels is biologically significant as ablating the L uORF in a recombinant E”OV reduces viral titers - to -fold in cell culture, severely impairs viral RN“ synthesis, and functions to maintain virus titers in cells treated with stress-inducing agents. These data suggest that a uORF in the E”OV L mRN“ regulates polymerase expression in response to the status of the cellular innate immune response and is required for optimal virus replication. It would be relatively easy to incorporate a combination of relevant miRN“s in a miRN“expression vector to test the utility of these miRN“s in genetically engineered VLP cell models in vitro that can be performed in a ”SL- facility, and then to extend these studies in animal models utilizing safe vectors in a ”SL- environment.

Utility of Potent Anti-viral MicroRNAs in Emerging Infectious Diseases http://dx.doi.org/10.5772/61687

Currently, there are several genetically engineered vaccines containing genes for surface proteins GP that are in clinical trial. The first among these is a vaccine that Ebola GP genes stitched into a weakened chimpanzee adenovirus that serves as a vector. The second vaccine contains the Ebola surface protein gene inside a weakened version of vesicular stomatitis virus VSV , which commonly infects farm animals. The potential dangers of employing of VSV are obvious: it can save men but potentially harm livestock in West “frica. The chimpanzee adenovirus will be a zoonotic event itself, and its potential danger cannot be underestimated [ , ]. The third vaccine uses a vector known as MV“, a modified version of the smallpox vaccine virus, and involves protection from an Ebola virus challenge months after the last vaccination. We noted that none of these three approaches mentioned a simple and well-tested method of human and animal vaccination. What happened to the simple, whole formalin-killed or UV-killed, less pathogenic E”OV vaccines that have been tried in so many viral vaccina‐ tions [ , ]? With viruses like the major Ebola strands, where the mortality rate is over %, it will be difficult to find a reasonable and ethical way to carry out an unbiased clinical trial. However, if one can prepare a dead Ebola virus with antigenicity intact, it would be easy to immunize high risk groups without utilizing unusual vectors as exemplified by harmless chimpan‐ zee adenovirus, VSV or MV“ modified smallpox virus , each with unknown long-term risk factors and accompanied by immediate concerns of viral vector-induced antigenic competition that may potentially quell proper immune responses to the Ebola antigens [ ]. We believe that a dead vaccine may induce the protective miRN“s and quell the pandemic. Increasingly, miRN“-induced intracellular immunity is being better understood, and several clinical trials are under way to treat viral diseases and cancers [ – ]. The cost of each of these vaccines would run into millions of dollars and would be prohibitively expensive to any of the individuals who are predicted to be infected with the virus in West “frican nations. In contrast to the proposed recombinant vaccines, each of the more traditional killed vaccines has been very inexpensive to produce and has benefited billions of humans [ ].

. Conclusions The current ongoing Ebola outbreaks in West “frica that began almost three years ago in March have already claimed , lives and over , cases. The rapid spread of the infection demands the need for rapid prevention methods. Currently, there are several vaccines that are in different phases of clinical trials. In this report, we highlight an alternative to the standard vaccine for Ebola prevention. We show that a preventive method based on miRN“s could be utilized and tested in nonhuman primates. Some of the lessons that we have learned from the recent West “frica Ebola outbreaks is to test the vaccine and other preventive methods that are currently available against Ebola before the major outbreaks occur. Therefore, we recom‐ mend that vaccines and preventive methods must be developed to the point that the measures

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correlate for human protection Phase I level , so when the outbreaks occur, the vaccine and other measures can be rolled out quickly to prevent the spread of the disease.

Acknowledgements We would like to thank Dr. Donald Gene Pace for his editorial assistance.

Author details Zhabiz Golkar , Donald G. Pace and Omar ”agasra * *“ddress all correspondence to: [email protected] Department of ”iology, School of Health and Natural Science, Voorhees College, Denmark, SC, US“ School of Humanities & Social Science, Claflin University, Orangeburg, SC, US“ School of Natural Science, Claflin University, Orangeburg, SC, US“

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Chapter 17

RNAi — Implications in Entomological Research and Pest Control Nidhi Thakur, Jaspreet Kaur Mundey and Santosh Kumar Upadhyay Additional information is available at the end of the chapter http://dx.doi.org/10.5772/61814

Abstract RN“ interference RN“i has progressed swiftly in the past decade to become a conven‐ ient and dominant genetic tool that has immense utility in diverse fields. The entomologi‐ cal research, ranging from functional genomics to agriculture, has gained enormous momentum due to this technology. RN“i tool helped to discover the functions of new genes and study the complicated genetic networks, thus providing an evolutionary in‐ sight into various processes. RN“i is also becoming a method of choice for controlling in‐ sect pest populations. It is envisaged as tailor-made insecticide, which is highly species specific. However, the efficiency of this mechanism is limited by various factors such as the stability of the trigger molecule, the candidate gene selection, delivery system adopt‐ ed and, most importantly, the choice of the target species. “part from the successful im‐ plication in diverse areas, there are certain drawbacks of this technology such as offtarget’ effects, lack of sensitivity of various species, etc. Further research would relieve these limitations and support the manifestation of this genetic tool with much more relia‐ bility. Keywords: RN“i, insects, pest management, efficiency of RN“i

. Introduction RN“ interference RN“i is a highly conserved, sequence-specific mechanism of gene silencing which is triggered by the presence of double-stranded RN“ dsRN“ . Since its discovery in , RN“i has attained the status of a powerful genetic tool [ ]. This reverse genetics technique is now immensely used in biomedical research, functional genetics and many other areas of biological research. ”roadly, all the reactions that take place for RN“ silencing are initiated when a long dsRN“ is processed into small dsRN“s of about to bp by the RNaseIII enzyme, called Dicer. These small dsRN“s are called small interfering RN“ siRN“ ,

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which when unwound using “TP-dependent activity are incorporated into the multi-subunit RN“-induced silencing complex RISC . Here, the siRN“ guides the RISC complex to degrade cellular RN“ molecules that are complementary to its sequence [ ]. Earlier this process was described in the experimental RN“i studies, and now it is the most accepted tool for gene knockdown studies. The advent of RN“i also revolutionised the entomological research, as novel gene functions were efficiently discovered. In , Kennerdell and Carthew were the first to use RN“i in vivo to study the genes Frizzled and Frizzled-2 in Drosophila melanogaster [ ]. The tremendous success of RN“i in model organisms has prompted its use for research in other insect species as well. In genomics and post-genomics era with the availability of a large amount of sequence information, RN“i further provides an opportunity to investigate the vital functions and crucial interactions that are of importance in both biomedical research and agriculture [ – ]. RN“i studies on insects of economic importance would provide new insights into unravelling the molecular interactions between various disease vectors and ultimately helping in the discovery of novel vaccine and drug targets. Disease vectors such as mosquito, ticks, mites, lice and others were studied extensively using RN“i [ ]. These insects cause many serious diseases in humans and animals. “mong other applications, this genetic tool is also gaining popularity as a promising technol‐ ogy in controlling a wide array of agricultural pests. There is a substantial amount of literature available which documents the success of RN“i as a feasible and sustainable strategy in managing the agricultural pests [ – ]. The core RN“i machinery being present in all the insects makes it possible to silence a wide variety of target genes to produce diverse physio‐ logical, developmental and reproductive restrains. The sequence specificity of endogenous RN“i pathway allows the targeted suppression of genes essential for insect survival and thus offers the development of a specific, logical and sustainable strategy to combat against insect pests. “gricultural pests are notorious and they cannot be efficiently managed by employing a single control agent or technique. Most commonly, the integrated pest management IPM strategies are utilised for combating the diversity of insect pests in the agro-ecosystem [ ]. “long with mechanical, cultural, biological and chemical methods, the transgenic technology should also be embraced in the IPM regime. In this regard, RN“i can play an important role along with the available insecticidal molecules. “mong various transgenic approaches to manage insect pests, Bacillus thuringiensis ”t toxin has shown spectacular success [ ] however, many important insect pests primarily sap-sucking pests are not amenable to ”t protection [ ]. RN“i can be harnessed to defend crops against insect pests. Successful application of RN“i technology in agricultural pest management requires i suitable candidate gene where its silencing can cause mortality of the insect and ii an effective method of dsRN“ delivery. Nevertheless, prior to field application, many aspects of this multi-faceted technology including safety and possible risks to environment need to be evaluated in detail. In this chapter, we discussed the potential of this technology in gene silencing experiments to study the gene function as well as on opportunity to combat against agricultural pests and other disease vectors. Further, the factors responsible for a successful RN“i experiment, the

RNAi — Implications in Entomological Research and Pest Control http://dx.doi.org/10.5772/61814

link with immune response and viral infections have been discussed, highlighting the possible shortcomings of this strategy.

. RNAi in insects RN“i offers species-specific molecules that can be flexibly manipulated and used in under‐ standing various complicated biochemical pathways. The research application of RN“i in entomology has elucidated the functions of several genes. Decrease in the mRN“ levels of a candidate gene due to introduction of a complementary dsRN“ fragment, and the study of the corresponding phenotype, illuminates a gene function. RN“i has been used to study various mechanisms related to insect development embryonic and post-embryonic , repro‐ duction, behaviour and other complicated biosynthetic pathways [ ]. Various insect orders have demonstrated amenability to RN“i-mediated silencing. Species of orders Coleoptera, Lepidoptera, Diptera, Hemiptera, Orthoptera, ”lattodea and Hymenoptera have been studied for various aspects using RN“i technique [ ]. The silencing efficiency ranges from % to % in different insects. “ large majority of the target genes were gutspecific genes however, genes from salivary glands, brain and antennae have also been targeted [ ]. RN“i-based studies can be carried out by either in vivo or in vitro studies. The former method is much easier and involves incubating the cells with the dsRN“ added to the medium. However, the in vivo approach is more useful in the field of functional genomics, especially in case of non-model organisms. Here, the dsRN“ dosage and developmental stage of insect can be specified. In addition, RN“i can be very helpful in identifying the mutant genes that are fatal to the organism,. The significance and compilation of various categories of RN“i experiments in entomology are summarised in Table [ – ]. Experiments

Insect

Gene and function

Parental RNAi

Tribolium castaneum

Zygotic genes [

Parental RN“i is an

Oncopeltus

Determination of the role of the gap genes, hunchback and

important method for

fasciatus

Kr(ppel [

analysing early

Gryllus bimaculatus

“ntenna and appendages [

RNAi in developmental biology

embryogenesis.

accessible or do not survive after

regeneration of insect legs [

], hedgehog, wingless and dpp in ]

Nasonia vitripennis

Various genes in demonstration of parental RN“i [

Tetranychus urticae

Distal-less was used, resulting in phenotypes with canonical

]

limb truncation as well as the fusion of leg segments [

microinjection. Embryonic RNAi

]

the initiation of proximodistal axis formation during the

It is crucial for many insects whose eggs are not

,

]

T. castaneum

Role of wingless wg in leg development [

]

]

343

344

RNA Interference

Experiments

Insect

Gene and function

N. vitripennis

Role of bicoid gene in the structural pattern of the anterior body region. “lso in the absence of bicoid gene, orthodenticle, hunchback and giant genes are responsible for proper head and thorax formation [

Oplegnathus. fasciatus

Hox genes and genes involved in segmentation and segment specification [

Larval/nymphal/pupal

T. castaneum larval RN“i

RNAi

]

]

To study the molecular basis of adult morphological diversity in various organs [

]

T. castaneum larval RN“i

Ubx/Utx during hindwing/elytron development [

T. castaneum larval RN“i

Laccase [

Bombyx mori pupal RN“i

Fatty acid transport protein ”m’F“TP [

Schistocerca

The eye colour gene of first-instar nymphs triggered a

Americana Nymphal RN“i

suppression of ommochrome formation in the eye [

S. Americana

]

] ]

]

Importance of early retinal genes eyes absent eya or sine oculis so in eye development [

]

Blattella germanica nymphal RXR/USP, along with EcR, of the RN“i

heterodimeric nuclear receptor of E [

G. bimaculatus G. bimaculatus

Mechanisms of leg regeneration [

]

period per gene for circadian-dependent locomotor activity rhythm [

G. bimaculatus

-hydroxyecdysone

]

]

Genes responsible for certain human disorders: fragile X mental retardation 1 fmr1 and Dopamine receptor DopR [

Regeneration-dependent

]

G. bimaculatus

Insect leg regeneration [

G. bimaculatus

Orthologs of Drosophila hedgehog Gb’hh , wingless Gb’wg

]

RNAi and decapentaplegic Gb’dpp are expressed during leg regeneration and play essential roles in the establishment of the proximal–distal axis [ RNAi in behavioural

Rhodnius

Nitrophorin

biology

prolixus

a decrease of anticoagulant activity and less efficient feeding behaviour [

Anopheles gambiae

]

“pyrase “g“py in the salivary glands shows important role in host probing behaviour [

D. melanogaster

]

]

-Hydroxy- -methylglutaryl Co“ reductase has been identified for the control of

RNAi — Implications in Entomological Research and Pest Control http://dx.doi.org/10.5772/61814

Experiments

Insect

Gene and function sexual dimorphism of locomotor activity in [ ]]

B. germanica

Neuropeptide pigment dispersing factor in the regulation of locomotor circadian rhythms [

Mechanism of insecticidal Mosquitoes

N“DPH cytochrome P

action

sensitivity of mosquitoes to pyrethroids [

]

reductase led to increased ]

Spodoptera litura and

“minopeptidase M led to decreased sensitivity to ”T toxin

Helicoverpa

[

]

armigera Understanding the

T. castaneum

biosynthetic pathway

Chitin synthases CHS1 and CHS2 are crucialexoskeleton and the midgut peritrophic matrix [

Bombyx mori

]

Bombykol is the main component of sexual pheromone, as well as pheromone-binding proteins and the receptor of the pheromone biosynthesis activator neuropeptide [

Epiphyas postvittana

]

Silencing of pheromone-binding protein of the antennae [

]

Table . RN“i in the study of gene function in insects.

“part from deciphering the function of genes involved in various metabolic pathways, RN“i also finds relevance in other aspects of insect science. It is quite beneficial in maintaining the beneficial insects and saving them from various parasites and pathogens. Certainly, this is useful in case of those parasites and pathogens, which have operative RN“i machinery. “ successful study in this regard shows the control of honey bee parasite Nosema ceranae. When the gene related to energy metabolism was silenced, it was observed that the honey bee population had reduced infestation of Nosema, and lower mortality [ ]. In another study, multiple genes of an ectoparasite of honey bee Varroa destructor were targeted [ ]. It poses a great threat to the health of bees, and its control is of utmost importance for the rearing industry. It is a blood-sucking parasite, so the bees were fed on a meal containing dsRN“ against the genes of Varroa. The RN“i-mediated control decreased the mite population by %, causing no evident damage to the bees. RN“i has also been useful in elucidating the impor‐ tance of various immunological pathways in D. melanogaster [ ]. Host–parasite relationships such as that of Anopheles–Plasmodium have also been studied by using RN“i. Early research was conducted on defensin and it was shown to be important for protecting mosquitoes against infections of Gram-positive bacteria [ ]. Later, the same group demonstrated how the development of Plasmodium is affected by Anopheles gambiae immune genes [ ]. Similarly, in Manduca sexta haemocytes, knockdown of haemolin a bacterial recognition protein decreased the ability of insects to clear Escherichia coli from the haemolymph. This eventually reduced their ability to engulf bacteria and highlighted the role of haemolin in the M. sexta immune response [ ].

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. RNAi in pest control Plants are damaged by a plethora of insect pests. The losses due to these pests and expenditure on the chemical pesticides amount to billions of dollars. In an attempt to reduce these losses, many reports have been published which demonstrate the successful application of RN“i technique in crop protection. Huvenne and Smagghe [ ] have summarised the reports on insects in which RN“i has been applied through feeding, and they discussed several factors that influence the success of RN“i on target insects, such as the concentration of dsRN“, the nucleotide sequence, the length of the dsRN“ fragment and the life stage of the target insects. The downregulation of expression of critical genes, caused by dsRN“/siRN“, eventually leads to death/growth retardation of the insect and forms the basis of pest control. In this view, the efficient delivery and uptake of the dsRN“ trigger is of prime importance. In contrast to the study of gene function, pest control strategies cannot depend on injection of the molecule. The deployment of pest controlling siRN“/miRN“ molecules would involve oral exposure, either as transgenic plants or as sprays. Most of the nutrients from gut lumen are absorbed in the midgut tissue of the insect therefore, this tissue is an attractive target for RN“i. “fter ingestion, the dsRN“ enters into the gut lumen of the insect. Insect gut is divided into foregut, midgut and hindgut. While both foregut and hindgut are covered with chitin, it is only the midgut that has exposed cell surfaces. It is the site of nutrient exchange between the haemolymph and the gut contents. Therefore, midgut epithelium is an attractive target as it is the primary tissue exposed to dsRN“ in the gut lumen. The stability of dsRN“ molecule and the efficiency of the silencing process discussed in section is determined by the gut pH and nucleases. “ breakthrough research in RN“i-mediated pest control was published in on the western corn rootworm, Diabrotica virgifera virgifera WCRW [ ], and cotton bollworm Helicoverpa armigera C”W [ ]. In the former study, a candidate gene was screened based on the complete cDN“ library. Out of a total of dsRN“s, Vacoular ATPase V-ATPase subunit “ was finally selected for the development of transgenic corn plants. The larvae reared on transformed plants caused much less damage to the roots and also showed the reduced expression of the target gene. The other study of Mao et al. [ ] targeted the pesticide detoxifying gene Cytochrome P45 CYP6AE14 , which provides gossypol tolerance to the insect. Transgenic plants express‐ ing dsRN“ corresponding to CYP6AE14 levels of this transcript in insect body was decreased and the larval growth was retarded. Following these two studies, many research groups started to consider RN“i as a feasible technique which could be employed in the transgenic ap‐ proaches to manage insects. “s we know from the various studies conducted, the ”t Bacillus thuringiensis toxins are effective on lepidopteran and coleopteran pests but fail to work against hemipteran pests like aphids, whiteflies, phyllids, etc. [ – ]. RN“i-engineered plants can be more useful in case of these phloem-feeding hemipteran pests, which are notorious not only because of feeding damages but also because of their ability to transmit plant viruses [ ]. The midgut genes of Nilaparvata lugens were downregulated by feeding on transgenic rice expressing dsRN“ against three separate genes, but no lethal phenotype was detected in this case [ ]. Pitino et al. [ ] demonstrated RN“i in aphid Myzus persicae directed towards the receptor of activated kinase Rack-1 gene by transgenic expres‐

RNAi — Implications in Entomological Research and Pest Control http://dx.doi.org/10.5772/61814

sion in Arabidopsis thaliana. “nother important pest, the whitefly, is also now amenable to RN“i [ ]. Its control was demonstrated both by feeding on artificial diet and by feeding on trans‐ genic tobacco plants expressing dsRN“ of V-ATPase A gene [ , ]. RN“i experiments conducted on agricultural pests as well as on insect vectors of several human diseases are summarised in Table [ – ]. Insect pests

Mode of delivery

Gene target Order Coleopteran

Diabrotica virgifera virgifera

“rtificial diet and

a-Tubulin, vacuolar “TPase subunit “ [

]

transgenic plant Phyllotreta striolata

Plant tissue

“rginine kinase [

Leptinotarsa decemlineata

“rtificial diet

Vacuolar “TPase subunit “ and E [

Diabrotica undecimpunctata

“rtificial diet

Vacuolar “TPase subunit “ and E, a-tubulin [

] ] ]

howardi Monochamus alternates

Injection

Laccase gene [

]

Order Diptera Aedes aegypti

”lood meal

Inverted repeat IR RN“ derived from the premembrane

“rtificial diet

protein coding region of the DENV- RN“ genome [

Feeding

V-“TPase “ [

]

“TP-dependent efflux pump [ Anopheles gambiae

Feeding

Chitin synthase [

]

]

Glossina morsitans morsitans Feeding

Midgut protein Tsetse EP transferrin [

Bactrocera dorsalis

Feeding and

Rpl

injection

elongase Noa small GTPase Rab

V-“TPase D subunit fatty acid

Order Hemiptera Acyrthosiphon pisum

Injection

Calreticulin, cathepsin-L [

Feeding

C

[

]

Hunchback [

]

V-“TPase [

]

aquaporin [

]

Cimex lectularius

Injection

Cpr gene[

Diaphorina citri

Topical

“bnormal wing disc [

]

In planta virus induced “bnormal wing disc [

]

Laodelphax striatellus

Feeding

Rhodnius prolixus

Injection

]

]

Disembodied [

]

Nitrophorins – [ a-Glucosidase [ Gap gene giant [

]

] ]

Phospholipase “ [

]

]

[

]

]

347

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RNA Interference

Insect pests

Mode of delivery

Gene target

Sitobion avenae

Feeding

Catalase [

In planta

CbE E [

Injection

Inhibitor of apoptosis gene I“P [

Injection

Polygalacturonase PG [

Oncopeltus fasciatus

Injection

Hunchback [

Riptortus pedestris

Injection

Circadian clock gene, mammalian-type cryptochrome ”la [ ]

Injection and

Gland nitrophorin

Lygus lineolaris

] ] ]

]

]

NP2 [

]

feeding Nilaparvata lugens

Feeding and transgenic

Hexose transporter carboxypeptidase trypsin-like serine

Feeding

protease [

Feeding and

“TP synthase subunit [

injection

Trehalose phosphate synthase [

] ] ]

Cathepsin ”-like protease nicotinic acetylcholine receptors [ Myzus persicae

Transgenic plant

MpC

2 and Rack-1 [

Bemisia tabaci

Injection

Snap, Chickadee, CG

“rtificial diet

“ctin “DP/“TP translocase α-tubulin ribosomal protein L

Transgenic plant

V-“TPase [

]

Injection/feeding

]

]

V-“TPase subunit “ [ Bactericerca cockerelli

, G“T“d [

“ctin, V-“TPase [

]

]

Order Hymenoptera Athalia rosae

Injection

Ar white gene Order Isoptera

Reticulitermes flavipes

Feeding

Endogenous digestive cellulase enzyme, hexamerin storage protein [

]

Order Lepidoptera Ostrinia furnacalis

Spraying

LIM protein

myosin

light chain

chymotrypsin-like serine protease chymotrypsin-like protease C chymortypsin-like serine protease C hydroxybutyrate dehydrogenase Kazal-type serine proteinase inhibitor fatty acid binding protein caboxypeptidse Ostrinia nubilalis

Spraying

Feeding

unknown

]

Chitinase OnCht chitin synthase OnCHS2 [

Epiphyas postvittana

[

]

Carboxylesterase gene EposCXE1 pheromone binding protein [

]

]

RNAi — Implications in Entomological Research and Pest Control http://dx.doi.org/10.5772/61814

Insect pests

Mode of delivery

Gene target

Helicoverpa armigera

Feeding

“cetylcholinesterase AChE [

Transgenic plant

Cytochrome P

Transgenic plant

Ecdysone receptor EcR [

Feeding and transgenic

HaHR moulting factor [

]

gluthatione-S-transferase[ ,

]

] ]

plant Hyalophora cecropia

Injection

Haemolin [

]

Manduca sexta

Injection

Cadherin [

]

Spodoptera litura

Injection

Vitellogenin receptor [

Spodoptera exigua

Feeding

Chitin synthase gene “ [

Spodoptera littoralis

Injection

β-“ctin gene [

Spodoptera frugiperda

“llatostatin C

Feeding [

] ]

]

]

allototropin cytochrome Plutella xylostella

Feeding

Rieske iron–sulphur protein RISP [

]

Order Orthtoptera Gryllus bimaculatus

Injection

Delta Notch [

Injection

Insulin receptor insulin receptor substrate

]

Injection

phosphatase and tensin homolog

Injection

target of rapamycin PRS -p -protein

Injection

kinase forkhead box O epidermal growth factor receptor [

]

Nitric oxide synthase gene NOS [ Circadian clock gene per [ Sulfakinins [ Schistocerca americana

]

]

]

Eye colour gene vermilion [

]

Table . Different insect pests targeted by RN“i.

The choice of a suitable target gene is central to pest control strategy. The gene selection approaches can be based on choosing the gene with known function such as detoxification enzymes, cell synthesis, nutrition, metabolism and cytoskeleton structure. These types of genes can be selected as insect pest control targets. For this, expressed sequence tag EST library of European corn borer Ostrinia nubilalis was screened to find out that a chitinase gene OnCht and a chitin synthase gene OnCHS2 , which are very important in regulating the growth and development of this insect [ ]. Likewise, the EST library of Bemisia tabaci was also used to screen out few important genes for RN“i-mediated control [ ]. The cDN“ library screening approach was also used. Mao et al. [ ] constructed a cDN“ library from RN“s expressed in the midgut of fifth-instar larvae exposed to gossypol. Several cDN“ libraries of WCR D. virgifera virgifera were prepared and considered upon the underlying principle that genes encoding proteins with essential functions would be the best RN“i targets for causing lethality [ ].

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“s an extension of the cDN“ library screening approach, the next-generation sequencing NGS technologies have led to novel opportunities for expression profiling in organisms lacking any genome or transcriptome sequence information. It enables the direct sequencing of cDN“ generated from mRN“ RN“-seq [ , ]. Hence, it provides the de novo genera‐ tion of the transcriptome for a non-model organism, including various pests. Wang et al. [ ] adopted Illumina’s RN“-seq and digital gene expression tag profile DGE-tag to screen optimal RN“i targets from “sian corn borer “C” Ostrinia furnacalis . The same technique has been used for the grain aphid, Sitobion avenae and Spodoptera litura [ , ]. It seems likely that the combination of DGE-tag with RN“-seq is a rapid, high-throughput, cost-effective and easy way to select for candidate target genes for RN“i, which may not be only limited to the midgut tissue but can also be selected from the whole insect. The convenience of RN“i to target specific pests while not harming other species is a perfect method of pest management. However, target gene selection and efficient delivery methods are the two major cornerstones of pest management by RN“i. The candidate gene for RN“i can be tailored to be species specific or they can also have a broad spectrum. Specificity can be achieved by designing dsRN“s that target the more variable regions of genes, such as untranslated regions UTRs . It was first demonstrated in Drosophila where UTR of the gammatubulin gene was targeted and even closely related species could be targeted selectively [ ]. However, it can also be possible to target multiple organisms with a single gene. One such example is of V-ATPase gene, which is an effective target in B. tabaci, D. virgifera and B. dorsalis [ , , ] all of these insects belong to different orders. In this case, either a conserved region can be selected which could affect closely related species or a mixture of dsRN“ fragments from different genes belonging to different species can be selected. Delivery methods that ensure continuous supply of dsRN“/siRN“ will be applicable in the fields. “ more reliable and verified method would be transgenic plants as the dsRN“ can be applied as bait, sprays, or supplied through irrigation systems [ , ]. The application approach by spray could be quite practical like the spray of chemical pesticides. Gan et al. [ ] have also demonstrated the control of viral infection using dsRN“ spraying. Similar results were obtained with “sian corn borer, Ostrinia furnacalis. This study showed that larval lethality or developmental disorders can be achieved by gene-specific RN“i, and spraying can be an efficient method for continuous supply of dsRN“ [ ]. The coating of dsRN“ molecule with liposomes is used for delivering siRN“ to mammalian cells, specific tissues and some insects [ , ]. This coating prevents the degradation of the molecule and enhances its uptake ability spraying may also be explored for such particles. Zhang et al. [ ] used the chitosan nano‐ particle based RN“i technology to suppress the expression of two chitin synthase genes AgCHS1 and AgCHS2 in “frican malaria mosquito A. gambiae larvae. ”acterial expression or chemical synthesis allows large-scale production of dsRN“ at efficient costs [ , , ].

. RNAi: Link with immunity and viral infections The RN“i mechanisms evolved primarily as a defence mechanism against viruses and transposons [ ]. Research has established that RN“i pathway also contributes to the innate

RNAi — Implications in Entomological Research and Pest Control http://dx.doi.org/10.5772/61814

immunity of the insects against the viruses having either dsRN“ genome or such replicative intermediates. It was demonstrated that the Drosophila S cells utilise the endocytosis-mediated pathway involving the pattern recognition scavenger receptors for the uptake of dsRN“ from the surroundings. These receptors are key players in the innate immune responses of the cell [ , ]. Saleh et al. [ ] demonstrated the strong link of dsRN“ uptake pathway and the activation of the immune response in the infected cells. The normal cells used dsRN“ uptake pathway to internalise viral dsRN“ and subsequently showed manifestation of antiviral response in these cells. On the contrary, the mutant cells defective genes used for dsRN“ uptake did not show activation of any antiviral response. Mutants in the core siRN“ compo‐ nents Dicer- , “GO- and R D are more susceptible to viral infections [ ]. Further, it was also reported that receptors such as Sr-CI and Eater, which contribute to majority of dsRN“ uptake in the Drosophila S cells, were significantly down-regulated after pathogenic virus treatments, and significant changes in phagocytic activity were observed. The role of RN“i in antiviral defence has also been firmly established in mosquitoes [ ]. Viruses can also affect the availability of RN“i machinery for other candidate dsRN“ molecules. They can saturate the RN“i machinery and affect the efficiency of RN“i mecha‐ nism. Many viruses are known to produce viral suppressors of RN“ silencing VSRs , which bind to the key elements of the RN“i pathway rendering it unavailable. Many of the viral proteins viz. ” protein from Flockhouse virus, “ proteins from Drosophila C virus and cricket paralysis virus are known to interfere in the siRN“ pathway of RN“-mediated silencing [ – ]. These viral proteins may affect the biogenesis of the trigger molecule by binding to important enzymes such as dicer, which generate the siRN“ from long dsRN“ or affect the target cleavage by binding to RISC. The viral proteins may also sequester the dsRN“ signal molecule or form complexes with the replicative intermediates of the siRN“ pathway. Viruses also produce large amount of RN“s and small RN“s that accumulate in the infected cells. It has also been hypothesised that the occurrence of alternative and effective antiviral pathway may become important in controlling the viral infections and may supersede the RN“i pathway. Few of these possible pathways have been worked upon. Goic et al. [ ] have reported the potential interaction of nucleic acid-based acquired immunity with the core RN“i machinery in the study of persistent infection of S cells by Flock House Virus FHV . The insects also protect themselves from foreign nucleic acids by becoming refractory to RN“i. In the oriental fruit fly, Bactrocera dorsalis, orally administered dsRN“-targeting endogenous genes, resistance to RN“i was seen due to a blockade in the dsRN“ uptake pathway. “ very interesting hypothesis is presented by Swevers et al. [ ] about the possible impact of persistent viral infection in the insects. In their work, the authors have analysed various factors that determine the response to exogenous dsRN“ in the background of viral infection.

. Efficiency of RNAi Though RN“i is a conserved mechanism in eukaryotes, its efficiency is governed by various factors. The response of different insect species towards this mechanism of gene silencing is

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imperative for successful implementation in the study of gene function and more importantly in the pest management programs. “lso, the efficacy is governed by many factors which are not intrinsic to the organism such as the delivery, dosage and choice of the candidate gene. Comprehending these factors will provide a better insight into designing the experiments for successful application of RN“i. The available reports indicate that the lower Insecta species such as that of Blatella show a much robust and persistent RN“i response, while higher Insecta species belonging to the orders Lepidoptera and Diptera are non-compliant [ ]. The sensi‐ tivity could vary within and among the orders. For instance, many Lepidopteran insects are resistant to RN“i. Terenius et al. [ ] have reviewed various factors that may be contributing to the poor responsiveness of these insects. The efficiency can vary with insect species, target gene, developmental stage of the organism, expression of RN“i machinery, method of delivery, stability of dsRN“, etc. “ few factors that may be crucial in determining the efficiency of RN“i are discussed below. . . The RNAi machinery RN“i evolved in organisms as a defence mechanism against viral infections at the cellular level [ , ]. The differences in the expression of core RN“i machinery can be a prime reason affecting the adequacy of RN“i mechanism. The systemic RN“-interference-deficient sidprotein forms a gated channel which is selective for dsRN“ molecule. Its role is well established in the systemic spread of the RN“i signal in the model organism Caenorhabditis elegans [ ]. The presence of SID-1gene orthologs in insects varies with the insect orders [ ]. The dipterans lack this gene completely. The mosquito Culex quinquefasciatus also lacks sid- ortholog but shows the systemic spread of the dsRN“ trigger [ ]. On the one hand, honey bee Apis mellifera showed an increase in the expression of SID-1 during the RN“i experiments, indicating its role in the uptake pathway [ ]. On the other hand, in Tribolium castaneum, the silencing of all three orthologs of SID-1 casted no influence on the efficiency of RN“i [ ]. In Bombyx mori, three orthologs are present but no significant success has been found in this lepidopteran, while mosquitoes show systemic RN“i despite the absence of sid-1 in several species [ – ]. R D , a cofactor of dicer- enzyme which cleaves long dsRN“ into siRN“ for loading into the RISC, is absent in B. mori making the insect very insensitive to RN“i. “nother important enzyme RN“-dependent RN“ polymerase RdRP , which amplifies the primary siRN“ signal in C. elegans, is entirely not reported in the insects [ ]. T. castaneum showed a robust systemic RN“i, but a wide survey of RN“i related genes did not show any traces of RdRP [ ]. However, RdRP-like activity was substituted in Drosophila cell lines by certain other enzymatic pathways. In many cases, the absence of certain well-known genes of the RN“i pathway is directly responsible for the poor response of the organism while in several other examples, the absence is compensated by other genes/pathways which play key roles of their counterparts. . . The RNAi molecule The exogenous dsRN“ molecule is the trigger for initiating the RN“i pathway. These molecules are delivered in the form of dsRN“, siRN“ or hairpin RN“. “part from sequence

RNAi — Implications in Entomological Research and Pest Control http://dx.doi.org/10.5772/61814

specificity, other parameters are also crucial in determining the efficiency of RN“i experi‐ ments. The study on the administration of dsRN“ feeding by means of artificial diet, natural diet, droplet method, blood meal, transgenic plants, etc. in insects has used varied length of dsRN“ molecule. The nucleotide length used in these reports ranges from to bp, while most of the studies used – bp as the optimal length [ ]. However, silencing effects have also been observed in the case of single siRN“ synthesised chemically administration in H. armigera or a cocktail of siRN“ obtained by using dicer enzyme to chop the dsRN“ molecule [ ]. In the cell line experiments done on the Drosophila S cell line, bp was found to be the optimum length of dsRN“ that could be absorbed by the cells [ ]. Not only the length but also the specificity of the RN“i molecule is a concern. It can be understood by the reports on Drosophila that feeding specific sequence of V-ATPase dsRN“ caused no silencing in non-target species [ ], which implies the specificity of the process. On the contrary, various other studies report non-specific silencing. Off-target effects were reported in Rhodnius prolixus [ ] and Colorado potato beetle Leptinotarsa decemlineata [ ]. Single mismatches are known to impair the RN“i effect in the mammalian cell lines [ ]. Further studies will clarify whether single mismatches show similar impact in insects as well. In case of pest management programs, long dsRN“s > bp are generally used which generate many probable siRN“ maximising the RN“i response [ ]. The dosage of the dsRN“ molecule also plays an important role in determining the efficiency of the process. Higher doses are required in case of feeding experiments as compared to injection. The silencing effect in R. prolixus was enhanced by multiple doses [ ]. Therefore, it follows that doses and types of administration oral or injectable also need to be optimised according to the life stage of the organism and the target tissue. . . Delivery of the molecule/uptake of silencing signal One of the most decisive factors for inducing RN“i is the efficient delivery of the dsRN“ molecule. The common methods of delivery are by microinjection, soaking, oral delivery and transgenic technique [ ]. Microinjection-based delivery is the most commonly used techni‐ que in studying gene functions. It has proven to work well for Tribolium, Drosophila and many other lepidopteran insects. “lthough it works well for larger insects, success with smaller insects is limited due to the invasive nature of this technique. The survival of aphids after microinjection procedure is highly dependent on the injected volume [ ]. Further, factors such as needle choice, optimal volume and place of injection are very crucial considerations and tend to vary with organisms and laboratories. Feeding-based experiments involve either in vitro synthesised or bacterially expressed dsRN“ molecules. The success of oral delivery methods indicates the possible employment of RN“i technique for target pest control. However, the stability of the molecule will always be a concern in the gut lumen. “rtificial diet mixed with dsRN“ could not induce RN“i in Drosophila spp. Ingested dsRN“ against a gut-specific aminopeptidase N gene also failed to develop RN“i response in Spodoptera litura [ ]. Therefore, it can be suggested that oral delivery is not equally suitable for all species. “nother convenient method of delivery is soaking. Nevertheless, this method is more appli‐ cable for cell line experiments rather than whole insects.

353

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“fter the delivery of the molecules, the next step is the uptake of the molecules by insects. Huvenne and Smagghe [ ] have elaborately reviewed the basic mechanisms involved in the uptake of dsRN“ in the insects. The spreading of the RN“i signal, i.e., systemic RN“i, is an important determinant of the efficiency of RN“i. In cases of functional genetics, cell autono‐ mous RN“i has been successfully employed to study the function of genes however, for implementation in the pest control programs, non-cell autonomous RN“i is important. The systemic spread of the silencing signal is absent in the most studied model insect Drosophila. In contrast, the most studied insect Tribolium shows a powerful systemic silencing effect [ ]. . . Potency of the silencing signal The manifestation of the RN“i effect also depends on the stability and persistence of the dsRN“ molecule. In Acyrthosiphon pisum, the silencing effect on the aquaporin gene began to reduce after five days [ ]. The early stability of dsRN“ molecule may be disrupted by the non-specific nucleases as reported in many of the lepidopteran insects [ ]. These are extracellular enzymes different from dicer and digest the trigger molecule, thereby preventing the RN“i cascade. In certain cases, the activity of dsRN“ degrading enzymes have been studied and their levels were measured in different stages, which was found related to the developmental stage. The dsRNase activity is also found in the digestive juices of Bombyx mori, saliva of Linus lineolaris and in haemolymph of Manduca sexta [ , , ]. The existence/ stability/mode of action of these enzymes are not sufficiently studied and future research in this direction needs to be carried out to comprehend the stability of dsRN“ molecule in the in vivo studies. The choice of gene can also decrease the strength of the silencing signal. Ideally, the protein whose function is to be silenced should have a short half-life, whilst the mRN“ turnover number should be high. The stability of protein explained the weak RN“i response in both D. melanogaster and T. casteneum [ ]. However, such studies have not been conducted for the majority of the genes and therefore it can be concluded that expression of RN“i is limited by many uncovered phenomena.

. Conclusions The advent of RN“ interference has been a crucial phase of the modern day science. The wide array of applications in the entomological research has led to many momentous findings. The functionality of many genes has been understood by this technique. Its implication in func‐ tional genomics is not only restricted to the study of a given set of genes but is also used to unveil the interaction of different genes in a particular metabolic pathway. The rapid pace of RN“i-based research suggests that it would soon facilitate better understanding of evolution, circadian rhythms, behavioural pattern, reproductive biology and interaction between host and parasites/pathogens. However, successful manifestation of RN“i is dependent on several factors. The insect species might lack the basic RN“i machinery [ ] or may rapidly degrade alien dsRN“. Such factors could be intrinsic to the concerned tissue or gene. The gene might have high transcription rate and could evade the effect of RN“i or the target mRN“ may be too transient.

RNAi — Implications in Entomological Research and Pest Control http://dx.doi.org/10.5772/61814

“s happens with every phenomenon, this mechanism can also undergo selection pressure. Viruliferous insects that also have RN“i suppressors would be able to thrive on RN“iprotected crops. Furthermore, single nucleotide polymorphisms SNPs that result in lower effectiveness of the RN“i could potentially be selected for and lead to the evolution of resistance [ ]. Genetic variations among insect species are already a challenge for RN“i. Therefore, parallel research must be carried out to develop strategies, which would minimise the resistance development and selective pressures. RN“i has proved its utility as a futuristic tool of insect pest management. However, there are several issues that need to be addressed before the implementation of this technology in fields. The knowledge gaps underlying large-scale implications of pesticidal RN“i-based crops on the environment should be identified and bridged. The off-target gene silencing is a serious concern where unintended organisms are adversely affected [ ]. The non-target effects can be categorised as off-target gene silencing, silencing the target gene in non-target organisms, immune stimulation and saturation of the RN“i machinery [ ]. “ balanced approach should be taken with maximum effects on the target pests with minimal effects on non-target organisms.

Acknowledgements SKU is thankful to the Department of Science and Technology, India, for DST-INSPIRE faculty fellowship and Panjab University for facility.

Author details Nidhi Thakur , , Jaspreet Kaur Mundey and Santosh Kumar Upadhyay * *“ddress all correspondence to: [email protected] Plant Molecular ”iology Lab, CSIR-National ”otanical Research Institute, Lucknow, India “cademy of Scientific and Innovative Research, “nusandhan ”hawan, New Delhi, India Department of ”otany, Panjab University, Chandigarh, India

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] Thakur N, Upadhyay SK, Verma PC, Chandrashekar K, Tuli R, Singh PK. Enhanced whitefly resistance in transgenic tobacco plants expressing double stranded RN“ of v-“TPase a gene. PLoS One : ,e . DOI: . /journal.pone. .

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] Zhu F, Sams S, Moural T, Haynes KF, Potter MF, Palli SR. RN“ interference of N“DPH-cytochrome P reductase results in reduced insecticide resistance in the bed bug, Cimex lectularius. PLos One : , e . DOI: . /journal.pone. .

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] Wan P-J, Jia S, Li N, Fan J-M, Li G-Q. RN“ interference depletion of the halloween gene disembodied implies its potential application for management of planthopper Sogatella furcifera and Laodelphax striatellus. PLos One : ,e . DOI: . / journal.pone. .

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] “raujo RN, Soares “C, Paim RM, Gontijo NF, Gontijo “F, Lehane MJ et al. The role of salivary nitrophorins in the ingestion of blood by the triatomine bug Rhodnius prolixus Reduviidae: Triatominae . Insect Biochemistry and Molecular Biology : , – . DOI: . /j.ibmb. . . .

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] Defferrari M, Lee D, Fernandes C, Orchard I, Carlini C. “ phospholipase “ gene is linked to Jack bean urease toxicity in the Chagas’ disease vector Rhodnius prolixus. Biochimica et Biophysica Acta : , – . DOI: . /j.bbagen. . . .

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] Deng F, Zhao Z. Influence of catalase gene silencing on the survivability of Sitobion avenae. Archives of Insect Biochemistry and Physiology : , – . DOI: . / arch. .

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] Xu L, Duan X, Lv Y, Zhang X, Nie Z, Xie C, Ni Z, Liang R. Silencing of an aphid car‐ boxylesterase gene by use of plant-mediated RN“i impairs Sitobion avenae tolerance of Phoxim insecticides. Transgenic Research : , – . DOI: . / s - .

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] Liu PZ, Kaufman TC. Hunchback is required for suppression of abdominal identity, and for proper germband growth and segmentation in the intermediate germ band insect Oncopeltus fasciatus. Development : , – . DOI: . /dev. .

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] Ikeno T, Numata H, Goto SG. Circadian clock genes period and cycle regulate photo‐ periodic diapause in the bean bug Riptortus pedestris males. Journal of Insect Physiolo‐ gy a: , – . DOI: . /j.jinsphys. . . .

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] “raujo RN, Santos “, Pinto FS, Gontijo NF, Lehane MJ, Pereira MH. RN“ interfer‐ ence of the salivary gland nitrophorin in the triatomine bug Rhodnius prolixus Hemiptera: Reduviidae by dsRN“ ingestion or injection. Insect Biochemistry and Mo‐ lecular Biology : , – . DOI: . /j.ibmb. . . .

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] Zha W, Peng X, Chen R, Du ”, Zhu L, He G. Knockdown of midgut genes by dsRN“-transgenic plant-mediated RN“ interference in the hemipteran insect Nila‐ parvata lugens. PLoS One : ,e . DOI: . /journal.pone. .

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] Chen J, Zhang D, Yao Q, Zhang J, Dong X, Tian H et al. Feeding-based RN“ interfer‐ ence of a trehalose phosphate synthase gene in the brown planthopper, Nilaparvata lugens. Insect Molecular Biology : , – . DOI: . /j. . . .x.

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] Zhang Y, Wang X, Yang ”, Hu Y, Huang L, ”ass C et al. Reduction in mRN“ and protein expression of a nicotinic acetylcholine receptor α subunit is associated with resistance to imidacloprid in the brown planthopper, Nilaparvata lugens. Journal of Neurochemistry. . DOI: . /jnc. .

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] Wuriyanghan H, Rosa C, Falk ”W. Oral delivery of doublestranded RN“s and siR‐ N“s induces RN“i effects in the potato/tomato psyllid, ”actericerca cockerelli. PLoS One : ,e . DOI: . /journal.pone. .

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] Wang Y”, Zhang H, Li HC and Miao XX. Second generation sequencing supply an effectiveway to screen RN“i targets in large scale for potential application in pest in‐ sect control. PLoS One : ,e . DOI: . /journal.pone. .

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] Khajuria C, ”uschman LL, Chen MS, Muthukrishnan S, Zhu KY. “ gut-specific chiti‐ nase gene essential for regulation of chitin content of peritrophic matrix and growth of Ostrinia nubilalis larvae. Insect Biochemistry and Molecular Biology : , – . DOI: . /j.ibmb. . . .

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] Kumar M, Gupta GP, Rajam MV. Silencing of acetylcholinesterase gene of Helicover‐ pa armigera by siRN“ affects larval growth and its life cycle. Journal of Insect Physiol‐ ogy : , – . DOI: . /j.jinsphys. . . .

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] Porta H, Jimenez G, Cordoba E, Leon P, Soberon M, ”ravo “. Tobacco plants ex‐ pressing the Cry “bMod toxin suppress tolerance to Cry “b toxin of Manduca sexta cadherin-silenced larvae. Insect Biochemistry and Molecular Biology : , – . DOI: . /j.ibmb. . . .

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] Tian H, Peng H, Yao Q, Chen H, Xie Q, Tang ” et al. Developmental control of a lepi‐ dopteran pest Spodoptera exigua by ingestion of bacteria expressing dsRN“ of a nonmidgut gene. PLoS One : ,e . DOI: . /journal.pone. .

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] Gong L“, Yang XQ, Zhang ”L, Zhong GH, Hu MY. Silencing of Rieske iron-sulfur protein using chemically synthesised siRN“ as a potential biopesticide against Plu‐ tella xylostella. Pest Management Science : , – . DOI: . /ps. .

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] Moriyama Y, Sakamoto T, Karpova SG, Matsumoto “, Noji S, Tomioka K. RN“ in‐ terference of the clock gene period disrupts circadian rhythms in the cricket Gryllus bimaculatus. Journal of Biological Rhythms : , – . DOI: . / .

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] Meyering-Vos M, Muller “. RN“ interference suggests sulfakinins as satiety effec‐ tors in the cricket Gryllus bimaculatus. Journal of Insect Physiology : , – . DOI: . /j.jinsphys. . . .

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] Winston WM, Molodowitch C, Hunter CP. Systemic RN“i in C. elegans requires the putative transmembrane protein SID- . Science : , – . DOI: . / science. .

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] Scott JG, Michel K, ”artholomay LC, Siegfried ”D, Hunter W”, Smagghe G et al. To‐ wards the elements of successful insect RN“i. Journal of Insect Physiology : , – . DOI: . /j.jinsphys. . . .

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] Wu H, Ma H, Ye C, Ramirez D, Chen S, Montoya J et al. Improved siRN“/shRN“ functionality by mismatched duplex. PLoS One : ,e . DOI: . /jour‐ nal.pone. .

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Chapter 18

Management of Insect Pest by RNAi — A New Tool for Crop Protection Thais Barros Rodrigues and Antonio Figueira Additional information is available at the end of the chapter http://dx.doi.org/10.5772/61807

Abstract The fast-growing human population requires the development of new agricultural tech‐ nologies to meet consumers´ demand, while minimizing environmental impacts. Insect pests are one of the main causes for losses in agriculture production, and current control technologies based on pesticide application or the use of transgenic crops expressing Ba‐ cillus thuringiensis toxin proteins are facing efficacy challenges. Novel approaches to con‐ trol pests are urgently necessary. RN“ interference RN“i is a gene silencing mechanism triggered by providing double-stranded RN“ dsRN“ , that when ingested into insects can lead to death or affect the viability of the target pest. Transgenic plants expressing dsRN“ version of insect specific target genes are the new generation of resistant plants. However, the RN“i mechanism is not conserved among insect orders, and its elucidation is the key to develop commercial RN“i crops. In this chapter, we review the core RN“i pathway in insects and the dsRN“ uptake, amplification, and spread of systemic silenc‐ ing signals in some key insect species. We also highlight some of the experimental steps before developing an insect-pest-resistant RN“i plant . Lastly, we review some of the most recent development studies to control agricultural insect pests by RN“i transgenic plants. Keywords: ”iotechnology, dsRN“, Entomology, Gene silencing, Insect control, RN“ in‐ terference

. Introduction “griculture has to continually adapt to rising environmental concerns in conjunction with meeting the increasing consumers´ demand. The fast-growing human population creates the need for the sustainable intensification of agriculture throughout the world which can be acomplished by adopting mechanization and new technologies to close yield gaps while

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minimizing environmental impacts. In the past few decades, insect pest control has been mainly conducted by the application of chemical pesticides because of the low cost and efficacy but their indiscriminate use has caused escalating problems with the evolution of insect resistance to the pesticides together with secondary pest outbreaks. The development of new biotechnological approaches, with the introduction of transgenic crops expressing Bacillus thuringiensis ”t Cry toxin proteins, also known as insect-resistant transgenic ”t-plants, decreased pesticide utilization in certain key crops, such as cotton and maize, and brought economical and environmental benefits [ - ]. ”ut once again, insect resistance has arisen, now against the ”t toxins, and outbreaks of nontarget pests have emerged [ , ], which makes necessary the development of novel approaches to control selected agriculture pests. RN“ interference RN“i is a gene silencing mechanism at the cellular level triggered by double-stranded RN“ dsRN“ and is likely to be the new approach underlying the next generation of insect-resistant transgenic plants. In some studies, successful delivery of dsRN“ molecules to insects by ingestion resulted in the expected essential gene target silencing [ , ], which led to death or affected the viability of the target insect, resulting in control of the pest. In general, long dsRN“s are processed by species-specific RNase-III-like enzymes, resulting in smaller double-stranded molecules. These shorter RN“s are loaded into RN“ complexes as a guide for finding target mRN“s that are either cleaved or blocked for translation in posttranscriptional silencing, or inducing histone modifications when involved in transcrip‐ tional silencing response [ , ]. However, the RN“i systemic spreading mechanism is not conserved across organisms, and its elucidation is an essential step in developing an efficient method to control agricultural pests by RN“i technology. In this chapter, we review how the RN“i mechanism occurs in insects, highlighting the core RN“i pathway and components, and new developments regarding dsRN“ uptake, amplifi‐ cation, and the spread of systemic silencing signals in some key insect species. We also discuss the critical experimental steps before developing an RN“i plant protected against a specific insect pest, with consideration to application. Lastly, we review some of the most recent published studies to control agriculturally important insect pests based on RN“i transgenic plants.

. RNAi mechanism RN“i is an important and natural antiviral defense mechanism, protecting organisms from RN“ viruses, or even avoiding the random integration of transposons [ ]. Over time, with the discovery of some aspects of the mechanism, RN“i has become a widely used tool to knock down and analyze the function of genes. Most of the RN“i pathways have dsRN“ as the precursor triggering molecule that vary in length and origin [ , ]. In addition, the RN“i pathways differ not only in the RN“ precursor molecule, but also in genes, enzymes, and effector complexes involved throughout the process. However, some key steps are conserved. ”riefly, RN“ duplexes are processed into short RN“ duplexes, which are then used to guide the recognition of their target, either to cleave a complementary mRN“, or to repress their

Management of Insect Pest by RNAi — A New Tool for Crop Protection http://dx.doi.org/10.5772/61807

target translation at a posttranscriptional silencing level, or to modify the chromatin structure at the transcriptional level [ , ]. The RN“ precursor molecules from the RN“i pathways, all of which are already identified in insects, are small RN“s, categorized into three classes [ , ]: the first two classes are small interfering RN“s siRN“s – nucleotides and microRN“s miRN“s – nucleotides [ ] Figure . ”oth miRN“s and siRN“s share a common RNase-III processing enzyme, Dicer, and closely related effector complexes and both can regulate gene expression at the posttran‐ scriptional level [ , , ]. Conversely, the third class of small RN“s, the PIWI-interacting RN“s piRN“s – nucleotides , are generated independent of the Dicer activity [ ]. piRN“s have been reported to play an essential role in germ-line development, stem cell renewal, transposon silencing, and epigenetic regulation [ - ]. These piRN“s originate from a diversity of sequences, including repetitive DN“ and transposons, and they seem to act both at the posttranscriptional and chromatin levels [ ]. The mechanism that generates and amplifies piRN“s is not well-understood, but involves slicer activities “rgonaute proteins associated with cleaving activity [ , , ]. Considered a specialized subclass of piRN“s, repeat-associated siRN“s rasiRN“s - nucleotides were identified in the Drosophila genome [ ], and suggested by Meister and Tuschl [ ] to be involved in guiding chromatin modification in this insect species Figure . The most recognized RN“i pathways are the siRN“ and miRN“ despite being triggered by different molecules, both precursors are long double-stranded RN“s dsRN“s . Naturally in a cell, long dsRN“s can derive from RN“ virus replication, from the transcription of conver‐ gent cellular genes or mobile genetic elements, or from self-annealing cellular transcripts [ ]. In the siRN“ pathway, these long dsRN“ are processed by Dicer into siRN“ duplexes. ”y contrast, in the miRN“ pathway, miRN“s are generated from endogenous transcripts primary miRN“s pri-miRN“s that form stem–loop structures [ , ]. In the nucleus, these hairpin regions are recognized and cleaved into precursor mirRN“s pre-miRN“s by Drosha, another RNase-III-family enzyme, and the pre-miRN“s are transported to the cytoplasm through the nuclear export receptor Exportin- Expo- [ , ]. Subsequently, the premiRN“ undergoes another endonucleolytic cleavage, now catalyzed by Dicer, generating an miRN“ duplex [ , ] Figure . The siRN“ and miRN“s duplex containing ribonucleoprotein particles RNPs are subse‐ quently rearranged into effector complexes. “lthough it is difficult to assign distinct functional labels, an siRN“-containing effector complex is referred to as an RN“-induced silencing complex RISC , and an miRN“-containing effector complex is referred to as an miRNP [ ]. In these complexes, the regulation is at a posttranscriptional level and every RISC or miRNP contains a member of the “rgonaute “go protein family [ ]. For the regulation at the transcriptional level as guided by rasiRN“s, a specialized nuclear “rgonaute-containing complex, known as the RN“-Induced Transcriptional Silencing complex RITS mediates gene silencing [ ]. In general, one strand of the short-RN“ duplex the guide strand is loaded onto an “rgonaute protein at the core of the effector complexes. During loading, the nonguide strand is cleaved by an “rgonaute protein and ejected. The “rgonaute protein then uses the guide RN“ to associate with target RN“s that contain a perfectly complementary sequence

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Figure . RN“-mediated gene silencing pathways. In the nucleus, primary miRN“ transcripts pri-miRN“s are proc‐ essed to mirRN“ precursors pre-miRN“ by the RNase-III-like enzyme Drosha. The pre-miRN“ is exported through the export receptor Exportin- Exp- to the cytoplasm, and then processed by Dicer to microRN“ miRN“ . These miRN“s are unwound and assembled into miRNP miRN“ containing effector complex of RiboNucleo Protein parti‐ cles or RISC RN“ Induced Silencing Complex , triggering silencing responses by translational repression of target mRN“s or mRN“-target degradation, respectively. In the cytoplasm, long dsRN“ are processed to siRN“ small in‐ terfering RN“ by the RNase-III-like enzyme Dicer. The short dsRN“s are unwound and assembled into RISC or RITS RN“-Induced Transcriptional Silencing complexes. The siRN“ guides the RN“ cleavage by the RISC complex, while rasiRN“ repeat-associated short interfering RN“ guides the condensation of heterochromatin by the RITS complex. mG: -methyl guanine ““““: poly-adenosine tail Me: methyl group P: ’- phosphate. “dapted from [ ]

and then catalyzes the slicing of these targets, either to be cleaved by RISC, to be blocked for translation in miRNP or by inducing histone modifications in RITS [ ] Figure . The mecha‐ nism of miRN“-guided translational regulation is not as well-understood in the case of target-

Management of Insect Pest by RNAi — A New Tool for Crop Protection http://dx.doi.org/10.5772/61807

RN“ cleavage, and to make things more complicated, miRN“s can act as siRN“s, and siRN“s can act as miRN“s [ ]. Dicer is one of the enzymes involved in RN“i mechanism that is encoded by a variable number of genes and presents distinct specificity among organisms [ ]. For instance, mammals and Caenorhabditis elegans, the best characterized animal for RN“i, have each a single Dicer responsible for functions in both siRN“ and miRN“ pathways [ ], while Drosophila mela‐ nogaster has two paralogues: Dicer- Dcr- that preferentially processes miRN“ precursors, and Dicer- Dcr- required to process long dsRN“ into siRN“s [ ]. However, in Tribolium castaneum, a model organism among insects for systemic silencing by RN“i, Dcr- showed an important role at the RN“i pathway, whereas Dcr- is suggested to have an involvement in wing development, most likely through the miRN“ pathway [ ]. “nother gene family involved in RN“i pathways is the “rgonaute proteins “go . “go is a central protein component of silencing complexes RISC, RITS, miRNP that acts in mediating target recognition and silencing [ ]. “rgonaute proteins contain two domains: a P“Z domain involved in dsRN“ binding, and a PIWI-domain responsible for RNase activity [ ]. In Drosophila, “go- is involved in the miRN“ pathway “go- in the siRN“ pathway while Piwi, “ubergine “ub , and “go- are associated in transcriptional silencing [ , - ]. In Tribolium, a single class of “rgonaute was identified Tc-“go- in the miRN“ pathway, while two classes of “go- paralogues Tc-“go- a and Tc-“go- b were found in the siRN“ pathway, probably deriving from gene duplication in the beetle lineage [ ]. This duplicated Tc-“go- might lead to higher amounts of “go- protein, potentially with the enhancement of the RN“i response [ ]. In the silkmoth Bombyx mori, a Lepidoptera species in which the RN“i response is considered much less robust [ , ], AGO genes from all three main RN“i pathways were identified BmAGO-1 – miRN“ BmAGO-2 – siRN“ BmAGO-3 – piRN“ , which were shown to be involved in the RN“i response in ”m cells [ ]. Taken together, these findings, and other reviews [ ], support the idea of a function overlap of the three main RN“i pathways in B. mori [ ]. . . Systemic RNAi Systemic RN“i is described as a silencing signal transmitted widely throughout a treated organism [ , ]. The knowledge about the systemic RN“i mechanism in insects is important as it may affect the approaches adopted to develop RN“i-mediated pest control because the systemic mechanism is not conserved among those organisms. Systemic RN“i has two important steps to be considered: the uptake of dsRN“ by the cells and the systemic spreading of the signals. Some of the main genes involved in systemic RN“i are presented below and discussed for the model organisms. 2.1.1. dsRNA uptake In insects, two types of dsRN“ uptake mechanisms have been identified [ ]. The first one involves a multi-transmembrane domain protein, Systemic Interference Defective Sid . In C. elegans, Sid- is essential and sufficient to mediate uptake and systemic spread of RN“i signal

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in both somatic and germ-line cells [ , ] conversely, in insects, Sid- -like Sil proteins appear to be variable across orders [ ]. For instance, Diptera do not present SIL genes, while Tribolium and B. mori presented three SIL homologues [ , ]. However, these Tribolium genes share more identity with another C. elegans gene, TAG-13 , not required for systemic RN“i in C. elegans compared to that with SID-1 [ ]. The second dsRN“ uptake mechanism involves endocytosis, specific for dsRN“s acquired from the environment, known as environmental RN“i [ , ]. First discovered in Drosophila S cells and later in C. elegans, this uptake of dsRN“ by endocytosis appears to be evolutionary-conserved [ - ]. However, we should be cautious to conclude that all organisms have a dsRN“ uptake mechanism based on endocytosis. For instance, Ulvila and colleagues [ ] working with Drosophila S cells, which are hemocyte-like, described high rates of endocytosis as compared to the majority of other cell types in this species [ ]. Other important proteins for systemic RN“i were identified in C. elegans but are specific only to germ-line cells, such as Rsd- , Rsd- , and Rsd- [ ]. The Rsd- protein contains no particular known motifs but interacts with Rsd- that has a Tudor domain, suggesting that these two proteins act together [ ]. The RSD-3 gene encodes a protein that contains an epsin aminoterminal homology ENTH domain, found in proteins involved in vesicle trafficking, sug‐ gesting the involvement of endocytosis in systemic RN“i [ ]. In Tribolium, a homologue for RSD-3 Tc-RSD3 has been found, but in Drosophila, which does not exhibit a systemic silencing response, a homologous Rsd- protein Epsin-like was identified [ ]. So, the presence of Rsd- does not seems to determine whether or not systemic RN“i occurs in insects, and it is possible that the expression level and/or tissue specificity of this gene may affect the degree of RN“i efficiency and the dsRN“ uptake from the environment [ ]. 2.1.2. RNAi systemic spreading: amplification and maintenance of dsRNA Once the dsRN“ overcomes all the uptake barriers, the silencing signal should be transported from treated cell to other cells, and spread to other tissues. Further, dsRN“ should be con‐ stantly produced, e.g., either by the amplification of dsRN“ by an RN“-dependent RN“ polymerase RdRP , and/or constantly acquired for the maintenance of the silencing responses. In C. elegans, primary siRN“s processed by Dicer are used as a template for an RdRP activity to produce secondary dsRN“s [ ]. The RdRP activity is key for the RN“i signal amplification. However, so far in insects, no RdRP-related protein has been found [ , ], suggesting that strong RN“i response in insects does not rely on amplification of the trigger dsRN“, and it must be based on a different mechanism yet to be identified. “lternatively, constant supply of dsRN“ may be provided by RN“i-plants to provide continuous effects. The presence of the main genes involved in systemic RN“i and amplification in Drosophila and Tribolium fail to explain the respective absence and presence of systemic RN“i [ ]. Never‐ theless, the several differences in the number of these core component genes found between species suggests an interesting avenue for further investigation [ ]. For instance, “go protein that have already been shown to determine RN“i efficiency [ ], was found to be duplicated in the Tribolium genome, while Drosophila carries only a copy of AGO-2 gene, suggesting a relationship between number of AGO copies and RN“i response [ ].

Management of Insect Pest by RNAi — A New Tool for Crop Protection http://dx.doi.org/10.5772/61807

“nother component that might affect RN“i efficiency in different insects are the proteins containing the dsRN“-binding motif dsR”M , which help small molecules to properly load inside of the silencing complexes [ ]. These proteins act together with Dicer, and seem to be responsible for determining Dicer specificity in Drosophila [ ]. In the T. castaneum genome, two R2D2-like genes a particular dsR”M were found TcR2D2 and TcC3PO , which might help Tribolium to be hypersensitive to dsRN“ molecule uptake by the cells [ ]. However, in the Anthonomus grandis transcriptome, only an R2D2 contig was identified [ ]. The presence of an additional R D -like protein in Tribolium might also allow a longer-lasting RN“i effect, once dsR”M proteins are known to bind to dsRN“s, and might be involved in the maintenance of dsRN“ in cells [ ].

. Factors affecting the silencing effect and RNAi efficiency as an insect control method The RN“i approach to control insect pests had been considered for many years, but application of this technology was just realized after it was shown that ingestion of dsRN“ would trigger RN“i. The concept of RN“i-plant mediated pest control was demonstrated in by the development of transgenic plants producing dsRN“s against specific insect genes, with the consequent effect on the target species [ , ]. The main prerequisites to generate successful RN“i insect-resistant transgenic plants are: i identification of a specific gene with an essential function in the insect that can cause developmental deformities and/or larval lethality when knocked down or knocked out and ii dsRN“ delivery by oral ingestion that must be uptaken by the insect cells, and spread systemically. The insect must uptake the dsRN“ version of a target gene region by feeding. To silence the target gene, this specific dsRN“ must be taken up from the gut lumen into the gut cells as what is considered as environmental RN“i. If the target gene is expressed in a tissue distinct from the digestive system, the silencing signal should successfully spread via cells and tissues as a systemic RN“i. ”oth environmental and systemic RN“i are considered noncell-autonomous RN“i, which means that the interfering effect takes places in tissues/cells different from the location of application or production of the dsRN“. Conversely, in the cell-autonomous RN“i, the silencing process is limited to the cell in which the dsRN“ is introduced [ ]. However, the mechanism of ingested dsRN“ uptake and systemic spreading of the silencing signal in the insect have yet to be fully characterized and understood. Some factors can affect the efficiency of the dsRN“ uptake and systemic silencing spread in different insects. Here, we highlight important points that must be considered in developing an RN“i approach against insect pests. . . Target gene The choice of the target gene should be carefully considered. Each gene requires particular effort to be silenced. Terenius and colleagues [ ] reviewed more than RN“i experimental

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results from RN“i of lepidopterans involving genes, from which only % were silenced at a satisfactory level, while % failed to be silenced, and % were silenced at insufficient levels. “mong the target genes, those involved in immunity were more effectively silenced, and, in contrast, genes expressed in epidermal tissues seem to be most difficult. Differences for RN“i sensitivity among genes in the same tissue was described in [ ]. . . dsRNA design The design of the dsRN“ determines the one particular target gene to be silenced, but offtarget effects can occur if siRN“s have some sequence similarity with unintended genes. Tobacco plants expressing Helicoverpa armigera ecdysone receptor EcR dsRN“ improved resistance to another insect, Spodoptera frugiperda, due to the high identity shared between the nucleotide sequence of HaEcR and SeEcR genes [ ]. “lthough this result implies that an RN“iplant can control two or even more lepidopteran pests, this can also affect nontarget insects, becoming a biosafety issue. . . dsRNA length The length of the dsRN“ fragments plays an essential role in the effectiveness of molecular uptake in insects, which is directly involved in the success of the target gene silencing. In most of the RN“i experiments, the insects are fed with long dsRN“s [ ]. Some experiments showed that long dsRN“s are more efficiently uptaken than siRN“s [ , ]. This may be due to the fact that a long dsRN“, with % match of the target mRN“, after processing into siRN“ will provide a greater diversity of siRN“s available to cause specific suppression of target gene and increase the desired effect, and, additionally, reduce the likelihood of developing resist‐ ance [ ]. In contrast, other studies reported suppression of genes in different insects via incorporation of siRN“ in diet instead of dsRN“ [ , ]. . . dsRNA concentration Optimal concentration of dsRN“ delivered to the insect is required to induce sufficient gene target silencing. It is noteworthy to mention that exceeding the optimal dsRN“ concentration may not result in more silencing [ , ]. However, higher concentration of dsRN“ decreased the duration of dsRN“ exposure to reach % mortality of Diabrotica virgifera virgifera [ ], suggesting an inverse relationship between dsRN“ concentration and duration of exposure. In cricket Gryllus bimaculatus , the highest concentrations of dsRN“ yielded more efficient gene knockdown [ ]. The amount of dsRN“ sufficient to significantly reduce mRN“ levels of PER and CLK genes were one and two µM, respectively. These concentrations reduced the expression level of the targets, but a higher concentration µM for the CYC gene was required, suggesting that the sensitivity to dsRN“s also depends specifically on the gene [ ]. . . Controls Empty vector, empty cassette, buffer only, irrelevant or nonspecific control such as dsGFP – Green Fluorescent Protein gene region , or any other kind of negative control are essential to

Management of Insect Pest by RNAi — A New Tool for Crop Protection http://dx.doi.org/10.5772/61807

discriminate specific gene silencing from the simple induction of siRN“ processing machinery by exposure to a dsRN“. Mainly, a negative control should demonstrate the specificity of the dsRN“ designed for a target, not interfering in specific target expression, and even unspecific effects. “lso, any control should have similar size and concentration of the used dsRN“ [ ]. . . Molecular silencing confirmation “n efficient molecular confirmation of the RN“i silencing should be conducted, which includes target RN“ expression, and analyses of protein amount and/or enzyme activity. In RN“ analysis, additional care should be taken for expression analysis. The method of choice for RN“ expression analysis is the quantitative amplification of reversed transcripts or RTqPCR, considered a very sensitive and accurate method. To provide precision in RT-qPCR, some essential care is required, such as the choice of appropriate stable reference genes and primer pair design with sufficient amplification efficiency. The reference genes should exhibit stable expression among experimental conditions, providing reliable estimate of gene expres‐ sion results [ ]. “dditionally, primers should be designed flanking the region used to design the dsRN“ to ensure that the initial cleavage of the mRN“ could be detected, thus avoiding false-positives [ ]. Conventional care of RT–qPCR reactions defined by the Minimum Information for Publication of Quantitative Real-Time PCR Experiments MIQE guidelines must be strictly followed [ ]. . . Protein stability and phenotype analysis Proteins can exhibit a long half-life and interfere with the phenotypic changes. However, phenotype changes are not totally related to small decrease of protein levels haplo-sufficient genes produce proteins capable of performing the biological processes normally, even at half of the protein levels [ ]. Phenotype changes could be more difficult to be observed in RN“i responses if the protein product of the target gene has a long half-life. For example, the reduction of Da6 nicotinic acetylcholine receptor subunit expression in both D. melanogaster and T. castaneum exhibited weak phenotype responses [ ], which may be explained based on the long stability of the nicotinic acetylcholine receptors n“ChRs [ ]. Nevertheless, for most of the genes, mRN“ turnover and protein half-life are unknown and this lack of information presents one of the principal challenges for the RN“i experiments [ ]. . . Insect issues, life stage, nucleases, and gut pH Some insect characteristics should also be considered before starting an RN“i experiment including the developmental stage of insects. “lthough handling advanced developmental stages of insects is more efficient, silencing effects are more prominent in earlier stages. For instance, in second instar larvae of Rhodnius prolixus, the gene nitropin 2 was knocked down for %, but no silencing was observed in the fourth instar individuals, even though both larval stages were treated with the same concentration of dsRN“ [ ]. In Spodoptera frugiperda, fifth instar larvae presented higher gene silencing as compared to adult moths [ ].

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“nother consideration that can affect the RN“i silencing efficiency is the presence of insect nucleases and gut pH. For instance, feeding assays with Lygus lineolaris showed no mortality effects because the saliva of L. lineolaris contains dsRN“-degrading activity. Thus, dsRN“ ingested did not result in siRN“ fragments, rather were completely digested to monomers [ ]. “lso, dsRN“ degradation is reduced during molting period and further reduced by starvation in some insects [ ]. The stability of the dsRN“ in the midgut could be affected not only by enzymatic but also by chemical hydrolysis [ ]. In both cases, gut pH is an important factor particularly, it is quite variable among insect orders, with variation even among gut regions [ ]. For example, in general, lepidopterans exhibit a strong alkaline gut up to pH . in some species , which provides a highly hostile environment for dsRN“ [ ] therefore, this order is particularly recalcitrant to gene silencing by RN“i.

. Overview on the use of RNAi to control insects by transgenic plants Most of the current transgenic crops with specific control against insect pests are based on Bacillus thuringiensis ”t toxins, which act in gut epithelial cell membrane in susceptible insects. ”t toxins are highly specific against certain orders of insects, where the most successful use was achieved against Lepidoptera and Coleoptera. However, continuous exposure of those insects to ”t crops evolved field-resistance, affecting the efficiency in controlling those pests [ , ]. “lso, there are limitations of ”t transgenics to manage some other important agricultural pests, such as the sap-sucking insects Hemiptera . This encouraged the development of new strategies to help in controlling agricultural pests. In , two studies demonstrated the concept of plants expressing dsRN“s derived from hairpin vectors that directed dsRN“s to target gene regions of economically important agricultural pests: the cotton bollworm Helicoverpa armigera Lepidoptera [ ] and the Western corn rootworm WCR Diabrotica virgifera virgifera Coleoptera [ ] . “fter the demonstration of plants resistant to insects, the application of RN“i by transgenic plants became a potential new approach to control important agricultural pests, which led to the flourishing of a new field of research. So far, searches on the publication database Web of Science from Thomson Reuters “ugust , identified published studies based on the combination of the topics RN“i, plant and insect, and only one quarter was published before . To implement RN“i in agricultural pest control, the target insect should uptake the dsRN“ autonomously, e.g., from transgenic plants expressing dsRN“. This feeding should be continuous, since insects lack an amplification mechanism based on RdRP, such as C. elegans. Many of the main agricultural pest species have already been targeted by RN“i technology using various genes and delivery methods [ ]. However, three orders have been the major focus of the development of transgenic plants expressing target gene regions for RN“i: Coleoptera, Hemiptera, and Lepidoptera. Here, we list some of the most recently published RN“i transgenic plants studies performed against those insect orders Table .

Management of Insect Pest by RNAi — A New Tool for Crop Protection http://dx.doi.org/10.5772/61807

Specie

Order

Crop

Target Gene

Remarks

Reference

Diabrotica v. virgifera

Coleoptera

Zea mays

vATPase

Mortality

[

]

Leptinotarsa decemlineata Coleoptera

Solanum

β-actin, Shrub

Mortality

[

]

Molting defect and [

]

tuberosum Helicoverpa armigera

Lepidoptera

Nicotiana tabacum Nuclear receptor

Spodoptera exigua

complex of

-

larval lethality

hydroxyecdysone HaEcR Helicoverpa armigera

Helicoverpa armigera

Lepidoptera

Lepidoptera

Nicotiana tabacum Molt-regulating

Arabidopsis

Developmental

[

]

[

]

Progeny reduced

[

]

Progeny reduced

[

]

Inhibited

[

]

[

]

transcription factor

deformities and

gene HR3

larval lethality

HaAK

Developmental

thaliana

Deformities and larval lethality

Myzus persicae

Hemiptera

Arabidopsis

MpC

1, Rack1

thaliana and Nicotiana benthamiana Myzus persicae

Hemiptera

Arabidopsis

serine protease

thaliana Myzus persicae

Hemiptera

Nicotiana tabacum hunchback hb

reproduction Bemisia tabaci

Hemiptera

Nicotiana rustica

vATPase

Mortality

Table . Overview of the recently published studies on the use of plant-RN“i against different insect pests

. . Coleoptera The coleopterans are likely to be the first target to be controlled by the new generation of transgenics, the RN“i-plants. Diabrotica virgifera virgifera Western corn rootworm, WCR is one of the most important agricultural pests, and this species, along with other coleopterans, requires little effort to have genes silenced by RN“i, independent of the delivery method and gene target. The significant breakthrough was demonstrated when WCR presented significant larval stunting and mortality, causing less injury to maize roots that express a hairpin version of V-ATPase A [ ]. Since then, many studies have been conducted using various target genes, while not focusing only on pest control, but also to characterize the mechanism of action of the RN“i [ ]. “ recent study with WCR demonstrated that long dsRN“s of Dv V-ATPase C expressed in maize provides highly efficient root protection, but the siRN“ population generated in the transgenic plant does not lead to lethal RN“i responses when consumed by the insect [ ].

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Studies have also been performed in other species, such as the important potato pest Leptino‐ tarsa decemlineata Colorado potato beetle, CP” . So far, this is the first study to compare efficacy at controlling pest insects by dsRN“s expressed either in the chloroplast or in the cytoplasm. Transgenic potato plants expressing hairpin versions of β-actin and Shrub genes both insectidal dsRN“s in the chloroplasts transplastomic plants conferred the most potent insecticidal activity insects died after days , while the conventional expression from nuclear transgenics did not affect the beetles [ ]. “n explanation for this result is that since choroplasts do not have cellular RN“i machinery [ ], the dsRN“s produced inside these organelles are not cleaved by a plant Dicer and the beetles ingest almost entirely long dsRN“. In contrast, beetles fed on nuclear-transformed plants consumed mostly siRN“s previous dsRN“-feeding studies already indicated that ingested long dsRN“s were much more effective than ingested siRN“s. It should be high‐ lighted that all the other studies have been based on an efficient cytoplasm-derived dsRN“ in various crops, indicating that possibly potato plants process long dsRN“ more effective than the plants from those other studies Table [ ] . . . Lepidoptera Plants producing Bt proteins were the first generation of transgenic plants to control insects, and most of the lepidopterans were successfully managed by ”t crops for years. However, the durability of ”t technology appears to be unsure. The number of pest species that evolved ”t resistance in the field, reducing transgenic efficacy, increased from one in to five in [ ]. “mong those five species, four are lepidopterans [ ]. The lepidopterans could be the first and main targets for RN“i crops if they were not as recalcitrant to gene silencing, without any definite explanation for the limited and unstable RN“i responses [ , ]. For instance, the concentration of dsRN“ necessary to knock down a specific gene by feeding Diatraea saccha‐ ralis Lepidoptera neonate larvae is much higher than the one required for the same larval stage of D. v. virgifera Coleoptera . The first successful RN“i plant protected against a lepidopteran Helicoverpa armigera was demonstrated by silencing the CYP6AE14 gene, necessary for detoxifying gossypol from cotton Gossypum hirsutum [ ]. Studies have been conducted to explore alternative target genes to control and understand the RN“i mechanism in lepiopterans. Larval lethality and molting defects were detected in H. armigera fed with transgenic tobacco plants expressing dsRN“ targeted to the nuclear ecdysone receptor complex HaEcR , absolutely required for insect development [ ]. The transgenic tobacco expressing dsRN“ of HaEcR had an improved resistance to another lepidopteran pest, Spodoptera exigua. This cross-species effect might indicate a risk to affect nontarget insects, highlighting the importance of biosafety studies that should be carefully conducted [ ]. The expression of dsRN“ in both Escherichia coli and transgenic tobacco plants to silence a molt-regulating transcription factor gene HR3 of H. armigera resulted in developmental deformity and larval lethality [ ]. Transgenic Arabidopsis thaliana plants expressing dsRN“ targeted to arginine kinase AK of H. armigera HaAK led to a % mortality rate in first instar larvae, while retarding growth in surviving larvae [ ]. However, no lethal phenotypes were

Management of Insect Pest by RNAi — A New Tool for Crop Protection http://dx.doi.org/10.5772/61807

observed for the third instar larvae, although transcript levels of HaAK were distinctly suppressed [ ]. . . Hemiptera Hemipterans are characterized as piercing/sucking insects, representing major agricultural pests that inflict direct damages by sucking sap, or indirectly by acting as a vector of several viruses and bacterial infections. Since hemipterans feed through sucking the phloem, only systemic chemical insecticides are effective against these insects, resulting in high residual toxicity. The problem is further aggravated as no ”t toxin has been identified as exhibiting adequate insecticidal effects against hemipterans. Transgenic crops based on RN“i offer a large potential to control hemipteran, requiring expression of target gene dsRN“s on the phloem. One first report was published in about developing transgenic Nicotiana benthamiana and A. thaliana expressing dsRN“ targeting genes expressed in Myzus persicae gut RACK1 and salivary glands MpC 2 [ ]. “ reduction of the expression level of the target genes and a decrease in M. persicae fecundity were observed, but no lethal effects [ ]. The same phenotype was observed when M. persicae fed on A. thaliana expressing dsRN“ of a serine protease gene MySP , with no lethal effects, but reduced fecundity [ ]. Once again, reduced reproduction, but no lethal phenotype was observed when M. persicae was fed on tobacco plants expressing dsRN“ targeting the hunchback gene [ ]. “ possible explanation for not achieving the expected phenotypes mortality after target gene depletion is that the dsRN“ level produced by RN“i transgenic plants could not be sufficient for an efficient uptake of dsRN“ or siRN“ by sap-sucking insects [ ]. However, more recently, mortality rates were observed in Bemisia tabaci fed in tobacco plants expressing dsRN“ of v-ATPaseA [ ]. This result provides a proof-of-concept that plants expressing dsRN“, at an efficient level and targeting crucial genes, could resist the attack of Hemipteran pests [ ].

. Conclusion and future perspectives Since the concept of a transgenic plant expressing dsRN“ targeted to a specific essential gene in the insect that affects its viability was first demonstrated in , the technology has been extended to a large number of insect species from various orders. Elucidating the various mechanisms and components of the RN“ interference pathway has progressed, but many aspects remain to be clarified. Many differences in components and mechanisms among insect orders and between insects and other organisms still need to be worked out. Some of these differences e.g., genes involved, gene number, and level of expression may explain variation in recalcitrance among insect species and need to be further investigated. Of particular interest are the mechanisms of dsRN“ uptake, signal amplification, and systemic spread in the major pest species. “dditional insect- or order-specific characteristics, such as gut pH, presence of dsRN“-degrading activity in digestive system, among others that could be associated with differences in recalcitrance to RN“i need to be dissected and clarified.

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Due to the variety of RN“i response to RN“i in insects, no single protocol is suitable for all species. Issues related to the choice of effective target genes, including determining the size of optimal dsRN“ length and ideal gene region. “ssuming that the method of choice to deliver dsRN“ is transgenic plants, a major question still to be addressed is the impact of plant dsRN“ processing in the effective RN“i-induced silencing. There is still a need for investigation in this area. The choice of a suitable inducible promoter for expressing the dsRN“ construct is another point barely explored. ”ased on the recent publications reviewed in this chapter, the progress in developing RN“iplants to control important insect pests widely demonstrated the potential of this technology to complement or replace ”t crops, providing resistance against a broad variety of insect pests. However, to be applied on a commercial level, several issues related to the RN“i mechanism and biosafety still need to be addressed. “s a new technology, risk assessments and govern‐ ment regulations still have to be developed. However, RN“i transgenic crops are expected to have wider acceptance and reduced biosafety requirements for RN“i traits, in comparison to a protein incorporated into a plant, such as a ”t transgenic [ ]. Thus, RN“i-mediated pest control will open a new paradigm in insect pest management.

Author details Thais ”arros Rodrigues* and “ntonio Figueira *“ddress all correspondence to: [email protected] Centro de Energia Nuclear na “gricultura CEN“ , Universidade de São Paulo, Piracicaba, SP, ”razil

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] Griebler M, Westerlund S“, Hoffmann KH, Meyering-Vos M. RN“ interference with the allatoregulating neuropeptide genes from the fall armyworm Spodoptera frugiper‐ da and its effects on the JH titer in the hemolymph. J Insect Physiol. : . DOI: . /j.jinsphys. . .

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] Price DRG, Gatehouse J“. RN“i-mediated crop protection against insects. Trends Bio‐ technol. : . DOI: . /j.tibtech. . .

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Chapter 19

RNA Interference – Natural Gene-Based Technology for Highly Specific Pest Control (HiSPeC) Eduardo C. de Andrade and Wayne B. Hunter Additional information is available at the end of the chapter http://dx.doi.org/10.5772/61612

Abstract RN“i technologies are more environmentally friendly, as the technology provides great‐ er specificity in pest targeting, while reducing the potential negative effects on ecosys‐ tems and leaving beneficial insects and other organisms unharmed in crop ecosystems. Consequently, the increase in native fauna improves the efficacy of biological control agents against pests and pathogens. “ growing understanding of the ubiquitous nature of RN“i, along with evidence for efficient, non-transgenic, topical applications has al‐ ready begun to garner support among organic and industry producers. Designing solu‐ tions to agricultural problems based upon the same mechanisms used in nature provides newer, safer solutions to pests and pathogens for all agricultural industries. Keywords: Future, Crops, Organic, Non-transgenic, RN“i

. Introduction “ phenomenon initially reported in plants [ ] called the attention of the scientific community, leading to the discovery of a sophisticated mechanism of gene regulation and protection against invasive nucleic acids [ – ]. Furthermore, the mechanism described in plants, referred to as post-transcriptional gene silencing PTGS , or virus-induced gene silencing VIGS , had been described in the s [ ] and was often referred to as pathogen-derived resistance [ ]. RN“i is a natural process of gene regulation and antiviral defense system of eukaryotic cells. RN“i is a mechanism that functions as a gene silencer by targeting specific RN“ sequences. RN“i results in degradation, and in some situations, translation inhibition, resulting in a reduction or complete elimination of the expression of a targeted RN“ [ ]. RN“i is also linked to suppressing gene expression at transcriptional level by directing epigenetic alterations on chromatin [ ].

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The basic RN“i process consists of the trigger molecule, a long endogenous or exogenous double-strand RN“ dsRN“ molecule that is expressed in, or introduced into, the cell, which is processed by Dicer, a ribonuclease III RNase III enzyme into small RN“ duplexes of – nucleotides in length. These duplexes are separated with one strand the guide strand , producing a protein complex known as the RN“-induced silencing complex RISC . The RISC complex uses the specific sequence of the guide strand to determine potential target messenger RN“s mRN“ . Once bound to the mRN“, the guide strand directs a RISC-bound endonu‐ clease [called slicer , an “rgonaute “GO protein] to cleave the mRN“ which has homology to the guide strand [ ]. Thus, the RISC complex can target the messenger RN“ mRN“ , an invasive virus RN“, or a transposable element transcript Please note that RN“i cannot eliminate transposons itself, but its transcripts. [ ]. These components appear to be cosmo‐ politan in their distribution across the RN“i spectrum of the eukaryotic phyla. This implies that a common ancestor had a functional RN“i pathway [ ], estimated to have occurred over a billion years or more ago [ ]. The generation of virus-resistant transgenic plants, by expressing fragments of viral genomes, was the first demonstration of the beneficial use of the RN“i [ , ]. However, with the demonstration that ingestion of dsRN“s can robustly silence genes in Caenorhabditis elegans [ ], RN“i became not only an important tool in genetic studies to identify gene function but also opened a new field of application in plant protection against insects, arthropods, and patho‐ gens. In this chapter, we outlined some aspects on the development of RN“i-based strategies to control insects, presenting some considerations and research steps that are important to be addressed.

. RNAi-based products for agricultural management of insect pest “dvances in genomics and initiatives to sequence genomes of agriculturally important organisms created big breakthroughs within the entomology fields of study, including taxonomy, insect physiology, toxicology, immunology, pest management, and microbe–host interactions. This new paradigm affected how research could be conducted, to make discov‐ eries and increase the speed by which these could be accomplished. With these breakthroughs, entomologists, pathologists, and biologists are rapidly advancing toward better, safer, and more specific pest and pathogen management. Development of gene-based methods was dependent upon having the knowledge to understand how the cells in a living organism respond to the threats from viral pathogens, how they regulate their own gene expression, and how they maintain these natural complex systems throughout their lives. The initial genomes which were sequenced and annotated to elucidate these very complicated interactions from: the nematode – C. elegans, the fruit fly – Drosophila melanogaster, the red flour beetle – Tribolium castaneum, the silkworm – Bombyx mori, the pea aphid – Acyrthosiphon pisum, the honey bee – Apis mellifera, to bumble bee – Bombus terrestris and including a few more examples from the world of agriculture [ ]. Current genome initiatives, like the i K

RNA Interference – Natural Gene-Based Technology for Highly Specific Pest Control (HiSPeC) http://dx.doi.org/10.5772/61612

“rthropod initiative [ ] and the ”eijing Institute of Genomics, ”GI, China, plan to sequence thousands of arthropod and plant genomes. The enormous amount of new information produced will also increase the understanding of the genetic basis of the mechanisms nature uses to solve problems at the cellular and organismal levels. The number of arthropod species which have had successful RN“i reports continues to increase, and this trend will carry agriculture into the future, covering five Classes, in four Subphyla of the “rthropoda phylum, which includes eight insect orders and over insect species [ – ].

. Pitfalls and solutions – Relevant considerations for development of RNAi in insects Though the use of RN“i strategies to control a desired insect pest seems to be very straight‐ forward however, some issues should be taken into considerations: for oral treatments the dsRN“ must survive ingestion, then be absorbed by the epithelial cells, and depending upon the target translocated through the hemolymph to reach other tissues. In insects, the dsRN“ is mainly introduced through feeding, but dermal application has already been reported to possibly bypass gut issues [ – ] once inside the body, the dsRN“ must enter into the cell to activate the RN“i mechanism. In insects, cell uptake of dsRN“ varies widely between species, because there are different mechanisms of systemic absorption and translocation of dsRN“s within and between cells, respectively, leading to differences in response and influencing the efficiency in silencing the target gene [ , ] once the RN“i mechanisms are triggered in a group of cells, it has been demonstrated that a systemic spread of silencing may also occur, so that other tissues / cells are also affected, which may increase the RN“ effect. Successful studies have shown that dsRN“ can circulate in the hemolymph, and cause a suppression of genes in tissues distant from initial entry sites in the insect gut, affecting cuticle formation, the nervous system, or ovaries[ – ]. However, in insects results can be highly variable and research efforts continue to elucidate the effects of systemic signaling. In theory, any cellular mRN“ can be inactivated in a precise and controlled manner. With this in mind, the use of the RN“i mechanism to manage an insect pest relies on the capacity to design the dsRN“. The sequence of the dsRN“ provides specificity and the researcher must determine the active concentration needed to obtain the desired RN“i outcome thus, proper design and evaluation of the dsRN“ becomes critical. Identification of vitally important i.e., with high mortality target genes of a particular insect is a crucial step toward development of RN“i-based control strategy. Thanks to world science development and increasing efforts of the research community, the identification of an essential target can be achieved by an extended literature search and analyses of available DN“/RN“ sequence databases [ ]. Once identified, candidate target sequences are used to design potent RN“i causing structures . Then, the dsRN“s must be experimentally validated for functionality, specificity, and stability, toward the specific RN“i target of interest. Furthermore, the development of an efficient delivery system for RN“i causing structures

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is another key step. There are several methods available that include, but are not limited to: microinjection [ ], soaking for mosquito larvae, and nematodes [ , ], and feeding chewing and piercing-sucking insects [ , , , , ]. One approach to identify potential target genes which will function under field conditions is to perform bioassays that closely mimic the conditions in the field, for example, using a bioassay that mimics the feeding of a hemipteran insect acquiring the dsRN“ during the natural feeding process performed on the crop plant. One problem with delivering dsRN“ through feeding is that, depending on the bioassay, it may be difficult to measure the dose of dsRN“ ingested, from the dose absorbed by the gut cells and the target cells [ ]. Oral delivery of dsRN“ through feeding can be performed by using artificial diet, detached plant parts leaves, buds, roots , or intact plants [ , , , ]. Delivering dsRN“ through the diet provides an easy procedure to screen large numbers of dsRN“s in insect larvae and adults [ , , , ]. In addition, it allows addressing different issues, such as effective length of dsRN“, determine regions of the gene to be target which may provide better suppression, and to determine the effective lethal concentration LC [ , ]. “lthough oral feeding provides a more natural screening system, it is important to take into consideration that for some insect species from across all taxonomic orders, they may not provide an effective RN“i response when conducting oral feeding bioassays regardless of the dosage of dsRN“, as the dsRN“ may not enter, or be detected in the insect’s body [ ]. In contrast, in these same insects when dsRN“ was injected directly into the insect’s body, a potent RN“i response was observed [ , , ]. Indeed, lack of positive results using feeding bioassays does not necessarily indicate that the insect is insensitive to RN“i, but in a majority of the situations, insects have nucleases in the saliva, in the midgut, or even in the hemolymph that degrades dsRN“ before it can be absorbed by the cells [ – ]. Wynant et al. [ ] discussed the interactions of enzymes and RN“i-causing dsRN“s in the alimentary tract and hemolymph of insects and other arthropods using oral delivery. The elucidation of the roles of microbes and host enzymes on RN“i efficacy across arthropod species continues to be a challenging field of research [ ]. Where information about absorption of dsRN“ or presence of nucleases is not available for a particular insect species, the use of reporter dsRN“s molecules are useful to clarify possible issues. It is essential that the dsRN“s sequence should not match with any insect’s transcript sequence. The dsRN“ movement can be monitored detected , or quantified in the insect’s body by RT-qPCR, showing that the insect has acquired the potent, fully functional, systemi‐ cally spreading dsRN“ during feeding plant, diet, drop of water, etc. Figure . When conducting a RT-PCR detection of reporter dsRN“ after insect feeding, it is important to sample a tissue other than the gut such as the hemolymph, fat body, or ovary. Careful collection of tissues which are not in direct contact with the gut provides evidence that the dsRN“ was truly absorbed by the cells, and you are not just detecting dsRN“ just in the digestive tract. With small insects, as the “sian Citrus Psyllid “CP Diaphorina citri whitefly or aphids , collection of material can be difficult without bringing in gut tissues, so another option is to

RNA Interference – Natural Gene-Based Technology for Highly Specific Pest Control (HiSPeC) http://dx.doi.org/10.5772/61612

Figure . Topically applied RN“, provides long dsRN“s for insect ingestion. If absorbed into the plant, then long dsRN“ persists in plant vascular tissues, xylem, and phloem, for several weeks due to lower metabolic activity, fewer enzymes, and microbes. Once the dsRN“ enters a plant or insect cell, the RN“i mechanism is triggered, and the dsRN“ processed [Image: Hunter, W.”., USD“-“RS, ].

let the insects feed on the source of the dsRN“ plant, diet, etc. for a period of – h, then transfer them to an untreated food source. “fter feeding for h, or more, the insect should excrete any food residue from the treated food source, which contains dsRN“. “fter this period, proceed with sample collection for dsRN“ detection. The reporter dsRN“ is designed so that the sequence does not match with any known mRN“ transcript in your insect. This is to avoid off target of other transcripts in the insect. Some commonly used dsRN“s which are used as negative controls in RN“i experiments are: green fluorescent protein GFP , β-glucuronidase GUS , and enhanced yellow fluorescent protein EYFP [ ]. When designing RN“i experiments, important questions arise regarding the design of dsRN“, including: the length of the molecule and the region targeted within the mRN“. The minimal required length to achieve an RN“i effect will vary depending on insect species [ ]. For example, in Tribolium castaneum, analysis showed that the dsRN“ length had a strong influence on the efficacy of the RN“i response, with longer dsRN“ proven to be more effective on curtailing gene expression [ ]. In T. castaneum dsRN“, it was observed that length was crucial for cellular uptake a minimum of nucleotides were necessary to achieve the desired interference. However, other studies in the potato/tomato psyllid, Bactericera cockerelli [ ], the pea aphid Acyrthosiphon pisum [ ], and the lepidopteran Manduca sexta [ ] have reported gene suppression using shorter dsRN“s, between and nucleotides in length. These molecules are called small interfering RN“s siRN“ . Overall, the majority of studies dem‐

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onstrate success with dsRN“ ranging from to nucleotides in length. Interestingly, Huvenne and Smagghe [ ] reported success using a dsRN“ , nucleotides in length. dsRN“s longer than nucleotides provide the advantage of resulting in many siRN“s, postcleavage against the targeted mRN“, thus maximizing the RN“i response and preventing the development of individuals with resistance due to the natural genetic variation. There is no consensus on the mRN“ region that the dsRN“ should match to e.g., ′ or ′ . For example, in the pea aphid, A. pisum, no difference in mortality was observed in groups of insects that received dsRN“ matching the ′ or ′ end of the hunchback hb gene [ ]. In the mosquito, Aedes aegypti greater RN“i effects were achieved when insect larvae ingested dsRN“ targeting the ′ end versus ′ end of an apoptosis gene, AaeIAP1 [ ]. These different results highlight the need to screen several dsRN“ molecules across the entire mRN“ [ ]. In the context of field applications of RN“i for insect management, dsRN“s can be designed to be highly specific to both the target gene and the insect species. If desired, the RN“s can be designed to have a broader spectrum to affect several pest species. For example, RN“i strategies can be designed to remove one aphid species from a cropping system, or be designed to remove multiple aphid species from that same ecosystem [ , ].

. Bioassays for dsRNA screening For RN“i research attempting the development of a viable pest management product. It is of utmost importance to identify the best delivery mechanisms i.e., topical sprays, baits, or transgenic plants as early as possible this will expedite the entire process and can cut years off of the development and commercialization timeline. The example outlined below highlights RN“i bioassays directed toward two citrus insect pests, each one with different feeding behaviors: piercing-sucking plant-feeding the “sian citrus psyllid Diaphorina citri, Hemiptera and a chewing beetle pest the weevil Diaprepes abbreviatus, Coleoptera . In both situations, the bioassays were designed to evaluate the efficacy of oral ingestion of dsRN“s under natural feeding conditions which mimic conditions the insects will encounter in the field. . . Bioassays for piercing-sucking insects The artificial feeding bioassay is being widely used for studies on insect nutrition, pathogen acquisition, toxicity, and RN“i Figure “ [ – ]. It is notable that liquid feeding bioassays dsRN“s mixed in a liquid diet or a sucrose solution frequently result in high mortality levels in the controls, and increased degradation of dsRN“ in the solution due to bacterial or fungal contaminations [ , ]. In addition, these bioassays require significantly high dsRN“ concentrations to achieve insect mortality. Concentrations up to µg/µL [ – ] cannot be reproduced inside plant vascular tissues. Hemipteran pests in citrus psyllids, leafhoppers, aphids, whiteflies have piercing-sucking mouthparts that are inserted into the plant vascular system to feed. The development of an

RNA Interference – Natural Gene-Based Technology for Highly Specific Pest Control (HiSPeC) http://dx.doi.org/10.5772/61612

Figure . RN“i feeding assays used at the USD“ lab, “RS, Ft. Pierce, FL , to successfully screen dsRN“ constructs rapidly and under controlled conditions: “ sucrose solutions within artificial membranes ng dsRN“/µL [ ], ” rooted okra plant cuttings, C citrus flush ng to ng dsRN“/ . g plant tissue . Plant cut‐ tings absorb dsRN“ directly providing systemic movement of dsRN“, D insects are given a feeding access period to ingest the dsRN“ across days to observe mortality Hunter and “ndrade, USD“-“RS [ – ].

RN“i control strategy against these insects relies on effective delivery of the dsRN“ through the vascular tissues. Demonstration of the first dsRN“ delivery into full-sized citrus trees and grapevines, without a delivery vector, expression vector, or transformation event was performed in [ ]. These results showed that two hemipteran insects, the xylem-feeding leafhopper H. vitripennis , and the phloem-feeding “sian citrus psyllid D. citri , tested positive for ingested dsRN“ after feeding from host plants treated with dsRN“ as either a foliar spray or root drench. These results along with other studies that demonstrated successful RN“i through oral ingestion in insects [ , , , , ] support the potential for exogenously delivered RN“i control strat‐ egies. Use of cut plant feeding bioassays for hemipteran pests enables the screening of a large number of dsRN“s molecules at a reduced cost of materials and time. The bioassay, can use leaf disks, whole leaf, new growth leaves and stem, or rooted cuttings, to absorb and deliver dsRN“s. In citrus, the flush , which are new growth foliar shoots, are collected from potted citrus seedlings grown in a glasshouse USD“-“RS, Fort Pierce, FL . The leaves and stem material are about – cm long. The plant material is washed in . % bleach water, for min. Then the base of each stem is cut at a degree angle while submerged in filtered water. The material is then placed into a . mL tube containing . mL water Figure ” and C . The dsRN“ solution, µL, is added to the water, the tube top is wrapped with plastic or Parafilm™ and placed under artificial lighting to stimulate absorption of dsRN“ solution. The next day the tube is filled with water using a gauge syringe needle and syringed filtered . µ . The treated cuttings are then placed into a cage and adult insects provided feeding access for days Figure D . The plant material can remain viable for up to days on average. While most bioassays may terminate after eight to days of observations for mortality, having a

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longer feeding access time enables observations on insect oviposition, egg viability, or nymph development. Each dsRN“ molecules has an optimal concentration. So each dsRN“ molecule is evaluated across a range of total concentrations i.e., , , , nanograms/ tissue . The bioassay permits screening for synergistic effects of multiple dsRN“s and to screen a single dsRN“ against multiple insect species. For example, the assay using citrus flush permits screening of dsRN“s designed against psyllids for off target effects in the citrus aphid Toxoptera citrici‐ dus and glassy-winged sharpshooter leafhopper Homalodisca vitripennis Germar , two closely related hemipterans, which also use citrus trees as a host plant. . . Bioassays for chewing insects For insects which are foliage feeders, the delivery of dsRN“ can be achieved as a foliar topical spray. In this scenario, the dsRN“s are evaluated similarly as topical insecticides. The dsRN“ solution is sprayed on leaves, and then fed to the insects. “n example of the effectiveness of this approach was reported by ”olognesi [ ] working with the coleopteran Diabrotica virgifera, in which dsRN“ administered through feeding, silenced genes in tissues far from the gut epithelium. Similar results were obtained while developing an RN“i strategy against the Diaprepes root weevil DRW , Diaprepes abbreviatus L., Curculionidae: Coleoptera “ndrade and Hunter . The adults feed and oviposition on mature citrus leaves. Topically applied dsRN“ was sprayed on a bouquet of citrus leaves for delivery to DRW adults Figure “ . RN“’s have been shown to move through the plant xylem and phloem [ ].

Figure . Citrus leaf bouquet” feeding bioassay for Diaprepes root weevil. A Fresh stems with leaves are washed in . % bleach water, rinsed with Nanopure™ filtered water, then the stems are freshly cut while submerged in water. The leaf bouquets are placed in water-filled containers and placed under artificial lighting for h prior to use. B Topical foliar treatment: the dsRN“ is mixed with water and applied using a low-volume aerosol sprayer. C “fter the leaves dry, the bouquets are caged with adult insects.

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“ test spray using only water established the volume needed to provide full coverage of leaf bouquets without excessive run off Figure ” . “fter the leaves have dried, they are caged with adult insects Figure C . Freshly treated citrus leaf bouquets replaced previous bouquets every five to seven days for a -week period. The total amount of dsRN“ to be sprayed over the leaf bouquet was determined by evaluating a range of concentrations in a pretest experi‐ ment for efficacy. The effects from RN“i in insects usually start to appear within to days post-ingestion, which suggests there may be a dose response [ ]. Since foliage feeding insects tend to eat a lot of leaf material each day, a low-dose spray may be able to deliver a significant amount of dsRN“.

. Final considerations on RNAi applied to agriculture Efficient delivery and increased stability of dsRN“ need to be developed if non-transgenic, topically delivered, RN“i strategies are to be established. Increased stability and superior delivery into some insects can be achieved using nanoparticle-mediated RN“i [ – ], traditional crop improvement strategies, in which plants express hairpin dsRN“s, will continue to be a mainstay of agricultural approaches [ , , ]. Transgenic plants have successfully used RN“i strategies to produce crops with improved virus resistance, increased nutrition and fiber content [ ] biotechnology companies are trying to move towards a faster, more natural process of topically applied RN“i. dsRN“ molecules are part of naturally occurring processes in all living organisms. They exist in our foods, and our bodies [ ]. The short persistence time of dsRN“ in the environment is demonstrated by the fact that analyses of soils and plant debris, treated with dsRN“ have consistently shown rapid breakdown of dsRN“s within – days [ ], also means less concerns about unintended contamination of water supplies, soils, or adverse air quality effects. Furthermore, since all living things have evolved to break down dsRN“ and use the nucleic acids as cellular nutrients, this technology will be safer than conventional chemistries for those who apply RN“i products, or eat the produce [ – ]. RN“i technologies have greater specificity in pest targeting, which reduces negative impacts on crop ecosystems by leaving more insects and other organisms unharmed in the field. The increased fauna consequently improves the efficacy of pollination, and biological control agents that help suppress a broad range of pests. The increased understanding of the ubiqui‐ tous nature of RN“i, along with evidence of efficient topical application, has already begun to garner support for this technology among members of the organic grower’s communities, which desperately need a truly natural, innovative breakthrough, to manage many of the pests and pathogens which plague the organic industries. . . Cost-effective methods for the mass production and formulation of dsRNA Cost-efficient methods for mass production of vast amounts of dsRN“ are being developed, and include bacterial, plant, and synthetic production [ ]. While small amounts of dsRN“ can be easily produced in the laboratory for research purposes, commercially available kits are

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not a viable, cost-effective method for the production of large quantities of dsRN“ [ ]. The costs associated with the commercialization and implementation of RN“i products are decreasing rapidly. The costs of dsRN“ production have dropped from $ , USD for g in to less than $ , USD for g today. For example, see [ ]. “s interests in commer‐ cialization of RN“i-based products increase, better production systems will be developed to meet the predicted demands of these growing markets [ , , ]. One of the most cost-effective methods for production is in bacteria, since for most countries bacteria-produced dsRN“ would provide an affordable production system which could advance RN“i as, The novel biological insecticide of the future! Most agricultural companies interested in the future of RN“i are working on developing their own technologies that will further reduce production costs predicted to be near $ USD per one gram by the end of . For example, see [ ]. . . Other applications Future applications of RN“i and other gene-based targeting biotechnologies will add value to existing beneficial insects pollinators, predators, parasitoids . “ real-world example is a study conducted over several years in which an RN“i product designed to reduce Israeli acute paralysis virus replication was fed to honey bees. The treated bees had significantly greater survival and produced significantly more honey [ ]. RN“i strategies have also reduced honey bee parasites, like Varroa mites [ , ], and internal microsporidian parasites [ ] without deleterious effects to the bees. The highly specific nature of RN“i approaches can be exploited to reduce pests with no harmful effects on non-target species. The use of RN“i in combination with beneficial pollinators and natural enemies has the potential to raise the level of all pest management efforts [ ]. ”iotechnology has demonstrated the safe production of plants which are more nutritious, less toxic, more resistant to drought, and more efficient for biofuel production [ , ]. RN“i has already been successfully used to produce crops which are virus- and drought-resistant [ ]. However, plants expressing dsRN“s while stable and safe take years to develop and millions of dollars to commercialize [ ]. Development of topically applied RN“i, which is a nontransgenic approach improves crop traits and provides a major step forward for environmen‐ tally sound crop management [ , ].

. Conclusions “s more insects and mites develop chemical resistance to one or more insecticides, now estimated to be over species with resistance to one or more products [ ], it is imperative that new types of pest control are developed. The public would like the world to be filled with environmentally friendly technologies, safe for human and animal consumption, technologies which are safe for use around animals and beneficial insects, safe for each type of ecosystem, forest, field, crop, or backyard, a technology that will not endanger water or food quality, a more natural solution, with a natural approach toward problem-solving. So enters RN“ interference!

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RN“ interference, or gene silencing, is a way to reduce specific mRN“s so that a particu‐ lar protein is either not made or it is reduced. The RN“i mechanism is a natural one which occurs in the cells of humans, animals, insects, and plants, and appears to have evolved as a primary defense system against virus replication [ ]. “ndrew Fire and Craig Mello, won the Nobel Prize in [ ] for explaining how the RN“i mechanism is triggered, when a cell encounters double-stranded RN“, and how this could be used to benefit humanity. Humanity’s greatest discoveries have come from observing the natural world while RN“i will not solve every problem, it certainly can help improve plant health, reduce insect pests and pathogens [ , , , ]. Some of the benefits from developing RN“i as topically applied products are: The rapid degradation of the molecules ensures low environmen‐ tal risks. “ll cells have the capacity to degrade dsRN“, and the salvage pathways to recycle these bases and nucleosides to form new nucleotides. Thus, cells constantly breaking down DN“ and RN“ into recycled nucleotides [ ]. Topical RN“i applications do not insert genes, so do not produce proteins. RN“i reduces the expression of the targeted proteins, a modulation effect of the natural system. RN“i can be designed and tested faster in about – years than producing transgenic crops, which can take to years and cost hundreds of millions of dollars [ ]. Finally, RN“i strategies as topical sprays would, for the first time, be able to remove one or two closely related insect species, while leaving all the other insects unharmed [ ]. The ability to design RN“i as highly specific pest control will finally provide relief to biological control agents and beneficial insects [ , , ], significantly improving integrated pest management programs. The advantages and promises from RN“i technology sound amazing. However, serious efforts in outreach and education are needed to better inform the different stake holders including the general public, and agricultural industry, leaders as well as decision makers in the regulatory and political communities to help expedite the release and adoption of RN“i products and technology.

. Disclaimer Mention of trade names or commercial products herein is solely for the purpose of providing specific information and does not imply recommendation or endorsement, to the exclusion of other similar products or services by the U.S. Department of “griculture. USD“ is an equal opportunity provider and employer.

Acknowledgements We thank Dr. Xiomara Sinisterra-Hunter, “gTec, LLC, Plant ”iotechnology Consultant, Port St. Lucie, FL, for critical reviews of the manuscript, and Maria Gonzalez, ”iological science technician, USD“, Fort Pierce, FL, for technical assistance.

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Author details Eduardo C. de “ndrade and Wayne ”. Hunter *“ddress all correspondence to: [email protected] Embrapa Cassava and Fruits, Rua Embrapa, s/n, Cruz das “lmas,”“, ”rasil USD“, “RS, U.S. Horticultural Research Laboratory, Fort Pierce, FL, US“

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] Zhou X, Wheeler MM, Oi FM, Scharf ME. RN“ interference in the termite Reticuli‐ termes flavipes through ingestion of double-stranded RN“. Insect ”iochem Molecul ”iol : – . DOI: . /j.ibmb. . . .

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Chapter 20

RNA Interference – A Powerful Functional Analysis Tool for Studying Tick Biology and its Control Remil Linggatong Galay, Rika Umemiya-Shirafuji, Masami Mochizuki, Kozo Fujisaki and Tetsuya Tanaka Additional information is available at the end of the chapter http://dx.doi.org/10.5772/61577

Abstract Ticks “cari: Ixodida are blood-sucking arthropods globally recognized as vectors of nu‐ merous diseases. They are primarily responsible for the transmission of various patho‐ gens, including viruses, rickettsiae, and blood parasites of animals. Ticks are second to mosquitoes in terms of disease transmission to humans. The continuous emergence of tick-borne diseases and acaricide resistance of ticks necessitates the development of new and more effective control agents and strategies therefore, understanding of different as‐ pects of tick biology and their interaction with pathogens is very crucial in developing effective control strategies. RN“ interference RN“i has been widely used in the area of tick research as a versatile reverse genetic tool to elucidate the functions of various tick proteins. During the past decade, numerous studies on ticks utilized RN“i to evaluate potentially key tick proteins involved in blood feeding, reproduction, evasion of host im‐ mune response, interaction with pathogens, and pathogen transmission that may be tar‐ geted for tick and pathogen control. This chapter reviewed the application of RN“i in tick research over the past decade, focusing on the impact of this technique in the ad‐ vancement of knowledge on tick and pathogen biology. Keywords: “cari, ticks, Ixodidae, RN“ interference, tick-borne diseases

. Introduction Ticks belong to the class of “rachnida together with spiders, scorpions, and mites. To date, there are about species of ticks, majority of which are hard ticks belonging to the Ixodidae family, as well as about species are soft ticks belonging to the “rgasidae family, and a single species belonging to the Nuttalliellidae family [ ]. Most of the ticks of medical and veterinary importance are hard ticks. Through their blood-feeding behavior, ticks can directly

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affect their host by causing anemia, irritation, and allergic reactions particularly in heavy infestation. The saliva of some tick species may also contain neurotoxic substances that may cause the condition termed tick paralysis [ ]. “dditionally, the transmission of pathogens including viruses, bacteria, and parasitic protozoa also occurs during blood feeding [ ]. Ticks are considered second to mosquitoes in terms of their impact on public health, but they are the most important vectors of different pathogens in both domestic and wild animals [ ]. Tick infestation and tick-borne diseases T”Ds continue to have great economic impact on livestock production, particularly on cattle and small ruminants, in several continents [ ]. The annual loss in cattle production worldwide due to ticks and T”Ds has been estimated to be worth billions of USD [ ]. The complete dependence of ticks to host blood for the completion of their life cycle and generation of offspring is the reason for their notoriety as vectors of several diseases. Depend‐ ing on the species, a tick may utilize one to three hosts during their life cycle. Most of the pathogens they transmit can be carried on throughout their life cycle through transstadial from one stage to the next transmission and to the next generation through transovarial from adults to eggs transmission [ ]. “ single tick may carry multiple pathogens [ ], thereby having the potential of infecting a host with a cocktail of pathogens. Most tick-borne infections are zoonotic in nature, and more of these are being recognized in recent years [ , ]. “mong the T”Ds that are well-known in the veterinary and medical field are anaplasmosis, borreliosis, rickettsiosis, ehrlichiosis, babesiosis, theileriosis, and tick-borne encephalitis. The significant impact of ticks and T”Ds underscores the importance of tick control. For several decades, the application of chemical acaricides has been the primary tick control method, and acaricides were used extensively in livestock production. However, the continuous emergence of resistant tick strains makes most chemical acaricides ineffective [ ]. Moreover, the increasing concerns for animal product and environmental contamination set limitations for this control method. To search for new and more effective means of controlling ticks and T”Ds, researchers have actively expanded the understanding on tick biology. RN“ interference RN“i is a reverse genetic approach for manipulation of genes that commonly utilizes double-stranded RN“ dsRN“ to induce post-transcriptional genespecific silencing [ ]. RN“i has been extensively employed in many studies on tick biology and pathogen interaction since the first report of RN“i application in the hard tick Amblyomma americanum [ ]. In fact, it is evident that a number of laboratories in different countries working on tick research are routinely performing RN“i, as shown by an increasing number of recent publications utilizing this technique. Typically, functional studies using RN“i involve gene knockdown with subsequent infestation and evaluation of phenotypes, such as blood feeding and reproduction success Figure . Indeed, RN“i has been particularly useful in searching for tick proteins that can be targeted for control of tick development and T”Ds [ ]. This chapter aims to show the extent of RN“i application in tick research, emphasizing the progress of advanced knowledge on tick biology and tick-pathogen interaction. We first discussed so far known RN“i mechanisms and the current RN“i inducing methods in ticks then, briefly described the studies on tick physiology, immunity, and pathogen interaction that employed RN“i, highlighting the prospects of applications of RN“i in tick research.

RNA Interference – A Powerful Functional Analysis Tool for Studying Tick Biology and its Control http://dx.doi.org/10.5772/61577

A

B

C

D

actin

E

Hlfer1

F

dsLuc

dsHlfer1

H

G

dsLuc

dsHlfer1

Figure . “ typical RN“i experiment in the hard tick Haemaphysalis longicornis. The double-stranded RN“ dsRN“ is introduced to adult ticks by microinjection. Unfed adult ticks, placed on a double adhesive tape attached on a glass slide “ , are injected with dsRN“ using a pointed microcapillary glass attached to a microinjector ” through the membrane of the fourth coxa under a stereomicroscope C . Successful silencing, as shown by the absence of a band for the target gene, such as Hlfer1, is confirmed around d post-injection through RT-PCR after adjusting the cDN“ level using an internal control, such as actin D . Ticks were infested on a host and allowed to feed to repletion E and after dropping, parameters such as engorged body weight F , survival, egg laying G, H , and hatch were compared.

. RNAi pathway in ticks The mechanism of RN“i has been well studied in the nematode Caenorhabditis elegans and the fruit fly Drosophila melanogaster [ , ]. RN“i begins with the uptake of dsRN“ by the cell, followed by its cleavage to produce small interfering RN“s siRN“s . Cleavage of dsRN“ is

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accomplished by an RN“se III enzyme called Dicer. The siRN“s are then incorporated into RN“-induced silencing complex RISC , which then drives the degradation or translational inhibition of the target mRN“ that results to gene silencing. This silencing signal may spread among the cells and different tissues, leading to systemic gene silencing in the whole organism [ ]. The mechanism of RN“i in ticks has not been fully elucidated, but the study of Kurscheid et al. [ ] revealed that several components of RN“i machinery in other invertebrates are also present in the ticks, and they proposed a putative tick RN“i pathway. Here, we briefly describe the available knowledge on key components of RN“i machinery in ticks, in comparison of other invertebrates. . . dsRNA uptake There are two recognized dsRN“ uptake mechanisms in invertebrates: a transmembrane channel-mediated uptake through systemic RN“ interference defective SID transmembrane proteins described in C. elegans, and an endocytosis-mediated uptake described in most arthropods [ , ]. Several SIDs identified in C. elegans have been shown to be involved in the spread of RN“i [ ]. SID- , SID- , and SID- , which have wide tissue distribution, are involved in the systemic spread of RN“i [ – ], whereas SID- , localized mainly in the gut, is involved primarily in the intestinal uptake of ingested dsRN“ [ ]. The multi-domain SID- along the plasma membrane facilitates the traffic of dsRN“ into and out of the cells. Homologs of SIDare present in some arthropods and vertebrates [ ]. ”oth SID- , a conserved tyrosine kinase, and SID- have intracytoplasmic localization, the latter being associated with late endosomes [ , ]. SID- has luminal localization in the intestinal cells, and it was also found in the lower levels of excretory duct cells [ ]. In addition to SIDs, endocytosis has also been also implicated as a dsRN“ uptake mechanism in C. elegans through a protein containing an epsin N-terminal homology ENTH domain [ ]. In D. melanogaster, dsRN“ uptake in cells is facilitated mainly by scavenger receptor-mediated endocytosis [ ]. Two scavenger receptors, Eater and Sr-CI, have been identified to be responsible for the majority of dsRN“ uptake. These scavenger receptors are mainly expressed in the plasmatocytes and have a primary role in the phagocy‐ tosis of bacterial pathogens [ , ]. SID homologues have not been identified in ticks. However, a homologue of ENTH, Epn-I, has been identified in the hard ticks Rhiphicephalus Boophilus microplus and Ixodes scapularis [ ]. “ class ” scavenger receptor identified in Haemaphysalis longicornis HlSR” has been demonstrated to mediate systemic RN“i in this tick [ , ]. Combined injection of dsRN“ against HlSRB and other target genes, Vitellogenin- HlVg-1 and Vitellogenin Receptor HlVgR effectively silenced these genes. However, silencing HlSRB prior to injection of dsRN“ against HlVg-1 and HlVgR inhibited the silencing of the latter two genes, suggesting that the uptake of the injected dsRN“ is dependent on HlSR” in ticks. Similar to D. melanogaster scavenger receptors, HlSR” is also involved in the phagocytosis of bacteria [ ], but it is expressed not only in the hemocytes but also in the other organs such as midguts, salivary glands, and ovary [ ]. The presence of ENTH homologue and scavenger receptor indicates that the uptake of dsRN“ in ticks is through endocytosis. “dditionally, the presence of scavenger receptor in different tick tissues strongly implies its involvement in systemic RN“i, particularly after

RNA Interference – A Powerful Functional Analysis Tool for Studying Tick Biology and its Control http://dx.doi.org/10.5772/61577

dsRN“ injection. Introduction of dsRN“ into the hemocoel of ticks directly exposes the different tick organs to dsRN“, and the scavenger receptor in these organs most likely mediates the entry of dsRN“ into the cells. . . dsRNA processing and RISC assembly The recommended length of dsRN“ to effectively induce silencing of the target gene in nonmammalian systems is more than bp [ ]. “ study in R. B. microplus showed, however, that short dsRN“s between and bp were also effective in inducing silencing of Ubiquitin-63E homologue, with minimal off-target effects, but short hairpin dsRN“s were not able to induce silencing effects [ ]. “fter cellular uptake, dsRN“s are cleaved into – nt siRN“s by an RN“se III enzyme called Dicer. In contrast to C. elegans and mammals that have only one Dicer, D. melanogaster and mosquitoes have two Dicers [ ]. Dicer- is the one involved in the generation of siRN“, whereas Dicer- acts on stem loop RN“ precursors to generate micro RN“ miRN“ . ”oth, however, are required for siRN“-induced gene silencing due to their distinct roles in siRISC assembly [ ]. Only a single putative Dicer has been identified so far in the hard tick I. scapularis, which is more similar to mammalian Dicer- [ ]. The RN“i inhibition of a target mRN“ is accomplished by RISC formed by siRN“s and “rgonaute “GO proteins. “GO proteins are highly conserved between species, encoded by multiple genes in most organisms. “ll “GO proteins are characterized by two domains: the P“Z domain and the PIWI domain [ ]. Upon “TP activation, “GO mediates RISC recognition of mRN“ target that are homologous to siRN“s, subsequently leading to the cleavage of the mRN“ target [ ]. In most insects, including D. melanogaster and mosquitoes, five “GO genes have been identified [ , ]. In ticks, a homologue of “GO- has been identified in I. scapu‐ laris and R. B. microplus, and a homologue of “GO- has been identified in I. scapularis [ ]. However, the functions of these tick “GOs remains to be confirmed. . . Amplification of RNAi signal The ability to spread throughout the whole organism, inducing total systemic silencing of the target gene in spite of introducing only a relatively small amount of dsRN“, is an important aspect of RN“i observed in plants and invertebrates. This systemic RN“i-induced gene silencing in both plants and C. elegans involves RN“-directed RN“ polymerase RdRP that amplifies the RN“i signal [ ]. RdRP function in RN“i has not been found in arthropods, but a putative homologue of RdRP EGO- protein of C. elegans has been identified in the hard tick I. scapularis, and a partial sequence was also identified from R. B. microplus [ ].

. Methods of introducing dsRNA in ticks . . Injection Direct injection is the most widely used technique for introducing dsRN“ for in vivo gene silencing, not only in ticks but also in insects [ , ]. Through this method, dsRN“ is usually

415

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RNA Interference

introduced directly into the hemocoel of ticks allowing the dsRN“ to circulate within the hemolymph. In most reports, a high concentration of at least µg dsRN“ per tick has been shown to be effective in inducing gene silencing [ ], but in some reports, lower concentration has been found to be similarly effective [ – ]. Injection has been accomplished using a – -gauge needle attached to a Hamilton syringe particularly in large tick species, such as Amblyomma americanum[ ], Dermacentor variabilis, and D. marginatus [ ], while microinjection using a microcapillary drawn to a fine point needle and inserted to a micromanipulator has been commonly employed in smaller tick species, including Ixodes [ ], and Haemaphysalis [ ] ticks. Different injection sites include the lower right quadrant of the ventral surface of the exoskeleton [ ], the groove between the basis capituli and the scutum [ ], the ventral torso of the idiosoma, away from the anal opening [ ], and the coxal membrane in the fourth coxae [ , ]. In some reports, dsRN“ was injected through the spiracle [ , , ] and anal pore the latter inducing midgut-specific silencing of the target gene [ ]. Injection of dsRN“ has been commonly performed in unfed adult ticks, subsequently allowed to recover for at least h before infestation or use in succeeding experiments. Exceptionally, dsRN“ has been also injected in engorged R. B. microplus [ , - ], A. americanum, I. scapularis, and D. variabilis adults [ ], which produced significant effects on the eggs and larvae, and microinjection has also been accomplished in I. scapularis [ - ] and engorged O. moubata nymphs [ ]. . . Soaking Soaking in dsRN“ has been previously employed to study RN“i in the cell lines of D. melanogaster [ , ]. In tick research, this method has been applied to induce in vitro RN“i not only in cell cultures [ - ], but also in some organs including whole salivary glands [ , - ] and midguts [ ]. Soaking live Varroa destructor [ ] and Dermanyssus gallinae [ ] mites, as well as Aedes aegypti larvae [ ] in a solution of dsRN“ has been already demonstrated in producing effective silencing of the target genes in these organisms. However, soaking whole ticks in a solution of dsRN“ has not been commonly performed. Soaking Haemaphysalis longicornis nymphs in a solution of dsRN“ for h resulted to significant transcript reduction of the target gene, although the effect on the phenotype was not observed in all the nymphs [ ]. In our laboratory, we have attempted to soak H. longicornis larvae, nymphs, and adults in a dsRN“ solution overnight, which resulted to a significant decrease in the mRN“ level of a targeted gene Galay et al., unpublished results . Soaking offers a simpler and less invasive method of introducing dsRN“ without injuring the ticks and is applicable to immature tick stages. Furthermore, it does not require injection equipment therefore, it is less laborious. . . Electroporation Electroporation is a technique that employs electric impulses to promote DN“ uptake of cells and has been primarily used with in vitro cell transfection [ ]. In tick research, this technique has been first applied to facilitate the introduction of dsRN“ in I. scapularis eggs and nymphs [ ]. “fter electroporation, fluorescein-labeled dsRN“ was visualized all over the nymph’s body and eggs, indicating the successful entry of dsRN“. In a more recent report, the wax coating of the eggs was first removed using heptane and hypochlorite prior to electroporation

RNA Interference – A Powerful Functional Analysis Tool for Studying Tick Biology and its Control http://dx.doi.org/10.5772/61577

[ ]. Using heptane alone did not significantly decrease the hatching rate. Thus, heptane may be more helpful in evaluating the effect of a particular dsRN“ to egg hatching. This technique also offers a less invasive method of dsRN“ introduction that can be applied to immature tick stages and eggs. . . Feeding Feeding dsRN“ in insects has been achieved in different species using diets mixed with dsRN“, liposome-embedded or lipophilic siRN“s, and bacteria and transgenic plants that can synthesize dsRN“ [ , ]. “lthough in vitro feeding assays have been shown to be useful in studying different tick molecules and tick-pathogen interaction [ ], its application in RN“i study in ticks has been limited. “ study on the Lyme disease vector I. scapularis employed capillary feeding of dsRN“ to nymphs to suppress anticomplement gene isac [ ]. In another study, adult R. B. microplus ticks were capillary fed with ubiquitin dsRN“ mixed in whole blood or Bm 6 dsRN“ mixed in bovine serum [ ]. In both cases, ticks were pre-fed in an animal host before capillary feeding was performed. While this method may be advantageous over injection due to very minimal injury, drawbacks may arise from the uncertainty whether an individual tick will ingest the amount of dsRN“ that will effectively induce silencing, and the possibility of variation in the amount of dsRN“ ingested by the ticks within a treatment group. Furthermore, capillary feeding is difficult to perform and may not be applicable in ticks with short hypostome.

. RNAi and study of tick physiology . . Genes related to salivary functions The saliva is an important arsenal of ticks containing hundreds of pharmacologically potent substances that facilitate attachment to their hosts and blood-sucking [ ]. Different salivary proteins have redundant functions in counteracting the hemostatic [ ], inflammatory, and immune mechanisms [ ] of the host. “side from its function in tick feeding, the salivary glands are also involved in osmoregulation and transmission of pathogens [ ]. Many studies on characterization of salivary proteins in the recent years employed RN“i Table . In fact, the first report on the application of RN“i in tick research described a tick inhibitor of inflammatory mediator, a salivary histamine-binding protein, wherein researchers induced in vitro RN“i by soaking salivary glands in dsRN“ [ , ]. Soluble N-ethylmalei‐ mide-sensitive factor attachment receptors SN“RE complex proteins, which mediate exocytosis in secretory pathways of the salivary glands, have been characterized in Amblyom‐ ma ticks. These include N-ethylmaleimide-sensitive fusion NSF protein, Synaptosomal “ssociated Protein of kDa SN“P[ ], Ykt [ ], and vesicle transport through inter‐ action with t-SN“REs Vti [ ]. Silencing various genes such as Salp , Salp pac [ ], Neuronal isoform munc - nSec [ ], and synaptobrevin [ ] affected the secretion of

417

418

RNA Interference

anticoagulant or the anticoagulant activity of salivary gland extracts, indicating that these genes are important in tick anti-hemostatic mechanism. Longistatin [ ] and acidic chitinases [ ] have been found to be important in the formation of blood pool and tick cement cone, respectively. The attachment site of longistatin-silenced H. longicornis ticks did not show pathological changes, such as hemorrhagic lesions correspond‐ ing to the blood pool, while the attachment site of ticks simultaneously silenced acidic chitinases exhibited blood leakage and these ticks can be easily removed. Various protease inhibitors that have roles in anti-hemostatic, anti-inflammatory, and immunomodulatory mechanism have been also characterized using RN“i, including a cystatin, sialostatine L [ ], a Kunitz type protease inhibitor, rhipilin [ ], and serine protease inhibitors serpin [ , ]. Other salivary proteins with immunomodulatory function, such as the anti-complement protein, isac [ ], and two proteins that can inhibit neutrophil function, ISL and [ ], have also been knockdowned in I. scapularis. Silencing of isac in nymphs, induced by capillary feeding of dsRN“, not only reduced blood feeding, but also decreased the load of the spiro‐ chete Borrelia burgdorferi in the tick. Meanwhile, the saliva of ticks devoid of ISL and had reduced ability in inhibiting host integrin. “n osmoregulatory protein aquaporin, characterized in I. ricinus through RN“i, showed that suppression of this protein impaired the concentration of blood meal due to failure in removing water [ ]. . . Genes related to digestion and midgut function The midgut of ticks houses various kinds of enzymes that act on a large amount of ingested host blood, which contains great quantities of hemoglobin [ ]. Functional studies on these enzymes and other midgut proteins using RN“i have expanded the understanding of tick digestive physiology Table . Silencing hemoglobinolytic enzymes, such as leucine amino‐ peptidase [ , ], longipain [ ], and cathepsin L [ , ] had negative impact on tick feeding. Moreover, the longipain of H. longicornis was found to have a protective role in Babesia infection through its babesiacidal activity [ ]. Other proteins important in tick digestion that have been characterized using RN“i are thrombin inhibitors that prevent blood coagulation and serine proteinase, which induce erythrocyte degradation. Silencing of thrombin inhibitor hemalin from H. longicornis [ ] and boophilin from R. B. microplus [ ] prolonged the blood feeding period and decreased the oviposition of these ticks, respectively. Silencing serine protease reduced the weight of ticks after blood feeding due to impaired erythrocyte degradation [ ]. . . Genes related to reproductive function Ticks are known for their high fecundity, laying hundreds of eggs per batch in the case of soft ticks and up to thousands in the case of hard ticks. “ series of physiological events takes place in female ticks during and after blood feeding that initiate ovarian maturation and subsequent oviposition. Vitellogenesis, the synthesis and oocyte deposition of the yolk pro‐ tein precursor vitellogenin , is a key process for ovarian development and oocyte matura‐ tion induced by blood meal in ticks [ ]. Three genes encoding vitellogenin have been identified and characterized in H. longicornis [ ].

RNA Interference – A Powerful Functional Analysis Tool for Studying Tick Biology and its Control http://dx.doi.org/10.5772/61577

Target gene

Tick species

RNAi Effect

Refs

“ltered feeding pattern and longer feeding

[

,

[

]

[

]

Inhibited secretion of anticoagulant stimulated [

]

Salivary proteins Histamine-binding protein Amblyomma americanum H”P

period decreased histamine-binding activity in the salivary glands

Salp / Salp pac

Ixodes scapularis

Impaired feeding, decreased post-blood meal weight, decreased anticoagulant activity of salivary gland extract

Neuronal isoform munc

- A. americanum

nSec

Decreased post-blood meal weight and prolonged feeding time, decreased anticoagulant secretion of salivary gland

Synaptobrevin

A. americanum

by PGE A. americanum

Cystatin

Decreased post-blood meal weight, mortality

[

]

[

]

[

]

[

]

[

]

[

]

[

]

[

]

[

]

[

]

Decreased egg weight and egg conversion ratio [

]

N-ethylmaleimide sensitive A. maculatum

Inhibition of engorgement, failure of

]

fusion protein NSF

oviposition

during feeding, low feeding success rate “nticomplement protein

I. scapularis

Decreased post-blood meal weight, decreased Borrelia burgdorferi infection

Isac Sialostatin L cystatin

I. scapularis

Failure to feed on the host, decreased postblood meal weight and failed oviposition

“quaporin

I. ricinus

Decreased post-blood meal weight, decreased volume of ingested blood

HlYkt

SN“RE

Haemaphysalis longicornis

Decreased post-blood meal weight, high mortality, supressed salivary secretion and anticoagulant activity

ISL

and

I. scapularis

Supressed PMN inhibitory activity of saliva from knockdowned ticks

Rhipilin Kunitz type

Rhipicephalus

Prolonged attachment time, decreased post-

protease inhibitor

haemaphysaloides

blood meal weight

Longistatin

H. longicornis

Mortality after attachment, failure to engorge, poor blood pool formation

Serine protease inhibitor

A. americanum

serpin

No effect on tick attachment, feeding and oviposition

R. haemaphysaloides

Decreased attachment rate and engorgement weight

Reprolysin

Rhipicephalus Boophilus microplus

[

]

419

420

RNA Interference

Target gene Synaptosomal “ssociated Protein of

Tick species

RNAi Effect

Refs

A. maculatum

Decreased post-blood meal weight, decreased

[

]

[

]

Decreased post-blood meal weight, egg weight [

]

kDa

egg weight, failure in hatching

SN“PVti SN“RE

A. americanum, A. maculatum Decreased post-blood meal weight and survival, failed oviposition

Glutaminyl cyclase QC

A. maculatum, I. scapularis

and hatch “V

A. americanum

Decreased post-blood meal weight

[

]

“cidic chitinase “ch

A. americanum

Leakage of blood from the mouthparts in late

[

]

feeding phase, loose attachment in the host’s skin Digestive activity Longepsin

H. longicornis

No effects reported

[

]

Leucine aminopeptidase

H. longicornis

Extended pre-oviposition period, decreased egg [

,

weight and egg conversion ratio, morphological abnormalities in the oocytes Hemalin thrombin

H. longicornis

inhibitor

Longer blood feeding period, failure to

[

]

engorge, decreased inhibitory activity of fibrinogen clot formation in the midgut

”oophilin thrombin

R. B. microplus

Decreased oviposition

[

]

H. longicornis

Suppressed erythrocyte degradation decreased [

]

inhibitor Serine proteinase

post-blood meal weight Longipain

H. longicornis

Impaired blood feeding, decreased post-blood [

]

meal weight, increased B. gibsoni infection level and transovarial transmission Cathepsin L

“stacin

I. ricinus

Decreased weight gain

[

]

H. longicornis

Decreased post-blood meal weight

[

]

R. B. microplus

Decreased egg weight and egg conversion ratio [

]

H. longicornis

Decreased egg conversion ratio

[

]

Failure of Vg uptake by oocytes failed

[

]

[

]

Tick reproduction Follistatin-related protein FRP Vitellogenin receptor VgR Dermacentor variabilis

oviposition H. longicornis

Suppressed oocyte maturation and failed oviposition, failure of B. gibsoni transovarial transmission

]

RNA Interference – A Powerful Functional Analysis Tool for Studying Tick Biology and its Control http://dx.doi.org/10.5772/61577

Target gene

Tick species

RNAi Effect

Refs

A. hebraeum

Suppressed oocyte maturation, long pre-

[

]

Failure to engorge and lay eggs in females fed [

]

oviposition period, Voraxin

A. americanum

with males injected with a combination of subolesin and voraxin dsRN“ Vitellogenin Vg

H. longicornis

Decreased post-blood meal weight, abnormal

[

]

oocytes, decreased egg conversion ratio G“T“ factor

H. longicornis

Disrupted egg development

[

]

S kinase

H. longicornis

Disrupted egg development

[

]

Decreased post-blood meal weight, mortality

[

]

H. longicornis

Decreased post-blood meal weight and survival [

]

I. scapularis

Decreased post-blood meal weight and

[

]

[

]

Decreased post-blood meal weight

[

]

Lower post-blood meal weight, low survival

[

]

[

]

Mortality after engorgement, leakage of blood [

]

Target of rapamycin TOR H. longicornis

after engorgement, Failure of oocytes to mature and failure to lay eggs Structural and metabolic function Glutamine:fructose- phosphate aminotransferase HlGF“T β-“ctin

oviposition Na+-K+-“TPase

I. scapularis

Decreased post-blood meal weight and oviposition

Valosin-containing protein H. longicornis HlVCP Cyclophilins

H. longicornis

Immunophilin

after blood feeding and failure to lay eggs after silencing cyclophilin “

Ribosomal protein P

H. longicornis

Decreased post-blood meal weight, low engorgement rate, and high mortality

Protein disulphide

H. longicornis

isomerases PDI Organic anion transporter

from the midgut, little egg output A. americanum

polypeptide O“TP Ferritins FER

Decreased post-blood meal weight, oviposition [

]

and egg conversion ratio I. ricinus

Decreased post-blood meal weight, oviposition [

]

and hatch H. longicornis

Decreased post-blood meal weight, survival,

[

,

[

]

oviposition and hatch Iron regulatory protein IRP

I. ricinus

Decreased post-blood meal weight and hatching of eggs

]

421

422

RNA Interference

Target gene

Tick species

RNAi Effect

Refs

Elongation factor -α

R. annulatus, R. B.

High post-blood meal mortality, decreased

[

microplus

post-blood meal weight and failure of

]

oviposition H. longicornis

Lysine-ketoglutarate

Longer blood feeding period, decreased post-

reductase/saccharopine

blood meal weight, longer pre-oviposition

dehydrogenase LKR/SDH

period, decreased oviposition and hatch after

[

]

SDH silencing higher volume of hemolymph after LKR silencing R. B. microplus

Ubiquitin

R. annulatus Glycogen synthase kinase- R. B. microplus

Shorter post-blood meal survival, decreased or [

,

absence of egg output, impaired embryogenesis

]

,

High mortality

[

]

Decreased oviposition and hatching

[

]

Inhibited feeding, low post-blood meal weight [

]

GSKCD

receptor

A. americanum

tender cuticle Insulin-like growth factor

A. americanum

Decreased post-blood meal weight

[

]

High mortality and very low post-blood meal

[

]

[

]

[

]

[

]

[

]

[

]

[

]

binding protein-related proteins Putative . S, ITS and

S A. americanum

rRN“

weight S ribosomal A. americanum

Putative ” protein L

e

Putative interphase

weight A. americanum

cytoplasm foci protein Putative threonyl-tRN“

High mortality and very low post-blood meal

High mortality and very low post-blood meal weight

A. americanum

synthetase

High mortality and very low post-blood meal weight

Putative

S ribosomal

protein L

a

Putative mitochondrial

A. americanum

S A. americanum

rRN“ Chymotrypsin inhibitor

% mortality

High mortality and very low post-blood meal weight

H. longicornis

HlChI

Mortality after attachment, retarded blood feeding and longer feeding period, decreased post-blood meal weight, decreased egg weight and egg conversion ratio

Scavenger receptor

H. longicornis

Decreased post-blood meal weight, mortality after engorgement, decreased oviposition and hatch inhibited bacterial phagocytosis of granulocytes

[

,

]

,

RNA Interference – A Powerful Functional Analysis Tool for Studying Tick Biology and its Control http://dx.doi.org/10.5772/61577

Target gene E-”P eIF E-binding

Tick species

RNAi Effect

Refs

H. longicornis

Decreased lipid accumulation in the midgut

[

]

[

]

protein

and fat bodies after long starvation period

Protein kinase ” “KT

H. longicornis

Inhibition of engorgement and growth of organs during blood feeding decreased expression of longepsin, HlMIF and HlVgs

R. B. microplus

Decreased cell glycogen content and viability,

[

]

and altered cell membrane permeability Spook Spo

Ornithodoros moubata

“rrested development and molting

[

]

Shade Shd

O. moubata

“bnormal ecdysis and delayed molting

[

]

Cystatin RHCyst

R. haemaphysaloides

Decreased attachment and hatching rate

[

]

Tropomyosin

H. longicornis

Longer feeding time, decreased engorgement

[

]

rate and post-blood meal weight, high mortality after blood feeding, failed oviposition Protective antigens Subolesin

D

I. scapularis

Decreased post-blood meal weight, oviposition [

,

]

,

,

and survival failure of embryogenesis silencing in eggs and larvae when dsRN“ injected to engorged females A. americanum

Decreased post-blood meal weight, oviposition [

]

and survival D. marginatus

Decreased post-blood meal weight, oviposition [

]

and survival D. variabilis

Decreased post-blood meal weight, oviposition [

,

]

and survival decreased fertility silencing in eggs and larvae when dsRN“ injected to engorged females R. sanguineus

Decreased post-blood meal weight, oviposition [

,

]

and survival more dramatic effect when simultaneously silenced with Rs R. B. microplus

High mortality, decreased post-blood meal

[

,

,

,

]

weight, oviposition and hatch in dsRN“injected adults and progeny of dsRN“-injected adults R. annulatus,

Decreased post-blood meal weight

[

O. erraticus

Decreased egg output

[

]

O. moubata

Decreased egg output

[

]

]

423

424

RNA Interference

Target gene

Tick species

RNAi Effect

Refs

Midgut protein Rs

R. sanguineus

Decreased post-blood meal weight and

[

]

oviposition Midgut protein Hl

H. longicornis

Decreased post-blood meal weight

[

]

Midgut protein ”m

R. B. microplus

Decreased number of engorging ticks, lower

[

]

post-blood meal body weight and survival after feeding in B. bovis-infected host, decreased egg weight Midgut protein Ree

R. evertsi evertsi

No significant effect

[

]

Midgut protein Ree“T“Q

R. evertsi evertsi

No significant effect

[

]

Longicin

H. longicornis

Decreased post-blood meal weight, increased B. [

]

gibsoni infection in the midgut and ovary, and transmission in the eggs α -macroglobulin proteins I. ricinus

Decreased phagocytic action of hemocytes

[

,

A. americanum

No effect on phenotypes

[

]

I. scapularis

Increased A. phagocytophilum infection level

[

]

Dual oxidase Duox

I. scapularis

Decreased level of B. burgdorferi

[

]

Peroxidase ISCW

I. scapularis

Decreased level of B. burgdorferi

[

]

Glutathione S-transferase

R. B. microplus

Decreased tick attachment and post-blood meal [

Macrophage migration

]

inhibitory factor Janus kinase J“K – signaling transducer activator of transcription ST“T pathway

]

weight

Selenoprotein W

R.sanguineus

Increased susceptibility to permethrin

R. B. microplus

Decreased tick attachment and post-blood meal [

[

] ]

weight Selenoprotein K

A. maculatum

Decreased oviposition

[

]

Selenoprotein M

A. maculatum

Decreased oviposition

[

,

Thioredoxin reductase

A. maculatum

Decreased native microbial load in midguts and [

]

]

salivary glands Rmcystatin

cysteine

R. B. microplus

Increased resistance to bacteria

[

]

Inhibited Anaplasma marginale infection in

[

,

protease inhibitor Pathogen acquisition/ transmission Subolesin

D. variabilis

salivary glands

]

RNA Interference – A Powerful Functional Analysis Tool for Studying Tick Biology and its Control http://dx.doi.org/10.5772/61577

Target gene

Tick species

RNAi Effect

Refs

R. B. microplus

Decreased A. marginale infection level in

[

]

salivary glands and tick cells I. scapularis

Salp

Decreased Borrelia burgdorferi transmission to

[

]

[

]

Reduced A. phagocytophilum acquisition

[

]

Decreased acquisition of B. burgdorferi after

[

]

the host I. scapularis

Salp

No effect on acquisition of A. phagocytophilum and B. burgdorferi in nymphs

Salp

I. scapularis

Salp D

I. scapularis

knockdown in salivary glands Varisin

D. variabilis

Decreased A. marginale infection level

[

Immunophilin

R. B. microplus

Decreased hatch, decreased larval survival,

[

]

[

]

]

increased B. bovis infection in larval progeny Kunitz-type serine protease R. B. microplus

Inhibition of engorgement, decreased egg

inhibitor Spi

weight

Glutathione S-transferase

D. variabilis

Inhibited A. marginale infection

[

]

H+ transporting lysosomal

D. variabilis

Inhibited A. marginale infection in the midgut

[

]

[

]

[

]

[

]

[

]

[

]

vacuolar proton pump

after acquisition feeding

v“TPase Selenoprotein M

D. variabilis

Inhibited A. marginale infection and multiplication in salivary glands

Putative von Willebrand factor

R. B. microplus

Will

Flagelliform silk protein

Decreased A. marginale infection level in salivary glands

R. B. microplus

Silk

Decreased A. marginale infection level in salivary glands and tick cells

Putative metallothionein

R. B. microplus

Increased A. marginale infection level in tick

Meth

cells

Tick salivary lectin pathway I. scapularis

Decreased load of B. burgdorferi and

inhibitor TSLPI

transmission to host

Kunitz-type serine protease D. variabilis

Increased rickettsial infection in the midgut

[

]

Decreased post-blood meal weight

[

]

Decreased post-blood meal weight, decreased

[

]

R. annulatus, R. B.

Decreased B. bigemina infection level Decreased [

]

microplus

post-blood meal weight in R. microplus

inhibitor DvKPI Kunitz-type protease

R. annulatus, R. B.

inhibitor

microplus

KTPI

Histamine release factor

I. scapularis

B. burgdorferi transmission TROSP“

425

426

RNA Interference

Target gene Serum amyloid “

Tick species

RNAi Effect

Refs

R. annulatus, R. B.

Decreased B. bigemina infection level

[

]

R. annulatus, R. B.

Decreased post-blood meal weight in R.

[

]

microplus

annulatus [

]

microplus Ricinusin

Calreticulin

R. annulatus, R. B.

Decreased B. bigemina infection level in R.

microplus

microplus decreased post-blood meal weight in R. annulatus

Chitin deacetylase-like

I. scapularis

protein IsCD“ “ntifreeze glycoprotein

No significant effect on B. burgdorferi

[

]

acquisition or transmission I. scapularis

Decreased survival and mobility of ticks in

[

]

extremely cold temperature decreased A. phagocytophilum infection level x-linked

I. scapularis

Increased A. phagocytophilum infection

[

]

R. B. microplus

Failure in transmission of A. marginale

[

]

inhibitor of apoptosis protein E ubiquitin ligase, XI“P Cytochrome c oxidase subunit III Table . Genes functionally characterized through RN“i in different tick species.

Silencing these genes through RN“i greatly reduced the reproductive capacity of female ticks, which showed immature and light-colored oocytes. The uptake of vitellogenin in the oocytes is facilitated by vitellogenin receptor, which has been characterized in D. variabilis [ ], H.longicornis [ ], and A. hebraeum [ ]. “side from the negative impact in oviposition consistently induced by RN“i in all these studies, silencing of H. longicornis vitellogenin receptor also reportedly inhibited the transovarial transmission of Babesia gibsoni. Three factors involved in the initiation of vitellogenesis, the G“T“ factor, S kinase [ ], and target of rapamycin TOR pathway [ ], have been also characterized in H. longicornis ticks using RN“i. The significance of other proteins to reproduction, such as a tick homologue of the human follistatin-related protein [ ] and the engorgement protein voraxin [ ] from the male gonad, has been also demonstrated using RN“i. . . Genes related to structural and metabolic functions Various gene encoding proteins important in cellular structure and metabolism have been characterized using RN“i. Due to their wide distribution and systemic function, knockdown of these proteins caused detrimental effects on different tick physiological functions and some even proved to be lethal Table . “mong these proteins is the multifunctional ubiquitin, which has been first targeted based on a homologous gene of D. melanogaster in a study investigating the components of tick RN“i pathway [ ]. Ubiquitin knockdown in R. B.

RNA Interference – A Powerful Functional Analysis Tool for Studying Tick Biology and its Control http://dx.doi.org/10.5772/61577

microplus shortened the post-blood meal survival of ticks and impaired egg viability and hatch. In separate studies, ubiquitin has also been the subject in examining off-target effects of RN“i [ ] and the feasibility of dsRN“ feeding in R. B. microplus [ ]. RN“i-mediated silencing of ribosomal proteins in A. americanum [ ], and ubiquitin, elongation factor- alpha and several other proteins in R. B. microplus and R. annulatus [ ] has been employed to screen potential antigens for tick control. In the hard tick H. longicornis, individual knockdown of glutamine:fructose- -phosphate aminotransferase [ ], cyclophilin “ [ ], the ribosomal protein P [ ], protein disulphide isomerases [ ], and tropomyosin [ ] resulted to decreased survival of ticks after engorge‐ ment. Two proteins with apparent roles in withstanding long starvation period, lysineketoglutarate reductase/saccharopine dehydrogenase LKR/SDH [ ] and E-”P [ ], have also been characterized in H. longicornis. LKR/SDH mRN“ expression is higher in starved ticks than in unfed ticks and knockdown of LKR resulted to high volume of hemolymph after blood feeding, suggesting its role in osmoregulation. Meanwhile, E-”P knockdown led to decreased lipid storage in the midguts and fat bodies of ticks during longer starvation period. “n interesting report on the application of RN“i in studying tick neurobiology targetedβ-actin and Na+-K+-“TPase of I. scapularis using fluorescently labeled dsRN“s to monitor the uptake in tick synganglia [ ]. The significance of proteins involved in iron metabolism to tick feeding and reproduction has been also demonstrated using RN“i in two hard tick species, I. ricinus [ ] and H. longicor‐ nis [ ]. Silencing two types of the iron storage protein ferritin greatly reduced the ticks’ capacity to engorge and produce eggs, also affecting post-blood meal survival due to occur‐ rence of iron-mediated oxidative stress [ ]. “n iron regulatory protein responsible for translation of iron binding proteins was characterized in I. scapularis, with its knockdown greatly reducing egg hatchability [ ]. Two enzymes, spook and shade, were characterized in the soft tick O. moubata and were shown to be important in ecdysteroidogenesis through RN“i [ ]. Silencing spook protein in nymphs caused arrested development and molting, whereas silencing shade delayed molting and led to abnormal ecdysis.

. RNAi studies on tick protective antigens and immunity The immune system of ticks has a vital role of protecting them from harmful substances in the blood, including components of their host’s immune system, and from various pathogens that they acquire in their blood feeding activity. Tick protective antigens, therefore, gain wide interest due to their potential as target for tick control. The highly conserved tick protective antigen subolesin, previously known as D , was first identified from I. scapularis through cDN“ expression library immunization ELI [ ], after which, it has been also identified in other hard tick species, and using RN“i, was found to be important in the success of blood feeding and reproduction [ ]. “n ortholog of subolesin has also been characterized in two soft tick species and RN“i demonstrated that subolesin is also important in the reproduction of soft ticks [ ]. The function of subolesin is unclear, but a report showed that subolesin

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RNA Interference

knockdown affected the expression of several genes involved in multiple cellular pathways, suggesting a role in gene expression by interacting with regulatory proteins [ ]. “side from being reported as a promising anti-tick vaccine antigen candidate in many studies, it has been also proposed that subolesin may be targeted in ticks that subsequently will be released for sterile acarine technique S“T [ ]. The membrane-bound glycoprotein ”m expressed mainly in the midgut of R. B. micro‐ plus [ ] is the first, and until recently, the only tick antigen that is commercially available as an anti-tick vaccine in some countries. The exact function of ”m , however, remains unclear yet. RN“i has been employed to knockdown Bm 6 and its homologues in other tick species, including R. sanguineus, H. longicornis, and R. evertsi evertsi, which in most cases affected the blood feeding and reproduction of adult ticks, except in R. evertsi evertsi wherein knockdown of two homologues did not yield significant effects [ ]. “ study in R. B. microplus also showed that knockdown of Bm 6 decreased the blood feeding capacity and survival of ticks after feeding on a B. bovis-infected host, suggesting that ”m may have a critical role in the fitness of ticks after feeding from an acutely B. bovis-infected host [ ]. The function of some components of immunity, such as α -macroglobulin proteins [ , ], antimicrobial peptides [ ], Janus kinase J“K -signaling transducer activator of transcription ST“T pathway [ ], dityrosine network [ ], and cysteine protease inhibitor in the hemocytes [ ] have been analyzed using RN“i. The α -macroglobulin proteins of I. ricinus, related to vertebrate complement system, were shown to be involved in the phagocytic activity of hemocytes against Gram-negative bacteria [ , ]. In contrast, a cysteine protease Rmcystatin identified in R. B. microplus was implicated as a negative modulator of tick immune response after its silencing greatly reduced the number of bacterial load in the ticks [ ]. The role of a defensin from H. longicornis, longicin, in ticks' immune defense against Babesia parasites was demonstrated through RN“i, as exhibited by a higher load of B. gibsoni in the midgut and ovary of longicin-silenced ticks after infestation in an infected host [ ]. Meanwhile, J“K-ST“T pathway was shown to be important in Anaplasma phagocytophilum infection in ticks after its knockdown increased the infection in the salivary glands of nymphs that fed on infected mice [ ]. “ dual oxidase and a peroxidase, ISCW , which together forms a dityrosine network, were separately silenced in I. scapularis, both resulting to reduced Borrelia burgdorferi persistence in ticks [ ]. The obligatory blood feeding lifestyle of ticks exposes them to high levels of pro-oxidants that may trigger oxidative stress. “ntioxidant enzymes function to protect them from the harmful effects of oxidative stress. Furthermore, these antioxidant enzymes provide detoxification mechanisms to counteract toxins that they encounter in the environment, such as chemical acaricides. RN“i has been very useful in evaluating the function of these antioxidants. Silencing a selenoprotein in R. B. microplus reduced the engorged body weight and egg output [ ]. In contrast, a study on A. maculatum showed that silencing two selenoproteins did not alter blood feeding, although the egg output was reduced. Interestingly, the total antioxidant capacities of the saliva from knockdowned ticks were higher, indicating that other antioxidant enzymes may have compensated for the absence of selenoproteins [ ]. In another study, silencing thioredoxin reductase, another selenoprotein, in A. maculatum did not have a negative

RNA Interference – A Powerful Functional Analysis Tool for Studying Tick Biology and its Control http://dx.doi.org/10.5772/61577

impact on blood feeding and reproduction. Likewise, variations in transcriptional expression of some antioxidant enzymes were also observed, suggesting compensatory mechanism in the absence of thioredoxin reductase [ ]. However, the more interesting finding in that study was the decreased microbiota population following thioredoxin reductase knockdown, possibly because of disturbed redox homeostasis balance. Meanwhile, silencing a glutathione S-transferase GST gene affected the attachment of ticks and reduced the post-blood meal bodyweight of R. B. microplus [ ]. It also made R. sanguineus ticks more susceptible to permethrin, although no significant effects on tick attachment, feeding and reproductive capacity were observed [ ].

. Understanding tick-pathogen interaction through RNAi RN“i has undoubtedly paved a way to better understand the different aspects of ticks' association with various pathogens. Numerous tick proteins with different functions have been found to be involved in the acquisition, establishment, and transmission of pathogens. Several proteins have been studied through RN“i to determine their importance in the development cycle of different pathogens. The knockdown of subolesin [ , ], GST, v“TPase, and selenoprotein M [ ] in D. variabilis, and putative von Willebrand factor, flagelliform silk protein and subolesin in R. B. microplus [ ] decreased the infection level of A. marginale in these hard ticks, implying that these proteins are significant in the establish‐ ment of infection of this rickettsia. RN“i also demonstrated that the Lyme disease agent B. burgdorferi can utilize several proteins of I. scapularis to facilitate its transmission to the host. These include salivary proteins such as tick histamine release factor [ ], Salp [ ], and the lectin complement pathway inhibitor TSLPI [ ] the latter two provide protection for B. burgdorferi against components of the host immune system. Salivary proteins Salp [ ], Salp [ ], and Salp D [ ] have been examined for their function in acquiring A. phagocytophilum or B. burgdorferi through RN“i. Knockdown of Salp did not affect the acquisition of either rickettsiae, whereas the knock‐ down of Salp and Salp D decreased the infection level of A. phagocytophilum and B. burgdorferi in the tick, respectively. “n interesting study on I. scapularis showed that A. phagocytophilum promotes cold tolerance through an antifreeze glycoprotein [ ]. In the absence of this antifreeze glycoprotein, the survival rate of ticks after exposure to extremely cold temperature and the infection level of A. phagocytophilum following exposure was reduced. Tick defensins, varisin from D. variabi‐ lis [ ], and ricinusin from Rhipicephalus ticks [ ] have been silenced to examine their functions in pathogen establishment the former reduced A. marginale infection level, while the latter did not have an effect on B. bigemina infection. Several reports also demonstrated the interaction of Babesia parasites and tick proteins through RN“i. Knockdown of the immunophilin gene in R. B. microplus had negative impact on the reproductive performance of the tick and also increased the infection rate of B. bovis in larval progeny [ ], while knockdown of TROSP“, serum amyloid “, and calreticulin reduced the

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infection level of B. bigemina in Rhipicephalus ticks [ ]. Silencing a Kunitz-type serine protease inhibitor from D. variabilis increased the rickettsial infection in the midgut [ ], whereas in R. B. microplus, silencing a Kunitz-type serine protease inhibitor, Spi, tended to increase the infection rate of B. bovis in larval progeny [ ], but silencing of another Kunitz-type protease inhibitor KTPI did not have any effect on Babesia infection [ ].

. Future directions in tick research and application in tick control Indeed, great progress in understanding tick biology has been already accomplished in the past. However, many aspects of tick physiology and host-tick-pathogen interaction need to be unraveled yet. Moreover, several optimizations can still be done to improve RN“i in tick research. While being the most widely used method of introducing dsRN“, the injection method particularly microinjection that requires elaborate equipment may not be accessible to all laboratories. Moreover, injection is mostly applicable to adult and sometimes nymphal stages, and may be injurious to the ticks, especially when performed by an inexperienced researcher. The soaking method is simpler, less invasive, and less laborious. Electroporation has been recently shown to be effective in introducing dsRN“ in eggs [ ] and may be useful in studying the function of genes that are involved in embryogenesis and physiology of immature tick stages. RN“i may also prove to be a promising tick control method and not just a research tool. In pest insect management, the possibility of using RN“i as a novel tool of pest control is already being explored by feeding liposome-coated dsRN“ or dsRN“ expressed in transgenic plants or bacteria [ ]. RN“i targeting several genes have been accomplished by feeding plants expressing dsRN“ in several species of economically important crop pests [ ]. Feeding dsRN“ to ticks is still an underdeveloped approach, which has been yet accomplished only by artificial feeding. Coating dsRN“s with liposomes or nanocarriers may increase dsRN“ stability that may make it feasible for administration to the host. Genes that are highly conserved across different tick species, and are of importance in tick survival are good candidate targets. These include proteins with structural and metabolic functions, such as ubiquitin, tropomyosin, and ferritin. However, the specificity of dsRN“ to the tick gene should be highly considered. “dditional consideration would be the establishment of a minimum effective dose, since the synthesis of dsRN“ is costly. “dditionally, RN“i has been proposed as an alternative method for the sterile insect technique in blood-sucking mosquitoes that will produce sterile males by feeding dsRN“ in mosquito larvae [ ]. Quite similarly, the application of RN“i for tick control was also proposed in a single report on D. variabilis, wherein the highly conserved subolesin was targeted leading to reproductive incapacity [ ]. In conclusion, the authors suggested that RN“i may be used to massively produce sterile ticks S“T that may be released in the field. Releasing subolesinsilenced ticks may also aid in the control of A. marginale, since it has been reported that subolesin knockdown reduced the infection level of this pathogen [ – ]. Introducing dsRN“ to eggs through electroporation described above may be a more convenient way of producing knockdowned ticks.

RNA Interference – A Powerful Functional Analysis Tool for Studying Tick Biology and its Control http://dx.doi.org/10.5772/61577

. Summary In this chapter, we have reviewed the application of RN“i in tick research and described the significant contribution of RN“i in advancing our knowledge on tick biology and tickpathogen interaction. RN“i has revolutionized the advancement of our understanding of various aspects of tick blood feeding and digestion, reproduction, metabolism, and immunity. “s a functional analysis tool, RN“i has become very handy in elucidating the functions of different proteins from more than hard tick species and a few soft tick species. It has been particularly helpful in screening potential target antigens for anti-tick and tick-borne pathogen vaccine development [ ]. Several methods of introducing dsRN“ in ticks have been employed but injection has remained to be the most widely used technique. The number of published research on ticks that involves the application of RN“i has been continuously increasing through the years, and it is expected to continue doing so. “ great majority of the published reports focused on hard ticks, but due to some physiological differences, more research using RN“i on soft ticks should be conducted. Finally, with numerous potential target genes already identified, the application of RN“i as a tick control method should be investi‐ gated in the future, starting with optimization of dsRN“ delivery method for practical use.

Acknowledgements This work was supported by the Japan Society for the Promotion of Science JSPS K“KENHI Grant Numbers , , and . We also thank our colleague, Melbourne R. Talactac, at the Laboratory of Infectious Diseases, Joint Faculty of Veterinary Medicine, Kagoshima University, for his assistance in writing this chapter.

Author details Remil Linggatong Galay , , Rika Umemiya-Shirafuji , Masami Mochizuki , Kozo Fujisaki and Tetsuya Tanaka * *“ddress all correspondence to: [email protected] Laboratory of Infectious Diseases, Joint Faculty of Veterinary Medicine, Kagoshima University, Kagoshima, Japan Department of Veterinary Paraclinical Sciences, College of Veterinary Medicine, University of the Philippines Los ”años, Los ”años, Laguna, Philippines National Research Center for Protozoan Diseases, Obihiro University of “griculture and Veterinary Medicine, Inada-cho, Obihiro, Hokkaido, Japan National “gricultural and Food Research Organization, Tsukuba, Ibaraki, Japan

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RNA Interference – A Powerful Functional Analysis Tool for Studying Tick Biology and its Control http://dx.doi.org/10.5772/61577

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