Oligonucleotide-Based Drugs and Therapeutics E-Book

• Herausgeber: John Wiley & Sons
• Kategorie: Wissenschaft und neue Technologien
• Sprache: Englisch

Veröffentlichungsjahr: 2018

Beschreibung

A comprehensive review of contemporary antisense oligonucleotides drugs and therapeutic principles, methods, applications, and research 

Oligonucleotide-based drugs, in particular antisense oligonucleotides, are part of a growing number of pharmaceutical and biotech programs progressing to treat a wide range of indications including cancer, cardiovascular, neurodegenerative, neuromuscular, and respiratory diseases, as well as other severe and rare diseases. Reviewing fundamentals and offering guidelines for drug discovery and development, this book is a practical guide covering all key aspects of this increasingly popular area of pharmacology and biotech and pharma research, from the basic science behind antisense oligonucleotides chemistry, toxicology, manufacturing, to safety assessments, the design of therapeutic protocols, to clinical experience.

Antisense oligonucleotides are single strands of DNA or RNA that are complementary to a chosen sequence. While the idea of antisense oligonucleotides to target single genes dates back to the 1970's, most advances have taken place in recent years. The increasing number of antisense oligonucleotide programs in clinical development is a testament to the progress and understanding of pharmacologic, pharmacokinetic, and toxicologic properties as well as improvement in the delivery of oligonucleotides. This valuable book reviews the fundamentals of oligonucleotides, with a focus on antisense oligonucleotide drugs, and reports on the latest research underway worldwide.

•    Helps readers understand antisense molecules and their targets, biochemistry, and toxicity mechanisms, roles in disease, and applications for safety and therapeutics

•    Examines the principles, practices, and tools for scientists in both pre-clinical and clinical settings and how to apply them to antisense oligonucleotides

•    Provides guidelines for scientists in drug design and discovery to help improve efficiency, assessment, and the success of drug candidates

•    Includes interdisciplinary perspectives, from academia, industry, regulatory and from the fields of pharmacology, toxicology, biology, and medicinal chemistry

Oligonucleotide-Based Drugs and Therapeutics belongs on the reference shelves of chemists, pharmaceutical scientists, chemical biologists, toxicologists and other scientists working in the pharmaceutical and biotechnology industries. It will also be a valuable resource for regulatory specialists and safety assessment professionals and an important reference for academic researchers and post-graduates interested in therapeutics, antisense therapy, and oligonucleotides.

Leseprobe

Oligonucleotide‐Based Drugs and Therapeutics

Preclinical and Clinical Considerations for Development

Edited by

Nicolay Ferrari and Rosanne Seguin

This edition first published 2018© 2018 John Wiley & Sons, Inc.

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Library of Congress Cataloging‐in‐Publication Data

Names: Ferrari, Nicolay, 1969– editor. | Seguin, Rosanne, editor.Title: Oligonucleotide‐based drugs and therapeutics : preclinical and clinical considerations for development / edited by Nicolay Ferrari, Rosanne Seguin.Description: Hoboken, NJ : John Wiley & Sons, 2018. | Includes bibliographical references and index. |Identifiers: LCCN 2018006576 (print) | LCCN 2018009683 (ebook) | ISBN 9781119070290 (pdf) | ISBN 9781119070306 (epub) | ISBN 9781118537336 (cloth)Subjects: LCSH: Oligonucleotides–Therapeutic use. | Antisense nucleic acids–Therapeutic use.Classification: LCC RM666.N87 (ebook) | LCC RM666.N87 O445 2018 (print) | DDC 572.8/5–dc23LC record available at https://lccn.loc.gov/2018006576

Cover Design: WileyCover Images: © nechaev‐kon/Getty Images;© Michal Sanca/Shutterstock

List of Contributors

Annemieke Aartsma‐RusDepartment of Human GeneticsLeiden University Medical CenterLeidenThe Netherlands

Scott A. BarrosSage TherapeuticsCambridge, MAUSA

Cindy L. BermanBerman ConsultingWayland, MAUSA

Kevin BrownFluidigm CorporationSouth San Francisco, CAUSA

Joy CavagnaroAccess BIOBoyce, VAUSA

Scott CormackOncoGenex PharmaceuticalsBothell, WAUSA

Beth E. DavisDepartment of MedicineUniversity of SaskatchewanSaskatoon, SaskatchewanCanada

Mehrdad DirinDepartment of Pharmaceutical ChemistryUniversity of ViennaVienna, Austria

Sheila M. GallowayMRL, Merck & Co., Inc.West Point, PAUSA

Gail M. GauvreauDepartment of MedicineMcMaster UniversityHamilton, OntarioCanada

Isabella GazzoliDepartment of Human GeneticsLeiden University Medical CenterLeidenThe Netherlands

Alain GuimondInSymbiosisMontrealQuebecCanada

Lydia HaileCentre for Drug Evaluation and Research, Office of New DrugsUS FDA Silver Spring, MDUSA

Jonathan HallETH‐ZurichZurichSwitzerland

Derek IrelandCentre for Drug Evaluation and Research, Office of BiotechnologyProducts, US FDASilver Spring, MDUSA

Aimee L. JacksonmiRagen TherapeuticsBoulder, COUSA

Cindy JacobsOncoGenex PharmaceuticalsBothell, WAUSA

Piotr J. KamolaGlaxoSmithKline R&DWareHertfordshireUKCurrent addressRIKEN Center for Integrative Medical SciencesYokohamaJapan

Lauren KaneGlaxoSmithKline R&DStevenageHertfordshireUKCurrent addressMRC Human Genetics UnitInstitute of Genetics and Molecular MedicineUniversity of EdinburghScotland

Peter KasperFederal Institute for Drugs and Medical Devices (BfArM)BonnGermany

Jeremy D. A. KitsonGlaxoSmithKline R&DStevenageHertfordshireUK

Doug KornbrustPreclinsightReno, NVUSA

Monica KriegerOncoGenex PharmaceuticalsBothell, WAUSA

Arthur A. LevinAvidity BiosciencesLa Jolla, CAUSA

Helen LightfootETH‐ZurichZurichSwitzerland

John MartucciCentre for Drug Evaluation and Research, Office of BiotechnologyProducts, US FDASilver Spring, MDUSA

Xin MingDivision of Molecular Pharmaceutics, UNC Eshelman School of PharmacyUniversity of North CarolinaChapel Hill, NCUSA

Nicolay Ferrari Centre de recherche du CHUM – Tour VigerMontreal, QuebecCanada

Frederick B. OlesonIndependent ConsultantConcord, MAUSA

John Paul OliveriaDepartment of MedicineMcMaster UniversityHamilton, OntarioCanada

Catherine C. PriestleyInnovative Medicines & Early DevelopmentAstraZenecaCambridgeUK

Montserrat PuigCentre for Drug Evaluation and Research Office of BiotechnologyProducts, US FDASilver Spring, MDUSA

Michael J. SchlosserMSR Pharma Services, Inc.Lincolnshire, ILUSA

Anneliese SchneiderPreclinical Services & ConsultingMunichGermany

Rosanne SeguinMontreal Neurological InstituteMcGill UniversityMontrealQuebecCanada

Zhanna SobolPfizer Inc.Groton, CTUSA

Patricia S. StewartOncoGenex PharmaceuticalsBothell, MAUSA

Kevin S. SwederForensic and National Security Sciences InstituteSyracuse UniversitySyracuse, NYUSA

Daniela VerthelyiCentre for Drug Evaluation and Research, Office of BiotechnologyProducts, US FDASilver Spring, MDUSA

Stefan VonhoffNOXXON Pharma AGBerlinGermany

Jonathan K. WattsRNA Therapeutics Institute and Department of Biochemistry and Molecular PharmacologyUniversity of Massachusetts Medical SchoolWorcester, MAUSA

Tacey E.K. WhiteAclairo Pharmaceutical Development Group, Inc., Vienna, VAUSA

Johannes WinklerDepartment of CardiologyMedical University of ViennaVienna, AustriaDepartment of Pharmaceutical ChemistryUniversity of ViennaVienna, Austria

Karen D. WisontOncoGenex PharmaceuticalsBothell, WA,USA

Preface

Development of oligonucleotide (ODN)‐based therapeutics is being progressed for a wide range of indications and using various routes of administration. There is a diversity of structures, chemistries, and mechanisms of actions for ODN therapeutics, but most of the members of this class of drug candidates can be categorized on the basis of whether they target either mRNA or proteins. ODN‐based therapy is distinct from gene therapy as it does not involve the modification of genes. Antisense ODN (ASO), short interfering RNA (siRNA), antagomirs, microRNA mimetics, and DNAzymes are part of the RNA‐targeting group, while immunostimulatory sequences (ISS), aptamers, and decoys are members of the protein‐targeting group.

Currently, six ODN‐based pharmaceuticals, including four ASO, have achieved marketing authorization in Europe and/or United States, and many more are undergoing late‐stage clinical testing. The first ASO drug, VITRAVENE (fomivirsen, Ionis Pharmaceuticals – formerly Isis), was approved in 1998 to treat CMV eye infections in HIV patients but within a few years was rendered obsolete by advances in antiretroviral cocktails for HIV therapy. The field waited 15 years for another approval. In 2013, the second ASO drug, KYNAMRO (mipomersen, Ionis Pharmaceuticals), was approved by the Food and Drug Administration (FDA) for the treatment of familial hypercholesterolemia. In 2016, out of 22 new drugs approved by FDA, 3 were for ODN therapeutics: DEFITELIO (defibrotide, Jazz Pharmaceuticals), a treatment for veno‐occlusive disease of the liver in individuals who have undergone bone marrow transplants granted in March; EXONDYS 51 (eteplirsen, Sarepta Therapeutics), a treatment for Duchenne muscular dystrophy granted in August; and SPINRAZA (nusinersen, Biogen), a treatment for spinal muscular atrophy granted in December. In addition, Atlantic Pharmaceuticals is developing alicaforsen, an ASO targeting ICAM‐1 for the treatment of pouchitis, and currently supplies alicaforsen in response to physicians’ requests under international named patient supply regulations for patients with inflammatory bowel disease. In January 2017, Atlantic announced it received agreement from the FDA to initiate a rolling submission of its New Drug Application for alicaforsen to treat pouchitis ahead of data from an ongoing phase III study, which is expected at the end of 2018.

The recent ODN approvals are indicative of the enthusiasm, vigor, and vitality of the field observed in recent years. There are currently over 100 companies combining hundreds of ODN programs. In 2015 alone, there were more than 35 Investigational New Drug submissions for ODN candidates. More than 145 ODN clinical trials are listed on ClinicalTrials.gov, 31 of which are active/recruiting. The diverse types of indications for which ODN therapies have been approved and for those currently in clinical development demonstrate that these therapies are not a “one‐off” development but rather are poised to claim their space in the apothecary of pharmaceuticals.

The advancement of a growing number of ODN programs, in particular ASO, in late stage of clinical development and the rapid pipeline expansion by various companies are testament of the progress, much of which was made in the 15 years between first and second drug approvals, in understanding the pharmacologic, pharmacokinetic, and toxicologic properties, as well as improving the delivery of ODN. There are now numerous examples of pharmacologic activity in animal models, and evidence of antisense activity in patients has been demonstrated in clinical trials.

The discovery of novel therapeutics is an inherently complex and interdisciplinary process, requiring close integration of scientists from several disciplines in an environment in which lessons are shared and taught across an organization.

The purpose of this book is to review the current state of knowledge of ODN and to examine the scientific principles and the tools utilized by scientists in preclinical and clinical settings as applied to ODN therapeutics.

Acknowledgments

We have embarked on this endeavor without anticipating the long twisting road that was ahead of us in putting this book together. We would like to give our heartfelt thanks to all authors. As editors, we were depending on their goodwill, commitment, and patience. We hope that their contribution will offer a useful review of the current understanding and recent advances in the field. In light of the challenges we are facing with this technology, we also hope the knowledge summarized in this book will provide guidance and will support those readers currently working in the field as well as the future developers that will further advance oligonucleotide therapeutics.

Nicolay Ferrari and Rosanne Seguin

1Mechanisms of Oligonucleotide Actions

Annemieke Aartsma‐Rus1, Aimee L. Jackson2, and Arthur A. Levin3

1 Department of Human Genetics, Leiden University Medical Center, Leiden, The Netherlands

2 miRagen Therapeutics, Boulder, CO, USA

3 Avidity Biosciences, La Jolla, CA, USA

1.1 Introduction

The promise of antisense oligonucleotide (ASO) therapeutics is the ability to design drugs that are specific inhibitors of the expression solely on the basis of Watson and Crick base‐pairing rules. The premise is that treatment of a patient with a DNA‐like oligonucleotide complementary to a disease‐related RNA (usually a messenger RNA) results in the formation of a heteroduplex that inhibits the function (generally translation) of that target RNA. Although antisense RNAs were first described in 1978 [1, 2], until recently the promise of selectivity and efficacy has always remained slightly out of reach for various reasons. Oligonucleotides are large molecules leading to synthesis and delivery issues. In addition, natural DNA and RNA oligonucleotides are rapidly degraded and cleared after systemic delivery. Over time many of the issues that have challenged developers of oligonucleotide‐based therapeutics have been addressed: Synthesis costs have been reduced by orders of magnitude over the past two decades, allowing more investigators to use the technology. Stability issues were addressed partially with the introduction of phosphorothioate backbones (reviewed in Ref. [3]) and later sugar modifications (reviewed in Ref. [4]), and, as a result, oligonucleotides now used clinically and preclinically have more conventional drug‐like properties [5]. In addition, fundamental discoveries have improved our understanding of the antisense mechanisms. We now know that target RNA structure and accessibility impacts activity of oligonucleotide therapeutics [6] and therefore pharmacologic activity. Apparently small changes in oligonucleotide chemistry can also have large pharmacologic effects as analyzed at the phenomenological level [7] and at the quantum level [8].

Our ability to design effective oligonucleotide‐based drugs has also been enhanced by studies of the molecular mechanisms of these agents. This chapter reviews the mechanisms of action, the chemistry, and the clinical applications of three broad categories of oligonucleotide therapeutics: antisense agents, splicing modifiers, and gene silencers that activate the RNA interference (RNAi) pathway.

Antisense technology has now produced dozens of clinical stage drugs and two approvals. That hybridization of an oligonucleotide to a pre‐mRNA could modulate the splicing of that RNA was described in 1993 [9], and the therapeutic potential of that mechanism is being exploited to treat Duchenne muscular dystrophy and now other diseases (see below). Running at first behind but more recently in parallel with applications of single‐stranded ASO agents is the use of double‐stranded RNA‐like molecules that activate the RNA‐induced silencing complex (RISC) to cleave targeted mRNAs or interfere with their translation. Synthetic small interfering RNA (siRNA) therapeutics relies on the same mechanism that is used by eukaryotic cells to control mRNA translation by endogenous microRNAs (miRNAs).

1.2 Antisense Oligonucleotide Therapeutics

1.2.1 Antisense Activity Mediated by RNase H

Zamecnik and Stephenson [1, 2] were the first to describe that a DNA strand complementary to a sequence of an mRNA prevented translation. They observed that an ASO prevented the accumulation of Rous sarcoma virus by inhibiting the translation of proteins encoded by the viral mRNA. A whole new potential field of therapeutics was launched with a single (understated) sentence: “It might also be possible to inhibit the translation of a specific cell protein” [1]. That RNase H was responsible for the inhibitor effects on translation was a conclusion reached by multiple investigators over a period of time. An elegant proof of the role of this specific enzyme in antisense activity was provided by Wu et al. in 1999 [10]; these authors showed that modulation of RNase H levels in cells or animals produces a coordinate change in antisense activity.

1.2.2 The RNase H Mechanism

Members of the RNase H family are ubiquitously expressed. The endonuclease mechanism of action and the crystal structure have been reviewed [10–15]. RNase H is approximately 20 kDa, and the isoforms in mammalian cells are known to have distinct functions. RNase H1 is necessary for transcription, and RNase H2 is thought to remove RNA primers in the replication of DNA [16]. The RNA binding domain of these enzymes is located at the N‐terminus. The catalytic activity is located in a C‐terminal domain and depends upon the presence of the 2′ hydroxyl on the ribose sugar for cleavage. The specificity of the enzyme is imparted by heteroduplex formation between a DNA and the targeted RNA. Thus, the enzyme does not cleave single‐stranded RNA in the absence of a heteroduplex nor does it cleave DNA in a double strand because of the absence of the critical 2′ OH.

Binding of RNase H to the heteroduplex results in hydrolysis of the RNA at a site distal to the binding region. The enzyme has a DNA recognition site into which a phosphate fits. This phosphate on the DNA strand is two base pairs distal to the cleavage site on the RNA. This DNA binding and recognition is a factor in the recognition of the heteroduplex. The heteroduplex landing site must contain at least five 2′ OH groups, and the position of RNA cleavage is approximately one helical turn from the binding domain [15]. The distance of the cleavage site from the RNA binding site is determined by a spacer domain between the binding domain and the catalytic domain [11–13]. The enzyme extends across a groove in the helix formed by heteroduplex to cut the RNA. Catalysis requires the presence of two metal ions (Mg2+ or Mn2+), which activate the nucleophile and stabilize the transition state during hydrolysis of the phosphodiester backbone of the RNA substrate. One metal ion serves to stabilize the transition state, and the other acts during strand transfer [15, 17].

Over a decade after RNase H antisense drugs had been in clinical trials, the identity of the specific RNase family member responsible for the mRNA cleavage remained unproven. By modulating the expression of human RNase H1 and RNase H2, Wu et al. [10] demonstrated that RNase H1 was associated with antisense activity in vivo. Antisense drug activity increased with RNase H1 overexpression and decreased with RNase H1 inhibition. The same was not true for RNase H2, demonstrating that the form of the enzyme associated with therapeutic activity is RNase H1.

1.2.3 Chemical Modifications to Enhance RNase H‐mediated Antisense Activity

RNase H is rather intolerant to chemical modifications to the DNA strand, and, as a result, ASO drugs that work through the RNase H mechanism must have a DNA‐like character in certain nucleotides. One modification tolerated by RNase H is the phosphorothioate linkage: a substitution of a nonbridging sulfur for the phosphodiester linkage between nucleotides. First described by Eckstein and due to the increased stability of the phosphorothioate linkage compared with the native phosphodiester linkage, the phosphorothioate is the most used chemical modification in ASO and siRNA agents. The substitution with sulfur increases the nuclease stability (reviewed in Ref. [3]) and has the added effect of increasing protein binding. This substitution also creates a chiral center at the phosphate. The increased nuclease resistance results from the fact that one of the two diastereomers is highly resistant to nuclease activity, probably as a result of the sulfur being in closer proximity to the metal ions of nucleases in the Sp configuration.

The phosphorothioate modification significantly alters the properties of an oligonucleotide compared with a native DNA oligonucleotide. Plasma half‐lives are extended in the phosphorothioate‐modified oligonucleotide due both to increased resistance to nucleases and to enhanced binding to plasma proteins. This later effect is both a blessing and a curse in that some of the acute toxicities of phosphorothioate oligonucleotides have been associated with binding to plasma proteins [18]. Ironically, whereas the phosphorothioate linkage is tolerated by RNase H, high concentrations of a phosphorothioate oligonucleotide are inhibitory to the enzyme’s activity [19, 20]. Thus phosphorothioate linkages must be used strategically to balance in delivery with toxicity to the organism and to the very enzyme that is responsible for the pharmacologic activity. A large number of chemical modifications to oligonucleotides have been tested with the goals of increasing potency to lower toxicity and reduce the potential for RNase H inhibition.

Because of the intolerance of the RNase mechanism for chemical modifications, a scheme was developed that ensured that the ASO retained a DNA‐like character. In the so‐called chimeric design [21], the central region has nucleotides with DNA‐like character (usually natural bases and sugars and a phosphorothioate backbone), and the flanking regions are modified with the aim of increasing affinity to the mRNA target and enhancing nuclease resistance. This modification pattern is also dubbed the gapmer design for the deoxy characteristic of the region between the modified termini (the gap). The size of the region required for RNase recognition and binding must be at least five nucleotides [22]. The minimal binding site size may be larger depending on the nature of the modifications flanking these deoxynucleotides. Crooke and his group have demonstrated that the nature of the 2′ sugar modifications (e.g. 2′methoxy ethyl) influences RNase H activity by changing the conformation of the oligonucleotide–mRNA heteroduplex. The conformational change in a heteroduplex is transmitted for some distance from the 2′ modification. A typical gapmer design has approximately 8–12 central DNA‐like residues. One of the factors that hamper the activity of phosphorothioate oligonucleotides that have been internalized by cells is protein binding and sequestration of the antisense molecule away from its target protein RNase H. Recent studies have begun the task of identifying these proteins, which is the first step to being able to exploit them for improving therapeutics [23].

Recognition and binding of the antisense drug to the RNA target are of course critical for the activity of antisense therapeutics. A host of different chemical modifications have been tested over the years with the goal of increasing binding affinity (reviewed in Ref. [24]). Addition of steric bulk at the 2′ position has the effect of producing a northern‐type sugar conformation. This conformation is inhibitory to RNase H but may allow for better hydrogen bonding, thus resulting in increases in affinity for the target RNA. Conformationally restricted nucleic acids, such as LNA, or bicyclic nucleic acids (BNAs) are extreme examples of conformational restriction that result in high affinity for a complementary RNA strand.

Wengel et al. [25] described a modification that has the opposite effect in that the sugar no longer cyclizes but is acyclic (or unlocked), which promotes flexibility. These unlocked forms can be useful when it is in the drug designer’s interest to reduce the potential for binding to an RNA target. These acyclic nucleotides support RNase H cleavage [26]. The 2′ arabino fluoro nucleotides also support RNase H binding and cleavage and are thus a potential modification that can be used anywhere in an ASO increase affinity to target mRNA [27].

1.3 Oligonucleotides that Sterically Block Translation

Single‐stranded ASOs may also act independently of RNase H to block translation or processing of pre‐mRNA. Subsequent sections of this chapter will discuss oligonucleotides designed to alter splicing. There are also reports of steric blockers that are inhibited in cell‐free translation systems and in cells; ASO modified to inhibit RNase H activity that hybridizes with the region that includes the AUG start codon very effectively block protein synthesis. More recently an alternative strategy for blocking mRNA function through the inhibition of polyadenylation was proposed by Gunderson [28]. By selecting an oligonucleotide that has homology to the U1 adapter small nuclear RNA and homology to the sequence in the 3′ terminus of the target mRNA, it is possible to get a duplex formed where polyadenylation should occur and subsequently block the polyadenylation step that is critical for mRNA function. Without polyadenylation the nascent mRNA is degraded.

1.4 Oligonucleotides that Act Through the RNAi Pathway

1.4.1 The RISC Pathway

Small interfering RNAs (siRNAs) and miRNAs are duplexes of 20–30 base pairs that regulate gene expression and control a diverse array of biological processes. These small RNAs exert their function through the formation of ribonucleoprotein complexes called RISCs that are instrumental in target transcript regulation. Therapeutic modulation of target regulation by siRNAs and miRNAs has the potential to impact diverse disease indications including viral diseases, cardiovascular disease, fibrosis, and cancer. Understanding of the function and modulation of these small regulatory RNAs has progressed at a rapid pace, resulting in translation to therapeutic development in only 10 years since their initial characterization.

In 1993, the cloning of lin‐4 in Caenorhabditis elegans marked the discovery that a short (~22 nt) RNA could function as a regulatory molecule and regulate translation via an antisense RNA–RNA interaction [29]. Within a few years, it became clear that endogenously expressed miRNAs are abundant and evolutionarily conserved and play diverse roles in gene expression in species from worms to humans [29–32]. The discovery by Fire and Mello in 1998 that short double‐stranded RNAs induce gene silencing in C. elegans[33] further revolutionized our understanding of gene regulation and the ability of RNAs to function as regulatory molecules. Shortly thereafter, short interfering RNAs (siRNAs) were shown to guide sequence‐specific target silencing in plants [34], Drosophila[35, 36], and mammalian cells [37, 38] in a conserved process termed RNAi. The ability of miRNAs and siRNAs to trigger specific gene silencing generated significant excitement of these small RNAs as a therapeutic modality, particularly for targets that are considered to be “undruggable” with small molecules.

miRNAs bind target mRNAs with partial sequence complementarity in the 3′ UTR, mostly involving residues 2–8 (the seed sequence) at the 5′ end of the guide strand [39]. Seed pairing has been shown to be both necessary and sufficient for target regulation by miRNAs in some contexts [40–43], although sequences in addition to the seed can also be important [44–47]. Because miRNAs do not require perfect complementarity for target recognition, a single miRNA can regulate expression from numerous mRNAs [48–50]. It is estimated that miRNAs as a class regulate the expression of 60% of genes in the human genome [51] to control differentiation, development, and physiology. Altered expression or function of miRNAs is linked to human diseases, giving rise to the idea that selective therapeutic modulation of miRNAs could alter the course of disease. The therapeutic inhibition of a miRNA or addition of a miRNA mimetic might produce a phenotype that is derived from a complex set of gene expression changes. The regulation of coordinated gene networks distinguishes miRNAs and their modulation as a therapeutic modality and provides a therapeutic advantage suggestive of combination therapy. Therapeutically, miRNA mimetics can be utilized to restore activity of miRNAs whose loss of function is linked to disease, whereas miRNA inhibitors (called antimiRs or antagomirs) can be used to block activity of miRNAs whose gain of function is linked to disease. A miRNA mimetic is a duplex oligonucleotide analogous to the mature miRNA. An antimiR is a single‐stranded oligonucleotide that is complementary to the miRNA and is designed to act as a steric block by binding with the miRNA to prevent it from interacting with target mRNA. Consequently, target transcripts are more highly expressed.

Both miRNAs and siRNAs are processed from double‐stranded RNA precursors by the RNase III enzymes Drosha and Dicer to yield the mature, approximately 22‐nt, double‐stranded RNA [52–54]. Mature miRNAs and siRNAs catalyze gene regulation in complex with a ribonucleoprotein complex called the RISC. The catalytic component of RISC is a member of the Argonaute (Ago) family. Because small RNAs in RISC must anneal to their complementary target mRNAs, one strand, termed the guide strand, is retained in RISC and provides the sequence specificity to guide mRNA silencing. The other strand, termed the passenger strand, is cleaved. The process of strand selection is termed RISC loading. Strand selection is not random. Strand choice is partly encoded in the intrinsic structure of the small RNA duplex, with thermodynamic properties being a major determinant [55, 56]. Unwinding of the duplex, selection of the guide strand and cleavage of the passenger strand are facilitated by the Argonaute protein [57, 58] in an ATP‐dependent process [59–63].

The most important domain of the guide strand is the seed sequence, which is the primary determinant of binding specificity for both siRNAs and miRNAs [39, 45, 49, 64–66]. Structural analysis of RNA associated with Argonaute provided insight into the role of the 5′ seed region of the guide strand in sequence‐specific pairing with target mRNA [67–69]. The phosphorylated 5′ end of the guide RNA serves as the anchor and is buried within a highly conserved basic pocket in the Mid domain of Argonaute. In contrast, the seed region is exposed and displayed in a prehelical structure that favors the formation of a duplex with the target mRNA. Systematic mutation analysis of siRNA guide strands elucidated distinct siRNA guide domains within Argonaute [70]. Consistent with the structural analysis, mismatches between position 1 of the guide and the target RNA do not impair catalytic activity of Argonaute [66, 70], whereas mismatches within the seed regions reduce target binding and hinder target silencing [70, 71]. Mismatches at the center of the seed region (positions 4 and 5) are more detrimental than mismatches at the periphery (positions 2, 7, and 8), perhaps explaining how some small RNAs, including miRNAs, can regulate targets through imperfect seed matching [45, 72].

Of the four Argonaute proteins in mammals, only one, Ago2, has endonuclease activity [73]. Target cleavage occurs at the nucleotide opposite positions 10 and 11 of the siRNA guide strand, and mismatches or chemical modifications at these positions considerably decrease catalytic activity [37, 38, 74–76]. Pairing with the guide strand positions the scissile phosphate of the target near the catalytic residues in the PIWI domain of Ago2 [37, 66, 74, 77, 78]. siRNAs tend to be perfectly complementary to the target mRNA, and this pairing might enable Argonaute to achieve a catalytically competent conformation [66]. miRNAs typically lack significant pairing in the 3′ portion of the guide strand, although such supplemental base pairing can compensate for a weak seed region [79].

1.4.2 Mechanisms of RISC‐mediated Gene Silencing

siRNAs and miRNAs guide RISC to target mRNAs in a sequence‐dependent manner and subsequently affect one of three facets of mRNA metabolism: cleavage/destabilization, translation, or mRNA localization. In Drosophila, the ultimate fate of the target mRNA depends in part on the Argonaute protein and in part on the small RNA associated with RISC. There does not seem to be a strict small RNA sorting system in human RISC loading, perhaps because the four Ago proteins in humans have largely redundant functions.

siRNAs guide Ago2‐containing RISC to complementary mRNA, whereupon the mRNA is degraded via endonucleolytic cleavage [80, 81]. The siRNA–RISC complex is subsequently released and able to bind and cleave another target mRNA molecule in a catalytic process. The power of RNAi arises from the discovery that the endogenous gene‐silencing machinery can be conscripted by synthetic siRNAs for selective silencing of a gene of interest [38, 74]. In theory, siRNAs can be designed to silence any gene of interest based solely on the sequence of the target mRNA. Efficacy and potency of target silencing can be enhanced by leveraging thermodynamics, 5′ nucleotide identity, and structure to bias for guide strand selection [55, 82]. Well‐designed siRNAs can achieve 95% silencing of the intended target.

Early reports suggested that siRNAs were absolutely specific for the target gene of interest. Target genes were silenced by complementary siRNAs but not unrelated siRNAs [83, 84], and silencing was abolished by single‐nucleotide mismatches at the cleavage site of the siRNAs [74, 77, 78]. Subsequently, unbiased genome‐scale expression profiling has revealed off‐target activity of siRNAs [85]. Off‐target silencing is mediated by limited target complementarity to the siRNA, primarily in the seed region [71], reminiscent of miRNA‐based target repression. Sequence analysis of off‐target transcripts revealed that the 3′ UTRs of these transcripts were complementary to the 5′ end of the siRNA guide strand containing the seed region [85]. Therefore, in addition to the intended, fully complementary target transcript, siRNAs can hybridize to and regulate the expression of transcripts with only partial sequence complementary to the siRNA. Interestingly, base mismatches in the 5′ end of a siRNA guide strand reduced silencing of the original set of off‐target transcripts, but introduced a new set of off‐target transcripts with complementarity to the mismatched guide strand [71]