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• W2795531081 abstract "RNA-targeted therapies represent a platform for drug discovery involving chemically modified oligonucleotides, a wide range of cellular RNAs, and a novel target-binding motif, Watson-Crick base pairing. Numerous hurdles considered by many to be impassable have been overcome. Today, four RNA-targeted therapies are approved for commercial use for indications as diverse as Spinal Muscular Atrophy (SMA) and reduction of low-density lipoprotein cholesterol (LDL-C) and by routes of administration including subcutaneous, intravitreal, and intrathecal delivery. The technology is efficient and supports approaching “undruggable” targets. Three additional agents are progressing through registration, and more are in clinical development, representing several chemical and structural classes. Moreover, progress in understanding the molecular mechanisms by which these drugs work has led to steadily better clinical performance and a wide range of mechanisms that may be exploited for therapeutic purposes. Here we summarize the progress, future challenges, and opportunities for this drug discovery platform. RNA-targeted therapies represent a platform for drug discovery involving chemically modified oligonucleotides, a wide range of cellular RNAs, and a novel target-binding motif, Watson-Crick base pairing. Numerous hurdles considered by many to be impassable have been overcome. Today, four RNA-targeted therapies are approved for commercial use for indications as diverse as Spinal Muscular Atrophy (SMA) and reduction of low-density lipoprotein cholesterol (LDL-C) and by routes of administration including subcutaneous, intravitreal, and intrathecal delivery. The technology is efficient and supports approaching “undruggable” targets. Three additional agents are progressing through registration, and more are in clinical development, representing several chemical and structural classes. Moreover, progress in understanding the molecular mechanisms by which these drugs work has led to steadily better clinical performance and a wide range of mechanisms that may be exploited for therapeutic purposes. Here we summarize the progress, future challenges, and opportunities for this drug discovery platform. The notion of creating “antisense” oligonucleotide-based drugs, first enunciated in 1978 (Stephenson and Zamecnik, 1978Stephenson M.L. Zamecnik P.C. Inhibition of Rous sarcoma viral RNA translation by a specific oligodeoxyribonucleotide.Proc. Natl. Acad. Sci. USA. 1978; 75: 285-288Crossref PubMed Google Scholar), is seductive because the forces that determine whether an oligonucleotide binds to its cognate RNA sequence, Watson-Crick hybridization, are well understood and because such drugs should, in principle, be much more specific than traditional small-molecule drugs. In common with other new platforms for drug discovery, the reduction of the concept to practice has taken three decades and numerous inventions (Crooke, 2008Crooke S.T. Antisense Drug Technology: Principles, Strategies, and Applications. CRC Press, 2008Google Scholar). With four RNA-targeting drugs now approved for commercial use and scores in clinical development, the technology has begun to yield the dividends that were originally contemplated. Serendipitously, as progress in developing these novel therapeutics was recorded, the opportunities for RNA targeting multiplied as new classes of RNAs and new roles for RNAs were discovered (Cech and Steitz, 2014Cech T.R. Steitz J.A. The noncoding RNA revolution-trashing old rules to forge new ones.Cell. 2014; 157: 77-94Abstract Full Text Full Text PDF PubMed Scopus (912) Google Scholar, Matsui and Corey, 2017Matsui M. Corey D.R. Non-coding RNAs as drug targets.Nat. Rev. Drug Discov. 2017; 16: 167-179Crossref PubMed Scopus (178) Google Scholar; see Figure 1). The purpose of this review is to summarize the current status of the technology and to consider future opportunities and challenges. In order to exploit the opportunity to use Watson-Crick hybridization of oligonucleotide analogs to RNA, the medicinal chemistry of oligonucleotides needed to be invented (Figure 2A ). Most modifications have focused on increasing the affinity per nucleotide unit for the cognate sequence and/or on enhancing resistance to nucleases, the enzymes that degrade these drugs (Bennett and Swayze, 2010Bennett C.F. Swayze E.E. RNA targeting therapeutics: molecular mechanisms of antisense oligonucleotides as a therapeutic platform.Annu. Rev. Pharmacol. Toxicol. 2010; 50: 259-293Crossref PubMed Scopus (739) Google Scholar, Levin et al., 2008Levin A.A. Yu R.Z. Geary R.S. Basic principles of the pharmacokinetics of antisense oligonucleotide drugs.in: Crooke S.T. Antisense Drug Technology: Principles, Strategies, and Applications. CRC Press, 2008: 183-215Google Scholar, Swayze and Bhat, 2008Swayze E.E. Bhat B. The medicinal chemistry of oligonucleotides.in: Crooke S.T. Antisense Drug Technology: Principles, Strategies, and Applications. CRC Press, 2008: 143-182Google Scholar). This effort has resulted in several chemical classes that have proven to be clinically effective and progressively more potent and better tolerated (Figure 2B). More recently, research has focused on enhancing productive delivery to specific cell types by conjugation of moieties that take advantage of high-capacity receptors such as the asialoglycoprotein receptor expressed by hepatocytes (Nair et al., 2014Nair J.K. Willoughby J.L. Chan A. Charisse K. Alam M.R. Wang Q. Hoekstra M. Kandasamy P. Kel’in A.V. Milstein S. et al.Multivalent N-acetylgalactosamine-conjugated siRNA localizes in hepatocytes and elicits robust RNAi-mediated gene silencing.J. Am. Chem. Soc. 2014; 136: 16958-16961Crossref PubMed Scopus (347) Google Scholar, Prakash et al., 2014Prakash T.P. Graham M.J. Yu J. Carty R. Low A. Chappell A. Schmidt K. Zhao C. Aghajan M. Murray H.F. et al.Targeted delivery of antisense oligonucleotides to hepatocytes using triantennary N-acetyl galactosamine improves potency 10-fold in mice.Nucleic Acids Res. 2014; 42: 8796-8807Crossref PubMed Scopus (199) Google Scholar, Prakash et al., 2016bPrakash T.P. Yu J. Migawa M.T. Kinberger G.A. Wan W.B. Østergaard M.E. Carty R.L. Vasquez G. Low A. Chappell A. et al.Comprehensive structure-activity relationship of triantennary n-acetylgalactosamine conjugated antisense oligonucleotides for targeted delivery to hepatocytes.J. Med. Chem. 2016; 59: 2718-2733Crossref PubMed Scopus (49) Google Scholar; see Figure 2B). Whether an RNA targeting oligonucleotide is single or double stranded has a considerable influence on performance. ASOs are single stranded, while siRNAs are double stranded, containing a sense and an antisense strand (Figure 2C). In an siRNA, the antisense strand is the pharmacologically active moiety and the sense strand can be considered a “drug delivery device” that transports the antisense strand to the intracellular RNA endonuclease Ago2 (Crooke et al., 2008Crooke S.T. Vickers T. Lima W. Wu H. Mechanisms of antisense drug action, an introduction.in: Crooke S.T. Antisense Drug Technology: Principles, Strategies, and Applications. CRC Press, 2008: 3-46Google Scholar). ASOs containing phosphorothioate (PS) substitutions distribute broadly after systemic administration (Geary et al., 2008Geary R.S. Yu R.Z. Siwkowski A. Levin A.A. Pharmacokinetic/pharmacodynamic properties of phosphorothioate 2′-O-(2-methoxyethyl)-modified antisense oligonucleotides in animals and man.in: Crooke S.T. Antisense Drug Technology: Principles, Strategies, and Applications. CRC Press, 2008: 305-326Google Scholar, Levin et al., 2008Levin A.A. Yu R.Z. Geary R.S. Basic principles of the pharmacokinetics of antisense oligonucleotide drugs.in: Crooke S.T. Antisense Drug Technology: Principles, Strategies, and Applications. CRC Press, 2008: 183-215Google Scholar). Single-stranded PS-substituted ASOs are amphipathic and bind to proteins in the serum, in the cell surface, and intracellularly. These interactions facilitate cell uptake and distribution such that ASOs of this type can be given by nearly all routes of administration in saline and distribute to most tissues in the body. In contrast, double-stranded siRNAs, which are also polyanions and hydrophilic, do not bind to serum proteins and are rapidly excreted; thus, they must either be formulated in lipids or other types of nanoparticles or be chemically modified and conjugated to a moiety that interacts with a high-capacity cell-surface receptor for effective tissue delivery (Dowdy, 2017Dowdy S.F. Overcoming cellular barriers for RNA therapeutics.Nat. Biotechnol. 2017; 35: 222-229Crossref PubMed Scopus (240) Google Scholar). Because individual RNA targeting oligonucleotides within a specific chemical class differ only in sequence, the members of each class have similar physico-chemical characteristics and thus common pharmacokinetic and biological properties. However, every chemical class differs, and even subtle modifications that appear to scientists not experienced in the field as chemically similar—e.g., 2′-O-methoxyethyl (2′-MOE) versus 2′-methoxy—can result in substantial changes in potency, pharmacokinetics, and generic chemical class effects (Bennett and Swayze, 2010Bennett C.F. Swayze E.E. RNA targeting therapeutics: molecular mechanisms of antisense oligonucleotides as a therapeutic platform.Annu. Rev. Pharmacol. Toxicol. 2010; 50: 259-293Crossref PubMed Scopus (739) Google Scholar). So, it is essential to precisely define the chemistry of RNA-targeted oligonucleotides. Figure 2 shows the major chemical classes that are in development and summarizes their properties. One informative way to think of oligonucleotides designed to bind to their cognate sequences in RNA is that they are agents that alter the complex intermediary metabolism of mRNAs; this begins with transcription of a pre-mRNA and includes RNA processing, transport, and utilization by translation followed by degradation (Crooke et al., 2008Crooke S.T. Vickers T. Lima W. Wu H. Mechanisms of antisense drug action, an introduction.in: Crooke S.T. Antisense Drug Technology: Principles, Strategies, and Applications. CRC Press, 2008: 3-46Google Scholar). That these agents are designed to alter the intermediary metabolism of mRNAs (Figure 3) implies that the rates of steps in mRNA intermediary metabolism and the rates of drug action are important in defining the ultimate pharmacological effects observed (Vickers and Crooke, 2015Vickers T.A. Crooke S.T. The rates of the major steps in the molecular mechanism of RNase H1-dependent antisense oligonucleotide induced degradation of RNA.Nucleic Acids Res. 2015; 43: 8955-8963Crossref PubMed Scopus (24) Google Scholar). This has proven to be true (see below). Double-stranded oligonucleotides (siRNAs) are pro-drugs. In fact, the sense strand of the siRNA meets the formal definition of a drug delivery device; it is non-covalently associated with the drug (the antisense strand), protects the antisense strand from degradation, and must be removed before the antisense strand can induce a pharmacological effect (Sigova and Zamore, 2008Sigova A. Zamore P.D. Small RNA silencing Pathways.in: Crooke S.T. Antisense Drug Technology: Principles, Strategies, and Applications. CRC Press, 2008: 75-88Google Scholar). Unfortunately, although the sense strand is important in facilitating interactions with the Ago2 loading complex, it is not an ideal drug delivery vehicle, as it impedes distribution and cellular uptake of siRNAs and is not inert. In fact, it may have pharmacological effects, and it is metabolized by the same enzymes, exo- and endo-nucleases, that degrade the antisense strand. The observation that double-stranded RNAs can lead to selective silencing of genes in C. elegans and plants resulted in the identification of a novel gene-regulatory pathway mediated by small RNAs (Fire et al., 1998Fire A. Xu S. Montgomery M.K. Kostas S.A. Driver S.E. Mello C.C. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans.Nature. 1998; 391: 806-811Crossref PubMed Scopus (10250) Google Scholar, Hamilton and Baulcombe, 1999Hamilton A.J. Baulcombe D.C. A species of small antisense RNA in posttranscriptional gene silencing in plants.Science. 1999; 286: 950-952Crossref PubMed Scopus (2087) Google Scholar). Fortunately, thanks to the efforts of many laboratories, the cellular pathway in which Ago2 participates and by which small RNAs reduce the levels of targeted RNAs is well understood (Daugaard and Hansen, 2017Daugaard I. Hansen T.B. Biogenesis and function of ago-associated RNAs.Trends Genet. 2017; 33: 208-219Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). This pathway includes a number of endonucleases that generate microRNAs from precursor RNAs (Xie and Steitz, 2014Xie M. Steitz J.A. Versatile microRNA biogenesis in animals and their viruses.RNA Biol. 2014; 11: 673-681Crossref PubMed Scopus (32) Google Scholar), a complex of proteins that load the microRNA into Ago2 (the loading complex), and Ago2, an RNA endonuclease that effects cleavage of a targeted mRNA (Liu et al., 2004Liu J. Carmell M.A. Rivas F.V. Marsden C.G. Thomson J.M. Song J.J. Hammond S.M. Joshua-Tor L. Hannon G.J. Argonaute2 is the catalytic engine of mammalian RNAi.Science. 2004; 305: 1437-1441Crossref PubMed Scopus (1776) Google Scholar, Meister et al., 2004Meister G. Landthaler M. Patkaniowska A. Dorsett Y. Teng G. Tuschl T. Human Argonaute2 mediates RNA cleavage targeted by miRNAs and siRNAs.Mol. Cell. 2004; 15: 185-197Abstract Full Text Full Text PDF PubMed Scopus (1234) Google Scholar); the latter is the primary mechanism by which pharmacological effects are induced by siRNAs (Soutschek et al., 2004Soutschek J. Akinc A. Bramlage B. Charisse K. Constien R. Donoghue M. Elbashir S. Geick A. Hadwiger P. Harborth J. et al.Therapeutic silencing of an endogenous gene by systemic administration of modified siRNAs.Nature. 2004; 432: 173-178Crossref PubMed Scopus (1716) Google Scholar). There are four known Argonaute proteins. Each can be loaded with microRNAs or siRNAs to result in alteration of translation and/or RNA stability, but siRNAs are designed to bind preferentially to Ago2 (Hutvagner and Simard, 2008Hutvagner G. Simard M.J. Argonaute proteins: key players in RNA silencing.Nat. Rev. Mol. Cell Biol. 2008; 9: 22-32Crossref PubMed Scopus (771) Google Scholar, Meister, 2013Meister G. Argonaute proteins: functional insights and emerging roles.Nat. Rev. Genet. 2013; 14: 447-459Crossref PubMed Scopus (486) Google Scholar). Ago2 is a member of the Ago family of proteins; it contains an RNase H domain, but cleaves RNA in an RNA-RNA duplex, not a DNA-RNA duplex. Loading of the antisense strand into Ago2 is efficient, but Ago2 imposes fairly rigid structural requirements if an antisense strand is to bind effectively. For example, a 5′ phosphate or phosphate analog is required, and a relatively limited range of 2′ modifications are tolerated at sites distal to the RNA targeting sequence, the seed sequence (Haraszti et al., 2017Haraszti R.A. Roux L. Coles A.H. Turanov A.A. Alterman J.F. Echeverria D. Godinho B.M.D.C. Aronin N. Khvorova A. 5′-Vinylphosphonate improves tissue accumulation and efficacy of conjugated siRNAs in vivo.Nucleic Acids Res. 2017; 45: 7581-7592Crossref PubMed Scopus (26) Google Scholar). The seed sequence is an 8-nucleotide sequence that identifies, by Watson-Crick hybridization, the mRNA to be degraded. The Ago2 mechanism has proven to be an efficient post-RNA-binding pharmacological mechanism with a number of important attributes. The pathway is well understood, the complex of proteins that loads Ago2 is defined, and it is clear that the Ago2 complex facilitates hybridization of the antisense strand to the target RNA (Salomon et al., 2015Salomon W.E. Jolly S.M. Moore M.J. Zamore P.D. Serebrov V. Single-molecule imaging reveals that argonaute reshapes the binding properties of its nucleic acid guides.Cell. 2015; 162: 84-95Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, Yao et al., 2015Yao C. Sasaki H.M. Ueda T. Tomari Y. Tadakuma H. Single-molecule analysis of the target cleavage reaction by the Drosophila RNAi enzyme complex.Mol. Cell. 2015; 59: 125-132Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). Once loaded, Ago2 retains the antisense strand for a prolonged period, so the duration of action can be quite prolonged. Because Ago2 is primarily localized to the cytoplasm, siRNAs effectively target cytoplasmic RNAs. As previously mentioned, the structural requirements for binding to Ago2 are known, as is the pathway facilitating drug discovery. However, from a drug discovery perspective, there are issues that must be better understood in order to develop siRNAs with optimal therapeutic indexes. The main issue is the potential lack of specificity that can result from the relatively promiscuous hybridization that an 8-nucleotide sequence generates. A second mechanism that might result in non-specific effects derives from the fact that siRNAs that can compete out microRNAs loaded onto Ago2 may lead to alteration of the half-lives of other cellular RNAs (Liang et al., 2013Liang X.H. Hart C.E. Crooke S.T. Transfection of siRNAs can alter miRNA levels and trigger non-specific protein degradation in mammalian cells.Biochim. Biophys. Acta. 2013; 1829: 455-468Crossref PubMed Scopus (24) Google Scholar). These issues are well understood at the cellular level, and to date they have not proven to be an issue in the clinic, though recent adverse events reported in short-term clinical trials have not ruled out these mechanisms (Alnylam Pharmaceuticals, 2017 RNAi Roundtable: Platform advances in RNAi Therapeutics). A number of post-binding pharmacological mechanisms have been shown to be available to single-stranded oligonucleotides. The mechanisms have been divided into effects that are induced simply by binding to a target RNA (occupancy-only mediated) and occupancy-mediated degradation of the target RNA (see Figure 3). Occupancy-only-mediated mechanisms include alterations in RNA processing, inhibition of translation, enhancement of translation, and obstruction of interactions of the target RNA with key proteins, among others (Crooke et al., 2008Crooke S.T. Vickers T. Lima W. Wu H. Mechanisms of antisense drug action, an introduction.in: Crooke S.T. Antisense Drug Technology: Principles, Strategies, and Applications. CRC Press, 2008: 3-46Google Scholar, Liang et al., 2016Liang X.H. Shen W. Sun H. Migawa M.T. Vickers T.A. Crooke S.T. Translation efficiency of mRNAs is increased by antisense oligonucleotides targeting upstream open reading frames.Nat. Biotechnol. 2016; 34: 875-880Crossref PubMed Scopus (45) Google Scholar, Liang et al., 2017bLiang X.H. Sun H. Shen W. Wang S. Yao J. Migawa M.T. Bui H.H. Damle S.S. Riney S. Graham M.J. et al.Antisense oligonucleotides targeting translation inhibitory elements in 5′ UTRs can selectively increase protein levels.Nucleic Acids Res. 2017; 45: 9528-9546Crossref PubMed Scopus (9) Google Scholar). To avoid unwanted cleavage of the target RNAs, ASOs must be modified such that the ASO-RNA duplexes are not substrates for RNase H1 or Ago2 cleavage (Bennett and Swayze, 2010Bennett C.F. Swayze E.E. RNA targeting therapeutics: molecular mechanisms of antisense oligonucleotides as a therapeutic platform.Annu. Rev. Pharmacol. Toxicol. 2010; 50: 259-293Crossref PubMed Scopus (739) Google Scholar). Such modifications include nucleoside moieties, morpholinos, and peptide nucleic acids (Figure 2). Recent examples of the value of these mechanisms are nusinersen and eteplirsen, two FDA-approved drugs that alter RNA splicing and are discussed below. ASOs also can be designed to take advantage of RNase H1 or Ago2 (Crooke et al., 2008Crooke S.T. Vickers T. Lima W. Wu H. Mechanisms of antisense drug action, an introduction.in: Crooke S.T. Antisense Drug Technology: Principles, Strategies, and Applications. CRC Press, 2008: 3-46Google Scholar). RNase H1 has proven to be a robust pharmacological mechanism that is active in both the cytoplasm and the nucleus (Liang et al., 2017aLiang X.H. Sun H. Nichols J.G. Crooke S.T. RNase H1-dependent antisense oligonucleotides are robustly active in directing RNA cleavage in both the cytoplasm and the nucleus.Mol. Ther. 2017; 25: 2075-2092Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). To take advantage of RNase H1, a central “gap” of 2′-deoxy nucleotides flanked by 2′-MOE wings is the design that best exploits the benefits of the 2′-MOE modification and the specificities of RNase H1 (Lima et al., 2008Lima W. Wu H. Crooke S.T. The RNase H mechanism.in: Crooke S.T. Antisense Drug Technology: Principles, Strategies, and Applications. CRC Press, 2008: 47-74Google Scholar). RNase H1 is highly selective for cleavage of RNA in an RNA-DNA duplex. The enzymology and cellular functions of RNase H1 are now well understood (Lima et al., 2008Lima W. Wu H. Crooke S.T. The RNase H mechanism.in: Crooke S.T. Antisense Drug Technology: Principles, Strategies, and Applications. CRC Press, 2008: 47-74Google Scholar), and to date, two ASOs that depend on RNase H1, fomivirsen and mipomersen, have been approved by the FDA. Two others, volanesorsen and inotersen, have been submitted for authorization to market (volanesorsen and inotersen are examples that are discussed later in this review). RNase H1 has proven to be quite a robust mechanism (Lima et al., 2014Lima W.F. Vickers T.A. Nichols J. Li C. Crooke S.T. Defining the factors that contribute to on-target specificity of antisense oligonucleotides.PLoS One. 2014; 9: e101752Crossref PubMed Scopus (20) Google Scholar). The kinetics of the major steps in the pharmacological effects of RNase-H1-based ASOs in cells have been defined, and they are, compared to small molecules, remarkably slow (20 minutes or more for each major step) (Vickers and Crooke, 2015Vickers T.A. Crooke S.T. The rates of the major steps in the molecular mechanism of RNase H1-dependent antisense oligonucleotide induced degradation of RNA.Nucleic Acids Res. 2015; 43: 8955-8963Crossref PubMed Scopus (24) Google Scholar). Although off-target hybridization and effects can and do occur, ASOs have been proven to be remarkably specific for on-target binding and RNase H1 degradation. Several factors contribute to this high target specificity. First, the inherent specificity of Watson-Crick base pairing, which is enhanced because the entire ASO sequence is used to identify and bind the target RNA, is obviously important. (Theoretical single-RNA specificity is achieved at 13 to 15 nucleotides in length, and typically ASOs are 18 to 20 nucleotides long) (Crooke, 2008Crooke S.T. Antisense Drug Technology: Principles, Strategies, and Applications. CRC Press, 2008Google Scholar). Second, RNase H1 is in limiting quantities; this, combined with the slow kinetics of drug action, increases specificity. Third, RNase H1 displays such high specificity for DNA-RNA duplexes that changes in ASO chemistry or mismatches can substantially reduce cleavage of an RNA. These factors, combined with RNA structure and RNA protein binding, have been shown to substantially limit potential off-target cleavages (Lima et al., 2008Lima W. Wu H. Crooke S.T. The RNase H mechanism.in: Crooke S.T. Antisense Drug Technology: Principles, Strategies, and Applications. CRC Press, 2008: 47-74Google Scholar, Lima et al., 2014Lima W.F. Vickers T.A. Nichols J. Li C. Crooke S.T. Defining the factors that contribute to on-target specificity of antisense oligonucleotides.PLoS One. 2014; 9: e101752Crossref PubMed Scopus (20) Google Scholar, Vickers and Crooke, 2014Vickers T.A. Crooke S.T. Antisense oligonucleotides capable of promoting specific target mRNA reduction via competing RNase H1-dependent and independent mechanisms.PLoS One. 2014; 9: e108625Crossref PubMed Scopus (22) Google Scholar). ASOs can also be designed to take advantage of Ago2, but the structural requirements of Ago2 impose limits on the types of chemical modifications that can be used. For example, 2′-methoxyethyl substituents that have proven to be highly useful for RNase-H1-type ASOs do not support binding to Ago2. Moreover, a very limited set of chemical modifications that stabilize the 5′ phosphate and 2′ modifications and meet the structural requirements of Ago2 has been identified (Lima et al., 2012Lima W.F. Prakash T.P. Murray H.M. Kinberger G.A. Li W. Chappell A.E. Li C.S. Murray S.F. Gaus H. Seth P.P. et al.Single-stranded siRNAs activate RNAi in animals.Cell. 2012; 150: 883-894Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, Prakash et al., 2016aPrakash T.P. Kinberger G.A. Murray H.M. Chappell A. Riney S. Graham M.J. Lima W.F. Swayze E.E. Seth P.P. Synergistic effect of phosphorothioate, 5′-vinylphosphonate and GalNAc modifications for enhancing activity of synthetic siRNA.Bioorg. Med. Chem. Lett. 2016; 26: 2817-2820Crossref PubMed Google Scholar, Prakash et al., 2017Prakash T.P. Lima W.F. Murray H.M. Li W. Kinberger G.A. Chappell A.E. Gaus H. Seth P.P. Bhat B. Crooke S.T. Swayze E.E. Identification of metabolically stable 5′-phosphate analogs that support single-stranded siRNA activity.Nucleic Acids Res. 2017; 45: 6994Crossref PubMed Scopus (0) Google Scholar, Yu et al., 2012Yu D. Pendergraff H. Liu J. Kordasiewicz H.B. Cleveland D.W. Swayze E.E. Lima W.F. Crooke S.T. Prakash T.P. Corey D.R. Single-stranded RNAs use RNAi to potently and allele-selectively inhibit mutant huntingtin expression.Cell. 2012; 150: 895-908Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar). Nevertheless, pharmacological activity of single-stranded RNAs has been reported, and work continues to optimize agents that cleave RNA via Ago2 continues. The focus of current work is to develop chemical modifications that enhance both in vitro and in vivo potency and activity in tissues other than the liver. The challenges of performing at a level better than current ASOs is daunting, but the mechanism and ASO design certainly merit continued research. An unmodified siRNA has a molecular weight of approximately 13 kD, and because the hydrophobic bases are shielded from water in a duplex structure, it is hydrophilic. Thus, activity following free uptake of unmodified siRNAs has not been rigorously demonstrated in any cell line (Dowdy, 2017Dowdy S.F. Overcoming cellular barriers for RNA therapeutics.Nat. Biotechnol. 2017; 35: 222-229Crossref PubMed Scopus (240) Google Scholar). To achieve cellular uptake, siRNAs must either be transfected with cationic lipids or other types of nanoparticles (Sahay et al., 2013Sahay G. Querbes W. Alabi C. Eltoukhy A. Sarkar S. Zurenko C. Karagiannis E. Love K. Chen D. Zoncu R. et al.Efficiency of siRNA delivery by lipid nanoparticles is limited by endocytic recycling.Nat. Biotechnol. 2013; 31: 653-658Crossref PubMed Scopus (355) Google Scholar) or take advantage of a high-capacity receptor by conjugation to a targeting ligand such as N-acetylgalactosamine (GalNAc). When a GalNAc moiety is attached to the siRNA, cellular uptake and distribution are mediated by the asialoglycoprotein receptors expressed on hepatocytes (Nair et al., 2014Nair J.K. Willoughby J.L. Chan A. Charisse K. Alam M.R. Wang Q. Hoekstra M. Kandasamy P. Kel’in A.V. Milstein S. et al.Multivalent N-acetylgalactosamine-conjugated siRNA localizes in hepatocytes and elicits robust RNAi-mediated gene silencing.J. Am. Chem. Soc. 2014; 136: 16958-16961Crossref PubMed Scopus (347) Google Scholar). Uptake by these receptors is primarily through clathrin-dependent endocytosis (D’Souza and Devarajan, 2015D’Souza A.A. Devarajan P.V. Asialoglycoprotein receptor mediated hepatocyte targeting - strategies and applications.J. Control. Release. 2015; 203: 126-139Crossref PubMed Scopus (125) Google Scholar). The mechanism(s) by which the siRNAs escape endosomal-lysosomal vesicles is currently unknown and remains a major area of investigation for all nucleic acid therapeutics. Because the nucleo-bases are exposed in a single-stranded ASO, these oligonucleotides are amphipathic. Phosphorothioate (PS) substitution of the phosphate groups reduces the hydrophilicity, stabilizes the ASOs to nuclease degradation, and enhances protein binding, which is critical to cell uptake and intracellular distribution (Crooke et al., 2017bCrooke S.T. Wang S. Vickers T.A. Shen W. Liang X.H. Cellular uptake and trafficking of antisense oligonucleotides.Nat. Biotechnol. 2017; 35: 230-237Crossref PubMed Scopus (116) Google Scholar, Eckstein, 2000Eckstein F. Phosphorothioate oligodeoxynucleotides: what is their origin and what is unique about them?.Antisense Nucleic Acid Drug Dev. 2000; 10: 117-121Crossref PubMed Scopus (307) Google Scholar, Zon and Geiser, 1991Zon G. Geiser T.G. Phosphorothioate oligonucleotides: chemistry, purification, analysis, scale-up and future directions.Anticancer Drug Des. 1991; 6: 539-568PubMed Google Scholar). Despite decades of effort, no other modification has been identified that results in the optimal protein binding that substitutions with PSs provide. Thus, essentially all ASOs except morpholinos and unmodified siRNAs protected in nanoparticulate complexes must have a significant number of PS substitutions to be pharmacologically active in vivo. In contrast to double-stranded siRNAs, PS ASOs are taken up by most cells in tissue culture without the need for transfection or targeting ligands (Juliano and Carver, 2015Juliano R.L. Carver K. 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• W2795531081 title "RNA-Targeted Therapeutics" @default.
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