nrd3625

Most currently marketed drugs are small molecules that target proteins such as enzymes and receptors, which represent a considerably small subset of total cellular pro- teins. By contrast, oligonucleotides are macro molecules that target pre-mRNA and mRNA — the carriers of genetic information before it is translated into proteins. Because mRNAs encode all cellular proteins, oligonucleo- tides targeting mRNA could prove to be effective for targets and diseases that are not treatable by current drugs. For example, genetic diseases — in which the defect in the gene can be best repaired by manipulating DNA or RNA rather than the protein — such as Duchenne muscular dystrophy (DMD) and spinal muscular atrophy (SMA) are two such diseases (discussed below). This Review covers the following three approaches that exploit oligonucleotides: RNA interference (RNAi), antisense oligonucleotides and steric-blocking oligonucleotides. These three approaches involve the binding of comple- mentary oligonucleotides to target RNA through base pairing, and therefore all three are — in essence — operating by an antisense mechanism. However, they differ substantially in their downstream mechanisms of action and the functional outcomes they produce. RNAi and antisense oligonucleotides — which will be discussed only briefly as they have been extensively reviewed in the literature (see REFS 1,2) — modulate gene expression by inducing enzymatic degradation of targeted mRNA and the removal of the disease-causing gene product, such as an oncogene or a pro-inflammatory cytokine. As cellular enzymes need to recognize these antisense compounds, they can only be chemically modified to a limited degree, which limits our ability to enhance their pharmacological qualities. Antisense compounds that modulate RNA function by blocking the access of cellular machinery to RNA, and so do not lead to degradation of the target RNA, are the main focus of this Review. This different mode of action leads to outcomes such as the repair of a defective RNA or the generation of a novel protein, which cannot be achieved using RNAi or antisense oligonucleotides. Furthermore, because RNA-blocking oligonucleotides do not need to exploit cellular enzymes for their activity, they can be subjected to more extensive chemical modifications that improve their drug-like qualities. The poor intracellular uptake of RNAi, antisense oligo nucleotides and steric-blocking oligonucleotides is a major impediment to their use as therapeutics. This is the main reason why the antiviral drug fomivirsen3 is currently the only approved antisense drug (although the drug was discontinued in 2004 as the market for the drug diminished). Recent advances in chemistries that improve the intracellular delivery of antisense oligonucleotides, as 1AVI BioPharma, 3450 Monte Villa Parkway, Bothell, Washington 98021, USA. 2Cold Spring Harbor Laboratory, 1 Bungtown Road, Cold Spring Harbor, New York 11724, USA. 3Department of Molecular, Cellular and Developmental Biology, 219 Prospect Street, Yale University, New Haven, Connecticut 06520, USA. Correspondence to R.K.  e‑mail: rkole@avibio.com doi:10.1038/nrd3625 Published online 20 January 2012 RNA interference (RNAi). A form of post- transcriptional gene silencing in which the expression or transfection of double- stranded RNA induces degradation by nucleases of the homologous endogenous transcripts, resulting in the reduction or loss of gene activity. RNA therapeutics: beyond RNA interference and antisense oligonucleotides Ryszard Kole1, Adrian R. Krainer2 and Sidney Altman3 Abstract | Here, we discuss three RNA-based therapeutic technologies exploiting various oligonucleotides that bind to RNA by base pairing in a sequence-specific manner yet have different mechanisms of action and effects. RNA interference and antisense oligonucleotides downregulate gene expression by inducing enzyme-dependent degradation of targeted mRNA. Steric-blocking oligonucleotides block the access of cellular machinery to pre-mRNA and mRNA without degrading the RNA. Through this mechanism, steric-blocking oligonucleotides can redirect alternative splicing, repair defective RNA, restore protein production or downregulate gene expression. Moreover, they can be extensively chemically modified to acquire more drug-like properties. The ability of RNA-blocking oligonucleotides to restore gene function makes them best suited for the treatment of genetic disorders. Positive results from clinical trials for the treatment of Duchenne muscular dystrophy show that this technology is close to achieving its clinical potential. R E V I E W S NATURE REVIEWS | DRUG DISCOVERY VOLUME 11 | FEBRUARY 2012 | 125 © 2012 Macmillan Publishers Limited. All rights reserved Antisense oligonucleotides Oligonucleotides that bind to complementary mRNA by base pairing and induce cleavage of targeted mRNA by ribonuclease H, an enzyme that degrades RNA in RNA–DNA duplexes. Small interfering RNAs (siRNAs). Synthetic, short, 21–22-nucleotide-long double-stranded RNAs with chemical modifications designed to increase their stability and cellular uptake. One strand of siRNA hybridizes to targeted mRNA and allows mRNA degradation. RNA-induced silencing complex (RISC). A multiprotein complex that, when combined with small interfering RNA (siRNA), affects mRNA degradation. A key component of RISC is an endonuclease, argonaute 2, which cleaves the targeted mRNA within the siRNA–mRNA duplex. well as differences that characterize the three technologies, are highlighted in this Review. Aptamers, which are more structurally complex than oligonucleotide RNA-based drugs, and interact directly with proteins rather than complementary RNA, are not covered in this article4. Oligonucleotides that degrade target mRNA RNAi. RNAi was first demonstrated in a nematode5 (Caenorhabditis elegans), in which the delivery of exo- genous, long, double-stranded RNA (dsRNA) effectively silenced the expression of a gene (encoding a myofilament protein) by inducing the degradation of a homo logous host mRNA. The mechanism that mediated gene silencing involved degradation of dsRNA into small interfering RNAs (siRNAs) — double-stranded RNA fragments that are 21–22 nucleotides long and interact with a multiprotein RNA-induced silencing complex (RISC). Within the RISC, the siRNA is unwound, the sense strand is discarded, and the antisense or guide strand binds to mRNA. When siRNA is fully complementary to its target, the endo nuclease argonaute 2 — a component of the RISC — cleaves the mRNA 10 and 11 nucleotides downstream from the 5′ end of the antisense strand6 (FIG. 1a). Although RNAi could not initially be detected in mammalian cells, later studies showed that these cells lacked the ability to cleave dsRNA into siRNA. The discovery that synthetic siRNA can enter the RISC and degrade targeted mRNA when delivered to cultured human cells7 led to a rapid increase in the amount of research related to siRNA. In 2002, the first full year after the discovery of siRNA, a query for siRNA in PubMed resulted in 234 citations; for the 2002–2010 period it lists 33,009 citations, of which 7,241 were from 2010. Notably, a first successful in vivo study was carried out as early as 2003, using naked siRNA to knockdown FAS mRNA in a mouse model of fulminant hepatitis8. The increase in the amount of RNAi research also resulted in the founding of companies — such as Alnylam Pharmaceuticals and Sirna Therapeutics — focused on the development of siRNA as a promising therapeutic platform. Systemically delivered unmodified siRNA is rapidly degraded by nucleases circulating in the bloodstream; it has a half-life in plasma of approximately a few minutes, and its uptake into target organs and cells is generally poor but it has shown some success in liver delivery8. Chemical modifications to promote metabolic stability and improve target cell penetration have been introduced to overcome such problems. However, it has become apparent that the interaction of siRNAs with the cellular RISC machinery presents a challenge for their use as therapeutics, because only limited chemical modifi- cations can be introduced into siRNA for it to remain functional within the RISC. In the antisense strand, phosphorothioate internucleotide linkages at the 3′ end, and 2′-O-methyl (2′-OMe) nucleotide substitutions (FIG. 2) in one or two internal nucleotides, are tolerated and improve the resistance of the siRNA to nucleases. The sense strand can be modified more heavily (that is, more internal nucleotides can carry 2′-OMe nucleotide substitutions) without substantially reducing efficacy (reviewed in REF. 9). Because of the difficulty in achieving intracellular delivery of siRNAs, they were administered locally in the majority of initial clinical trials. These included: intra- vitreal injection for the treatment of macular degener- ation (ClinicalTrials.gov identifier: NCT00363714); intranasal delivery for respiratory syncytial virus (ClinicalTrials.gov identifier: NCT00658086); and direct injections in skin lesions for pachyonychia congenita, a rare genetic skin disorder (ClinicalTrials.gov identifier: NCT00716014). It was noted that intranasal delivery in humans results in only minimal systemic distribution of the drug10, indicating that in the absence of delivery agents unmodified siRNAs have a limited bioavailability in humans. Two clinical trials that are currently underway aim to tackle the problems associated with systemic siRNA delivery by combining siRNA with delivery-enhancing agents. In one trial, a cocktail of two siRNAs, ALN-VSP02, has been formulated with lipid particles and targeted to mRNAs encoding kinesin spindle protein and vascular endothelial growth factor, which are essen- tial for tumour proliferation and tumour-supporting angiogenesis, respectively (see the 4 June 2011 press release on the Alnylam Pharmaceuticals website; ClinicalTrials.gov identifier: NCT00882180). In the second trial (of CALAA-01), siRNA has been formu- lated in a cyclodextrin–adamantane polyethylene glycol particle that includes a targeting component, human transferrin protein, which targets the siRNA to ribo- nucleotide reductase mRNA (ClinicalTrials.gov identi- fier: NCT00689065). These delivery-enhancing moieties should improve the cellular uptake of siRNA; if successful, these clinical studies will be of substantial interest. A series of lipid-based, liver-directed siRNA carrier particles was recently tested in a mouse model of haemo- philia and in healthy cynomolgus monkeys. Systemic delivery of the formulated siRNAs reduced the levels of factor VII mRNA and glyceraldehyde-3-phosphate dehydrogenase mRNA at remarkably low doses of 0.01 mg per kg in mice and 0.1 mg per kg in monkeys, respectively11. A single high dose of the preparation was well tolerated in both species. As the effects of siRNAs were examined only in the liver, it is not known whether they were taken up by other tissues. Nevertheless, these results represent dramatic improvements in siRNA delivery to specific target tissues — in this case the liver. Another problem associated with RNAi technology — which stems from mechanisms of RNAi — is the off- target effects of siRNAs. Short dsRNAs, termed micro- RNAs (miRNAs), are produced in mammalian cells and as a class they control the efficiency of the translation of mRNAs. Synthetic siRNAs may interfere with this pro- cess, thus mimicking the effects of miRNAs. Specifically, siRNAs can enter the RISC and bind with certain base pairs mismatched to untargeted mRNAs, thus acting like endogenous miRNAs and leading to off-target gene silencing12. In addition, siRNAs can activate an innate immune response via activation of Toll-like receptors, leading to undesirable side effects such as the induction of pro-inflammatory cytokines or interferon-α6,12. Taken together, these developments indicate that systemic R E V I E W S 126 | FEBRUARY 2012 | VOLUME 11 www.nature.com/reviews/drugdisc © 2012 Macmillan Publishers Limited. All rights reserved Nature Reviews | Drug Discovery Dicer RISC AGO2 RISC mRNA mRNA AGO2 RISC AGO2 a Small interfering RNA Double-stranded siRNA b Antisense gapmer oligonucleotides ASO gapmer Flank modifications siRNA complexed with dicer for loading into RISC siRNA guide strand in RISC Guide strand targets mRNA AGO2 in RISC cleaves targeted mRNA Transcription start site mRNA RNase H Degradation of ASO-targeted mRNA by RNase H RNase H c Translation-suppressing oligonucleotides d External guide sequence and RNase P RNase P TSO AUG mRNA AUG mRNA Ribosomal subunit Ribosomal subunit mRNA PPMO–EGS Cleaved mRNA Cleaved off RNA fragment PPMO–EGS Translation-suppressing oligonucleotides (TSOs). Modified oligonucleotides that block mRNA sequences near the initiation of the translation codon (AUG), interfere with the binding of ribosomes to mRNA and inhibit the translation of undesirable proteins. TSO–mRNA duplexes are not recognized by ribonuclease H or RNA-induced silencing complex, and the mRNA is therefore not cleaved. Figure 1 | Mechanisms of oligonucleotide-induced downregulation of gene expression. a | Small interfering RNA (siRNA). Synthetic double-stranded siRNA is complexed with components of the RNA interference pathway, dicer, argonaute 2 (AGO2) and other proteins, to form an RNA-induced silencing complex (RISC). The RISC binds to a targeted mRNA via the unwound guide strand of siRNA, allowing AGO2 to degrade the RNA. The RISC-bound siRNA can also bind with mismatches to unintended mRNAs, leading to significant off-target effects (see main text). b | Antisense gapmer oligonucleotides. These commonly have a phosphorothioate backbone with flanks that are additionally modified with 2′-O-methoxyethyl (2′-MOE) or 2′-O-methyl (2′-OMe) residues (highlighted in red in figure). Flank modifications increase the resistance of the antisense oligonucleotide (ASO) to degradation and enhance binding to targeted mRNA. The unmodified ‘gap’ in a gapmer–mRNA duplex is recognized by ribonuclease H (RNase H), a ribonuclease that degrades duplexed mRNA. c | Translation-suppressing oligonucleotides (TSOs). Phosphorodiamidate morpholino oligomers (PMOs) and their derivatives, or oligonucleotides fully substituted with 2′-MOE or 2′-OMe residues, are not recognized by RISC or RNase H and therefore do not lead to RNA degradation. Nevertheless, they lead to downregulation of gene expression via steric blockade of ribosome access to mRNA and suppression of protein translation. d | External guide sequences (EGSs) and RNase P. A peptide-conjugated PMO (PPMO) is designed to hybridize to targeted bacterial mRNA and form stem–loop structures such that the resulting duplex resembles tRNA. In bacteria, a tRNA-processing ribozyme — RNase P — recognizes this structure and cleaves mRNA. R E V I E W S NATURE REVIEWS | DRUG DISCOVERY VOLUME 11 | FEBRUARY 2012 | 127 © 2012 Macmillan Publishers Limited. All rights reserved Nature Reviews | Drug Discovery N O N O O P ON Base Base O N O N O O P O Base Base O NN H H N O N O O P ON Base Base O ON H RXRRXRRXRRXRX Acetylated O O P O S O O O Base Base O P O S O OCH3 O OCH3 O O P O O O O O Base Base P O O O OH OH O O P O S O O O Base Base OCH3 P O S O OCH3 O O P O S O O O Base Base P O S O O P O S O O Base Base O P O S O OO O DNA (PS) PMO PPMO PMOplus 2′-MOE 2′-OMe LNA RNA delivery of chemically modified siRNA is not very effective and that more work will be needed to achieve sufficient therapeutic activity and specificity in the absence of delivery agents. It has now been over a decade since the discoveries of RNAi and siRNAs but despite some promising clin- ical trial results the industry remains cautious about the therapeutic potential of RNAi-based drugs. For example, although Merck acquired Sirna Therapeutics for US$1.1 billion, it was quoted in 2009 as remaining sceptical about the development of siRNA-based drugs13; meanwhile, Roche, Novartis and  Pfizer14 decided to dramatically reduce or eliminate their RNAi research programmes, which raised the question “Is RNAi dead?” in a 2011 editorial15 (see the article on the GenomeWeb website, and the article on the Proactive Investors website). This is reminiscent of a previous question — “Does antisense exist?” — that was raised in a commentary in 1995 (REF. 16). The history of anti- sense oligonucleotides (BOX 1), which now seem to be well on their way to becoming drugs, suggests that RNAi will eventually succeed as a therapeutically viable technology. As a possible harbinger of further progress, an upcoming clinical trial will test chemically modified siRNA for the treatment of diabetic macular oedema (ClinicalTrials.gov identifier: NCT01445899). Antisense oligonucleotides. Since the first application of a short fragment of unmodified DNA in cell cul- ture as an antisense oligonucleotide, by Zamecnik and Stephenson17 in 1978, remarkable progress has been made in oligonucleotide drug development. Numerous chemical modifications that improve the drug-like prop- erties of DNA have been introduced (BOX 1), an antisense drug has been marketed and successful clinical trials are underway, as described below. Currently, a typical antisense oligonucleotide drug candidate is about 20 nucleotides long and has a phosphorothioate linkage (FIG. 2) between the nucleosides that form the backbone. In addition, five nucleotides at each flank are further modified (FIG. 2) to protect the antisense oligonucleotide from exonucleases, thus increasing its stability in vivo. Figure 2 | Oligonucleotide chemistries. All oligonucleotides are negatively charged. Phosphorothioate (PS) backbones, as well as 2′-O-methoxyethyl (2′-MOE) and 2′-O-methyl (2′-OMe) substituents, increase resistance to degradation and promote protein binding to target RNA. Locked nucleic acid (LNA) modification markedly increases the binding of the oligonucleotide to the targeted mRNA (see top panel). In phosphorodiamidate morpholino oligomers (PMOs), ribose (RNA) or deoxyribose (DNA) is replaced with morpholine rings, and the phosphorothioate or phosphodiester (RNA) groups are replaced with uncharged phosphorodiamidate groups, resulting in a compound that is neutral and very resistant to degradation (see bottom panel). Positively charged piperazine residues in positively charged PMOs (PMOplus), or positively charged arginine-rich peptides in peptide-conjugated PMOs (PPMOs), dramatically improve the intracellular uptake of the oligomers. R E V I E W S 128 | FEBRUARY 2012 | VOLUME 11 www.nature.com/reviews/drugdisc © 2012 Macmillan Publishers Limited. All rights reserved This design leaves a central 10-nucleotide phosphoro- thioate gap (hence the term ‘gapmers’) that allows the cleavage of targeted mRNA by ribonuclease H (RNase H)18 (FIG. 1b). The modified flanks also improve the binding of the antisense oligonucleotide to mRNA and reduce the side effects that are associated with the presence of phosphorothioate residues (BOX 1). In contrast to siRNA, which tolerates only limited modifications to remain RISC-compatible, more extensive chemical modifications in gapmers do not abrogate RNase H activity. One such modification, 2′-O-methoxyethyl (2′-MOE) ribonucleoside (FIG. 2), is present in two anti- sense oligonucleotides that have recently been successful in clinical trials: mipomersen (also known as ISIS 301012) and custirsen (also known as OGX-111)19,20. Mipomersen is a 2