Antisense December 2019

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Antisense RNA

Produced by: With support from: Nature Structural & Molecular Biology, Nature Reviews Molecular Cell Biology and Nature Communications MILESTONES IN ANTISENSE RNA RESEARCH

1970s Antisense cellular RNA and synthetic oligonucleotides inhibit mRNA translation (MILESTONE 1)

1990 RNA-mediated post-transcriptional gene silencing (PTGS) in petunia (MILESTONE 2)

1993 microRNAs emerge as novel post-transcriptional regulators of gene expression (MILESTONE 3)

1998 Double-stranded RNA mediates sequence-specific gene silencing in animals (MILESTONE 4)

FDA approval of the first antisense oligonucleotide drug (MILESTONE 5)

1999 Small RNAs trigger different types of PTGS in plants (MILESTONE 6)

2000 Elucidation of the molecular mechanism of RNA interference (MILESTONE 7)

2001 First demonstration that small interfering RNAs trigger gene-specific silencing in mammals (MILESTONE 8)

Discovery of PIWI-interacting RNAs (piRNAs) (MILESTONE 9)

2002 Advent of plasmid-based gene silencing and large-scale RNA interference screens (MILESTONE 10)

2005 Targeted gene silencing in vivo by antibody-mediated siRNA delivery (MILESTONE 11)

2012 Gene editing by CRISPR–Cas9, from bacteria to humans (MILESTONE 12)

2014 Chemical optimization enables targeted delivery of siRNA drugs to the liver (MILESTONE 13)

2016 FDA approval of an antisense oligonucleotide drug to treat spinal muscular atrophy (MILESTONE 14)

2018 First RNAi-based therapeutic agent approved by the FDA (MILESTONE 15)

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‘Waltz of the polypeptides’ by Mara G. Haseltine, Cold Spring Harbor Laboratory, NY, USA. Credit: Ivanka Kamenova / Springer Nature Limited

MILESTONE 1 First signs of antisense RNA activity

The first reports of antisense RNA activity in to sequestering the inhibitory RNA through replication and oncogenic transformation eukaryotes were published when the efforts base-pairing. The ideas put forward by of the cells. The chemically modified oligo to understand the mechanism of mRNA Ochoa — that small regulatory RNAs exist as nucleotide performed better, likely because the translation were well underway. At that time, double stranded structures and are generated modifications conferred resistance to nucleases cell-free in vitro translation systems were by endogenous RNase enzymes — have been in the culture medium. widely used to probe the function of different revisited in subsequent decades, as the mech- The most likely target processes of the translation co‑factors. These minimal systems, anisms for processing of small interfering complementary oligonucleotide were the cir comprised of a cell extract and a template RNAs and microRNAs were uncovered. cularisation of provirus DNA or the translation mRNA, proved instrumental in the initial Whereas the above reports described the of viral mRNA. Although the first possibility characterisation of cellular RNAs that could activity of cellular antisense RNAs, in 1977, was not ruled out, in a companion paper control mRNA translation through antisense Paterson et al. demonstrated that exogenous published in the same issue of Proceedings of recognition of their targets. plasmid DNA fragments complementary to the National Academy of Sciences, Stephenson Working in such a cell-free system, in an mRNA could also inhibit its translation in and Zamecnik demonstrated that the delivered 1975, Stuart Heywood and colleagues demon- vitro. A year later, Paul Zamecnik and Mary oligo inhibits translation of viral mRNA strated that short ribonucleotide sequences Stephenson reported the first synthetic DNA through sequence-specific hybridisation. purified from chicken muscle messenger ribo oligonucleotide delivered to cells, capable of Our knowledge of the mechanisms nucleoprotein and polysome fractions could inhibiting viral replication and oncogenic by which endogenous RNAs regulate key control translation of the mRNA encoding transformation caused by Rous sarcoma virus. processes in cell function and in devel- myosin. They called the RNAs ‘translation The 35S RNA of this retrovirus had recently opment has since grown exponentially. control RNAs’ (tcRNAs) and proposed that been found to contain a 20‑nucleotide repeat Yet, these early experiments, using highly tcRNAs act by binding to their mRNA targets sequence in its 5ʹ and 3ʹ ends. The authors purified components, provided the first in a sequence-specific manner. In follow‑up synthesized 13‑nucleotide-long DNA oligo- hints that cells produce small RNAs with work a decade later, Heywood demonstrated nucleotides complementary to part of the viral the capacity to affect translation, and that that one of these tcRNAs, tcRNA102, repeat sequence, which was hypothesized to be the complementarity of these RNAs to their recognises a sequence in the 5ʹ untranslated important for viral replication, and tested their target is important for their function. The region of chicken myosin mRNA, albeit with effects on the growth of chicken embryonic experiments by Zamecnik, Stephenson and imperfect homology. fibroblasts infected with Rous sarcoma virus. others laid the foundation of the field of anti- In the years immediately following Two synthetic oligonucleotide variants were sense RNA therapeutics and resulted in the Heywood’s original tcRNA discovery, several tested: one with free 3ʹ and 5ʹ termini, and one founding of Hybridon, the first biotechnology groups isolated similar small RNA species with chemical modifications. Strikingly, addi- company dedicated to developing synthetic from different organisms and demonstrated tion of either oligonucleotide to the cell culture oligonucleotides for therapeutic purposes. that these short RNAs could modulate trans- medium at the time of infection inhibited viral Ivanka Kamenova, Associate Editor, Nature Protocols lation. Notably, in 1977, Severo Ochoa and colleagues purified two distinct short RNAs MILESTONE STUDIES Bester, A. J. et al. Two classes of translational control RNA: their role in the regulation of protein synthesis. Proc. Natl Acad. Sci. USA 72, 1523–1527 (1975) | Lee-Huang, S. et al. Eucaryotic oligonucleotides affecting mRNA translation. Arch. Biochem. from a small crustacean, Artemia salina, and Biophys. 180, 276–287 (1977) | Paterson, B. M. et al. Structural gene identification and mapping by DNA–mRNA hybrid-arrested cell- showed that these RNAs exerted activating free translation. Proc. Natl Acad. Sci. USA 74, 4370–4374 (1977) | Zamecnik, P. C. et al. Inhibition of Rous sarcoma virus replication and cell transformation by a specific oligodeoxynucleotide. Proc. Natl Acad. Sci. USA 72, 280–284 (1978) | Stephenson, M. L. et al. Inhibition and inhibitory effects on A. salina mRNAs. of Rous sarcoma viral RNA translation by a specific oligodeoxyribonucleotide. Proc. Natl Acad. Sci. USA 75, 285–288 (1978). The activator RNA is complementary to the FURTHER READING Heywood, S. M. tcRNA as a naturally occurring antisense RNA in eukaryotes. Nucleic Acids Res. 14, 6771–6772 inhibitory RNA, and thus its stimulatory (1986) | Zamecnik, P. C. From protein synthesis to genetic insertion. Annu. Rev. Biochem. 74, 1–28 (2005) | Kresge, N. et al. The Discovery of tRNA by Paul C. Zamecnik. J. Biol. Chem. 280, e37 (2005). effect on translation was proposed to be due

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MILESTONE 2 Patterns of co‑suppression in plants

If cuttings were taken from the branches, the new individuals would have the same flower pattern as the …experiments branch from which they were cut. on pigmentation Back-crossing the transformed in petunias did plants to parental lines allowed not simply have the researchers to show that the unexpected altered pigmentation only occurred when the transgene was present results, and active. RNase protection assays but results using radiolabelled RNA probes completely antisense to the gene showed that opposite to messenger RNA levels for both what had been endogenous and transgenic CHS in transgenic plants were reduced or anticipated, essentially abolished. arguably It would have been very easy for marking the Jorgensen and his team to dismiss beginning of these results as a failed experiment, the field of RNA not least because a similar approach interference attempted by a group at the University of Amsterdam had not produced the dramatic phenotypes seen by the Californians. However, discussions between the two groups

Credit: blickwinkel / Alamy Stock Photo Stock Alamy / blickwinkel Credit: identified subtle differences in grow- ing conditions. Increasing the light It is often said that truly novel discov- by increasing the number of copies levels resulted in the Dutch petunias eries arise when experiments do not of the gene encoding the enzyme confirming Jorgensen’s observations. go as planned. This is certainly true chalcone synthase (CHS). CHS catal- These experiments showed clearly of one of the earliest observations of yses the condensation of one mole- that co‑suppression of gene expres- an RNA-mediated gene silencing cule of 4‑coumaroyl-CoA and three sion was dependent on high-copy- mechanism by Richard Jorgensen molecules of malonyl-CoA to form number transcription of a transgene and colleagues at the DNA Plant naringenin chalcone, from which a homologous to a native gene, but was Technology Corporation in Oakland, wide variety of flavonoid pigments not affected by the proximity of those California. Their experiments on are synthesised. genes in the plant genome. pigmentation in petunias did not Agrobacterium was used to It would take almost a decade simply have unexpected results, introduce multiple copies of the for the role of this form of RNAi as but results completely opposite to CHS gene, under the control of a a defence against plant viruses to be what had been anticipated, arguably cauliflower mosaic virus 35S pro- identified, and the complexities of marking the beginning of the field of moter, into three different petunia the underlying mechanisms are still RNA interference (RNAi). varieties. Not a single plant with a topic of research. In this respect, During the 1980s, the ability deepened colouration resulted from the 1990 paper was remarkably pres- to transform plant species using these transformations. Instead, cient in deducing from the “erratic Agrobacterium tumefaciens had many of the transformed flowers and reversible nature of the trans- opened up the possibility of creating were completely white, and others gene effect” that DNA methylation transgenic plants with traits desirable produced variegated patterns con- was involved. for agriculture, such as herbicide- taining more white regions than the Chris Surridge, resistant crops, by the introduction original plants. Chief Editor, Nature Plants of genes of bacterial origin. Another These patterns were stable, with MILESTONE STUDY Napoli, C. et al. Introduction goal was the creation of new varieties individual plants producing hun- of a chalcone synthase gene into Petunia results in of ornamental plants by modifying dreds of blooms, all patterned in the reversible co‑suppression of homologous genes in trans. Plant Cell 2, 279–289 (1990). their pigment synthesising pathways. same way. Very occasionally a plant FURTHER READING van der Krol, A. R. et al. Jorgensen’s group was working on would start producing differently Flavonoid genes in petunia: addition of a limited the popular annual Petunia hybrida, patterned flowers or reverting to number of gene copies may lead to a suppression of gene expression. Plant Cell 2, 291–299 (1990). attempting to deepen its colouration the parental form on a side branch.

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MILESTONE 3 microRNAs emerge as potent post-transcriptional gene regulators Credit: Vicky Summersby / Springer Nature Limited Nature Springer / Summersby Vicky Credit:

There is nothing more precise than lin‑4 did not encode a protein; just short of becoming adult worms. a Swiss watch — besides the pattern instead, it is transcribed into a small Lin-4 and let-7 were quite different of development of the nematode non-coding RNA with sequence … although from each other but, in 2000, worm Caenorhabditis elegans, one complementarity to the 3ʹ untrans- lin-4 binding did Ruvkun and colleagues found homo- of the most studied and useful lated region (3ʹ UTR) of lin‑14. not affect the logues in the genomes of Drosophila animal models. The postembryonic Lin‑4 was the first microRNA to be melanogaster and Homo sapiens. development of C. elegans entails discovered. overall mRNA Although humans have no hetero- passage through four accurately At the same time, Gary Ruvkun levels of lin‑14, chronic genes, fruit flies do, and the coordinated larval stages (L1–L4) and colleagues showed that binding it decreased temporal expression profile of let‑7 interspersed with moults. In the of lin-4 to the 3ʹ UTR is essential LIN‑14 protein was shown to be conserved between mid‑1980s, many scientists were for LIN‑14 downregulation. Both expression… worms and fruit flies. interested in genetic aberrations teams correctly hypothesised Since the discovery of lin‑4 and that could alter the precise timing of that lin‑4 pairs through antisense let‑7, many microRNAs have been C. elegans development. Genes that, complementarity to the 3ʹ UTR of identified. This family of small when manipulated, could delay or lin‑14, and forms an RNA duplex non-coding RNAs is involved in the advance the nematode’s cell cycle that leads to translational repression regulation of diverse biological pro- and developmental-stage progression of lin‑14. Although lin-4 binding did cesses, and includes many potential were called heterochronic genes. As not affect the overall mRNA levels of therapeutic targets — not bad for expected at the time, most of these lin‑14, it decreased LIN‑14 protein what were originally thought to be genes encode proteins. expression, subsequently causing mere worm time-keepers. In 1993, Victor Ambros and progression from L1 to L2. Anne Mirabella, colleagues demonstrated that down- This novel mechanism of Senior Editor, Nature Communications regulation of the protein LIN‑14 was post-transcriptionally regulating MILESTONE STUDIES Lee, R. C. et al. The crucial for the progression from the gene expression was shown, in both C. elegans heterochronic gene lin‑4 encodes small first larval stage (L1) to the second articles, to be conserved in several RNAs with antisense complementarity to lin‑14. larval stage (L2). Loss‑of‑function worm species, but at the time it was Cell 75, 843–854 (1993) | Wightman, B. et al. Posttranscriptional regulation of the mutations in lin‑14 cause C. elegans mostly thought to be a nematode heterochronic gene lin‑14 by lin‑4 mediates to skip a beat, starting development oddity. During the 1990s, a second temporal pattern formation in C. elegans. Cell 75, 855–862 (1993). from L2. On the other hand, muta- microRNA regulating C. elegans FURTHER READING Pasquinelli, A. E. et al. tions in another gene, lin‑4, halted development was identified and Conservation of the sequence and temporal developmental progression indefi- named let‑7. In the case of let‑7 expression of let‑7 heterochronic regulatory RNA. Nature 408, 86–89 (2000). nitely at the L1 stage. Surprisingly, mutant nematodes, larvae stopped

NATURE MILESTONES | ANTISENSE RNA DECEMBER 2019 | S7 Credit: Yustyna-Olha Shevchuk / Alamy Stock Photo to to 742 contributed to twitching the a effect, double-stranded RNA (dsRNA) gle-stranded RNA (ssRNA) or anddefects impaired motility. results inmore severe muscular complete loss of unc‑22 expression expressionthe of gene the unc‑22; phenotype caused by areduction in (RNAi). studied They twitching the what coined they ‘RNA interference’ uncover some of mysteries the behind Fire, Mello and colleagues aimed to that transmitted could be to offspring. resulted intranscription interference of either or sense antisense RNA elegansCaenorhabditis zling, however, that nematode inthe lines, plants and worms. It was puz- to regulate gene expression incell In 1998,antisense RNA was known Exogenous dsRNAsilencesgenesinC. elegans MILESTONES S8 mediated by dsRNA was further expression begins. 500 few RNA at cell per molecules the were to maintain expected only a was heritable,type although progeny adult inthe effect worm. This pheno- inproducingeffective atwitching consecutive injections, was highly andsense antisense RNA, rather than incremental co effects, Whereas ssRNA the produced only homologous dsRNA was compared. unc‑22to silence relative the to unc‑22 | DECEMBER 2019 To examine sin- whether That gene silencing was likely ‑ ‑ nucleotide ssRNA homologous cell stage,cell which is unc‑22 when

MILESTONE 4 was purified andwas purified its ability

, injection ‑ injection of

of mex‑3 antisense purified RNA from following injection of dsRNA derived mex‑3 transcript was not detectable hybridization. found They that the using easily detected can be in situ that is abundant inearly embryos and authorsthe mex‑3 , atranscript used targeted mRNA. responsethe to the was specific was not required 1:1,and to be that and endogenous the target mRNA stoichiometry dsRNA the between that dsRNA was involved, that the At point, this authors the deduced with well-characterized phenotypes. genes other homologous three to by dsRNA, but not ssRNA, that was reproduced couldsilencing be effects or intronic regions of unc‑22 Gene . related to or that targeted promoter control dsRNA that was either not after injection ofwas observed phenocopied No silencing. effect wormsthe with gel-purified dsRNA inferred by that fact the injecting To silencing the visualize effect, silencing bydouble-stranded RNA. their discoveryofRNAinterference —gene is awarded toAndrew Fire andCraig Mellofor The 2006Nobelprizeinphysiology ormedicine mex‑3. Surprisingly, injection

targeted mRNA specific to the response was … dsRNA… Development RNA leadstoeffectiveandspecificinhibitionofgeneexpression inC. elegans muscle. itive? FURTHER READINGNellen,W. & Lichtenstein,C.WhatmakesanmRNAanti-sense- stranded RNAinCaenorhabditis elegans. MILESTONE STUDY Fire, A. et al. Potent and specific genetic interference by double- Trends Biochem.Sci. 113, 503–514(1991). 18, 419–423 (1993) | Fire, A. et al. Production of antisense endogenous phenomenon. showed that RNAi is awidespread the RNAithe pathway underlying mechanism molecular of studies that characterized the laid ground-work the for adecade mediatedcan be by dsRNA. It show that strong gene silencing work seminal this was first to the RNAi remained unclear at time, the involvementthe of RNA transport. as well as inits progeny, suggesting somatic tissue of injected the worm gene inthe silencing was observed wormin the dsRNA was injected, finding was that regardless of where transcript levels. Another unexpected did not significantly mex‑3 affect w ww.nature.com/collections/antisense Although mechanism the of Nature 391, 806–811(1998). Faten Taki, Associate Editor, Communications Biology (MILESTONE 7) and and

Credit: Editorial / Alamy Stock Photo MILESTONES

MILESTONE 5 fomivirsen for the treatment of CMV retinitis in individuals with AIDS. In a pivotal phase III RCT, patients with First antisense drug is approved newly diagnosed CMV retinitis were randomly allocated to immediate intravitreal fomivirsen treatment or with fleeting success treatment deferral until progression. Median time to progression was Following the initial reports and (EMA; formerly the EMEA) for the 71 days in the immediate treatment characterization of gene silencing same indication in 1999. group, versus 13 days in the deferred …the success through endogenous and exogenous Fomivirsen is a synthetic treatment group. Progression after of [this drug] antisense RNAs, the first antisense 21‑nucleotide phosphorothioate treatment cessation occurred in oligonucleotide drug, fomivirsen, oligodeoxynucleotide designed to 44% of patients receiving immediate provided proof- received regulatory approval from be complementary to a sequence treatment, versus 70% of patients in of-concept of the United States Food and Drug in CMV mRNAs encoding the the deferred treatment group. Two the clinical Administration (FDA) in 1998. This major immediate-early region 2 additional RCTs demonstrated the promise of agent was indicated for the treatment proteins, which are essential for comparable efficacy of an intensive treatments of cytomegalovirus (CMV) retinitis CMV replication. In line with this and less-intensive regimen of intra- — a serious infection of the retina antisense mechanism of action, early vitreal fomivirsen for the treatment based on that can rapidly lead to blindness preclinical characterization revealed of CMV retinitis that had not been antisense oligo- — in carriers of human immuno that fomivirsen potently and selec- controlled by other drugs. nucleotides… deficiency virus (HIV) exhibiting tively disrupted CMV replication in However, despite the initial enthu- acquired immune deficiency syn- a dose-dependent manner in vitro, siasm and unmet clinical need in the drome (AIDS), who were intolerant providing the first indication of its late 1990s, the success of fomivirsen of, or had contraindications to, other efficacy. was ultimately fleeting. The drug treatments or were insufficiently Regulatory approval was largely was withdrawn by the FDA in 2001, responsive to previous treatments. based on three prospective rand- owing to the success of highly active Fomivirsen was subsequently omized controlled trials (RCTs) antiretroviral therapy in reducing the granted marketing authorisation led by the Vitravene Study Group, incidence of opportunistic infections by the European Medicines Agency which demonstrated the efficacy of in individuals with HIV in the early 2000s, which undermined demand for fomivirsen. The EMA followed suit in 2002, when the manufacturer (Novartis) voluntarily withdrew the drug from the market due to low demand. Nevertheless, the success of fomi virsen provided proof‑of‑concept of the clinical promise of treatments based on antisense oligonucleotides, which was undoubtedly valuable for the next wave of antisense drug approvals, beginning in 2013 with the FDA approval of mipomersen (an antisense oligonucleotide inhibitor of apolipoprotein B) for the treatment of homozygous familial hypercholesterolemia. Conor A. Bradley, Senior Editor, Nature Reviews Cross-Journal Team

FURTHER READING Drug Approval Package: Vitravene (US FDA, 2002); https://go.nature. com/2kfsM3N | Vitravene (EMA, 2002); https://go. nature.com/2kJcOze | Vitravene Study Group. A randomized controlled clinical trial of intravitreous fomivirsen for treatment of newly diagnosed peripheral cytomegalovirus retinitis in patients with AIDS. Am. J. Ophthalmol. 133, 467–74 (2002) | Vitravene Study Group. Randomized dose- comparison studies of intravitreous fomivirsen for treatment of cytomegalovirus retinitis that has reactivated or is persistently active despite other therapies in patients with AIDS. Am. J. Ophthalmol. 133, 475–83 (2002). Credit: Westend61 GmbH / Alamy Stock Photo Stock Alamy / GmbH Westend61 Credit:

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These RNAs were absent in control plants. In tobacco plants carrying a GUS transgene without sequence homology to endogenous sequences, 25‑nt antisense RNAs were also specifically detected in plants that exhibited PTGS. Furthermore, by inoculating a single leaf of a GFP-

Credit: BSIP SA / Alamy Stock Photo Stock Alamy / SA BSIP Credit: transgenic Nicotiana benthamiana plant with Agrobacterium tumefaciens containing GFP sequences in a T‑DNA vector, the researchers observed systemic silencing of GFP and detected 25‑nt GFP antisense RNAs in all affected tissues. Finally, in N. benthamiana infected by potato virus X, 25‑nt antisense RNAs were also found only in infected leaves. Thus, 25‑nt antisense RNAs were present in all types of PTGS examined. As 25‑nt RNAs are long enough to confer sequence specificity, but are short enough for transport through plasmodesmata, Hamilton MILESTONE 6 and Baulcombe proposed that these small RNAs are the likely triggers of PTGS. However, they did not know Small RNAs trigger silencing at the time how the small RNAs were generated. The antisense character of the 25‑nt RNA species and its in plants presence in RNA-virus-infected cells indicated that these short RNAs were By the late 1990s, many instances of or to viral sequences is suppressed, generated from an RNA template post-transcriptional gene silencing suggesting that the silencing mech- rather than a DNA template. Only a (PTGS) in plants had been reported. anism requires sequence specificity, …these studies little while later, scientists working The phenomenon was known to be a but the silencing trigger remained established in animal systems reported that long double-stranded RNA (dsRNA) is defence mechanism that could target elusive. low-molecular- endogenous sequences (for example, In 1992, Richard Jorgensen processed into 21–23‑nt dsRNA transposable elements), transgenes proposed that ectopic pairing of weight RNAs as fragments that guide mRNA cleavage or viral RNAs. When Fire and Mello homologous DNA might initiate the trigger of in vitro (MILESTONE 7). In vivo evi- described RNA interference in PTGS. This model was supported PTGS and RNA dence followed, demonstrating that Caenorhabditis elegans (MILESTONE 4), by subsequent studies, and antisense interference, long dsRNAs are cleaved to about it was immediately obvious that RNAs were suggested to initiate and showed 25 nt and that the antisense strand PTGS is the plant equivalent of RNA silencing by forming a duplex with triggers mRNA degradation by pair- that pairing of interference. the targeted mRNA, thereby facili- ing to target mRNAs. Together, these There are different types of PTGS tating its degradation or translation the antisense studies established low-molecular- in plants. Originally reported in petu- inhibition. However, no antisense strand with the weight RNAs as the trigger of PTGS nia, PTGS induced by a transgene RNA molecules with sizes compa- target mRNA and RNA interference, and showed with homology to an endogenous rable to typical mRNAs had been mediates mRNA that pairing of the antisense strand gene leads to ‘co‑suppression’ of both detected. degradation with the target mRNA mediates the transgene and the endogenous In 1999, Hamilton and Baulcombe mRNA degradation, but the molecu- gene (MILESTONE 2). Transgene- hypothesized that the antisense lar machinery involved remained to induced PTGS, however, can also RNA trigger might be too short for be discovered. occur when the transgene has no detection by standard RNA analysis Jun Lyu, homology to endogenous sequences, techniques. Therefore, they designed Senior Editor, Nature Plants causing silencing of only the trans- experiments to specifically hunt for gene. Systemic PTGS is first triggered short antisense RNAs in the different MILESTONE STUDY Hamilton, A. J. & locally, then spreads to other tissues. types of PTGS in plants. Strikingly, Baulcombe, D. C. A species of small antisense RNA in posttranscriptional gene silencing in plants. Finally, PTGS can also be mediated they detected 25‑nucleotide (nt) Science 286, 950–952 (1999). by viral RNAs. In all these types of antisense and sense RNAs in two FURTHER READING Jorgensen, R. Silencing of PTGS, cellular accumulation of RNAs tomato lines that exhibited transgene plant genes by homologous transgenes. Agbiotech News Info 4, 265–273 (1992). with homology to either a transgene co‑suppression of the ACO gene.

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MILESTONE 7 Pre-treatment of extracts with micrococcal nuclease abolished RISC activity, whereas DNase I had no effect, suggesting that RISC con- Mechanism of RNA tains RNA components. By purifying RISC activity using chromatography, the RNAi-active fraction was shown interference discovered to contain ~25 nt‑long small RNAs that were homologous to target Following the landmark discovery In 2000, two papers reported mRNAs. This was in line with the that exogenously provided double- the elucidation of the mechanisms presence of ~25 nt small RNAs stranded RNA (dsRNA) reduces underlying RNAi using biochemical … the RNAi- during post-transcriptional gene the expression of homologous approaches. These papers suggested active fraction silencing in plants (MILESTONE 6). mRNAs in Caenorhabditis elegans, that the key to RNAi is the conver- Zamore, Tuschl and colleagues was shown and the suggestion that such RNA sion of dsRNA into small RNAs, provided more direct evidence interference (RNAi) may have a which then guide specific cleavage to contain of the active role of small RNAs catalytic component (MILESTONE 4), of complementary targets. ~25 nt‑long in RNAi using D. melanogaster gene silencing triggered by dsRNA Work from the Hannon labo- small RNAs embryo lysates and radiolabelled was observed in many species ratory showed that incubation of that were RNAs. Both strands of long dsRNAs including Drosophila melanogaster, synthetic mRNAs with extracts of homologous to were processed into 21–23 nt small trypanosomes, fungi and plants. D. melanogaster S2 cells transfected RNAs. Interestingly, the cleavage However, the mechanism of with dsRNA recapitulates RNAi target mRNAs products of the target mRNAs were unwinding energetically-stable in vitro. The activity responsible produced at 21–23 nt intervals, the dsRNA to promote the search for for the sequence-specific mRNA same interval as during dsRNA complementary targets remained degradation was termed RNA- processing. These results suggested unknown. induced silencing complex (RISC). that small RNAs guide the cleavage of target mRNAs. Shortly thereafter, several labora- tories were able to identify enzymes and cofactors in the RNAi pathway using genetic and biochemical methods. These efforts revealed that dsRNAs are cleaved by the RNase III enzyme Dicer to generate small RNAs, which were termed small interfering RNAs (siRNAs). RISC comprises an Argonaute protein loaded with siRNA, which cleaves complementary mRNAs. The discovery of the mechanism of RNAi revolutionised experimental gene regulation. siRNAs targeting complementary mRNAs can be easily designed and rapidly synthesized. Of clinical relevance is the targeting of multiple mRNA molecules by the same siRNA molecule in consecutive rounds of base-pairing, compared with drugs that remain bound to their target molecule. Thus, the discovery of RNAi mechanisms has laid the groundwork for the development of RNAi-based therapeutics. Minju Ha, Associate Editor, Nature Communications

MILESTONE STUDIES Hammond, S. et al. An RNA-directed nuclease mediates post- transcriptional gene silencing in Drosophila cells. Nature 404, 293–296 (2000) | Zamore, P. D. et al. RNAi: Double-stranded RNA directs the ATP- dependent cleavage of mRNA at 21 to 23 nucleotide intervals. Cell 101, 25–33 (2000). Credit: imageBROKER / Alamy Stock Photo Stock Alamy / imageBROKER Credit:

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MILESTONE 8 Small interfering RNAs silence genes in mammals

The discovery that exogenous protect mice against fulminant hepa- double-stranded RNA (dsRNA) titis, which can result from excessive could silence specific genes in …this 2001 apoptosis in the liver. As hepatocytes Caenorhabditis elegans (MILESTONE 4) study uncovered express high levels of the FAS receptor, and are thus susceptible to FAS- was closely followed by the observa- the promise of tion that, in Drosophila melanogaster, mediated apoptosis, inhibiting Fas this phenomenon was dependent on siRNAs for the expression in hepatocytes was of the processing of the dsRNAs into study of gene interest for treating liver diseases. fragments of ~21–25 nucleotides function and Song et al. showed that tail vein (MILESTONE 7). This observation was highlighted their injection of siRNA targeting Fas pivotal to the development, in 2001, potential utility into mice could markedly reduce of dsRNAs that could silence specific the expression of FAS at the gene and for gene-specific genes in mammalian cells. protein level compared with mice Indeed, despite the efficiency of therapies injected with GFP siRNA; dsRNA-mediated gene silencing in these reductions were insect cells, dsRNAs of 38–1,662 base stable for 10 days pairs (bp) in mammalian cells did not after siRNA seem to silence specific genes, perhaps injection and because dsRNAs of >30 bp activate were achieved a cellular interferon response that at low doses. triggers non-specific degradation of Importantly, mRNA. In an attempt to circumvent whereas mice this problem, Elbashir et al. worked treated with downstream of the dsRNA-processing GFP siRNA step by directly generating 21‑bp small before or after interfering RNA (siRNA) duplexes injection with with symmetric, two-nucleotide Credit: Lara Crow / concanavalin A (to Springer Nature Limited 3ʹ overhangs. Transfecting siRNAs induce fulminant hepatitis) against a reporter gene into mouse, showed signs of liver damage, monkey and human cells repressed non-cognate reporter genes mice injected with Fas siRNA before reporter gene expression by 2–25‑fold, through the interferon response, or after concanavalin A treatment did indicating that siRNAs function in rendering their effect on the target not. Therefore, this study highlighted mammalian cells. Substituting uridine gene difficult to detect. the potential of siRNAs in both with thymidine in the 3ʹ overhang, The final confirmation that preventing and treating liver injury. to protect the siRNA from nuclease siRNAs could silence genes in The discovery that siRNAs could degradation and to reduce the cost of mammalian cells came from the silence genes in mammalian cells, siRNA synthesis, did not compromise observation that, 40–45 hours after and indeed in whole mammalian knockdown (repression) efficiency or transfection, siRNA specifically organisms, revolutionized the study specificity. Furthermore, an siRNA reduced the endogenous expression of gene function. Several advances in concentration of just 1.5 nM, which of the gene encoding lamin A/C siRNA technology have been made was several orders of magnitude below (by >90%) and of the genes encoding in the years since their discovery, the concentration of conventional lamin B1 and nuclear mitotic appara- including tangible improvements in antisense molecules required for gene tus protein (to low levels). Therefore, siRNA delivery (MILESTONES 11, 13), silencing, could silence target genes. although the underlying mechanism with the result that siRNAs are still Together, these findings suggested that was not yet clear, this 2001 study widely used in the laboratory today. siRNAs were powerful tools for gene uncovered the promise of siRNAs Katharine H. Wrighton, Team Leader, silencing in mammalian cells. for the study of gene function and Nature Reviews Cross-Journal Team Comparing siRNAs with dsRNAs highlighted their potential utility for of 50 bp and 500 bp in gene targeting gene-specific therapies. MILESTONE STUDIES Elbashir, S. M. et al. Duplexes of 21‑nucleotide RNAs mediate RNA assays confirmed that the short length The first proof‑of‑principle that interference in cultured mammalian cells. Nature of siRNAs was key to gene-specific siRNAs could be harnessed to treat 411, 494–498 (2001) | Song, E. et al. RNA silencing in mammalian cells. Longer disease came from a study published interference targeting Fas protects mice from fulminant hepatitis. Nat. Med. 9, 347–351 (2003). dsRNAs triggered the degradation of in 2003, showing that siRNAs could

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PIWI-clade proteins — Piwi, MILESTONE 9 Aubergine (Aub) and Argonaute 3 (Ago3) — revealed that each protein piRNAs — guardians of the germline binds to specific piRNA populations: Piwi-bound and Aub-bound piRNAs were mainly antisense to transposon sequences and harboured a strong preference for having a 5ʹ terminal uridine. Ago3‑associated piRNAs, on the other hand, were biased for transposon sense strands and had a preference for an adenine at nucleotide 10, with no preference for uridine at the 5ʹ end. Most strikingly, the 5ʹ ends of Ago3‑bound piRNAs were typically offset by precisely ten nucleotides from the 5ʹ ends of complementary Aub-bound piRNAs. This suggested a model in which an antisense piRNA, complexed with Aub, would recognize and cleave a sense transposon transcript. The cleaved product would then be processed into an Ago3‑bound sense piRNA, which could seek out target transcripts. Ago3‑directed cleavage Credit: imageBROKER / Alamy Stock Photo Stock Alamy / imageBROKER Credit: triggers generation of the original antisense piRNA, capable of both By 2001, RNA interference — the to germ cell and stem cell mainte- silencing the target element and sequence-specific inhibition of nance and to meiosis, although their further amplifying the response. The gene function by homologous dou- biochemical function remained This finding majority of the initial antisense pre- ble-stranded RNA (dsRNA) — had unknown. led to the cursor RNAs were derived from dis- been observed in a wide range of At the time, the related AGO- crete genomic loci, so‑called ‘piRNA realization eukaryotes. The phenomenon had clade subfamily of Argonaute clusters’, which are comprised mainly been linked to transposon-repression proteins had been shown to act in that piRNAs of defective transposon sequences in and anti-viral defence, especially RNA interference and microRNA- represented the fly. in plants, but the full spectrum and mediated gene regulation using a novel class Together, these studies established functional relevance of this mecha 21–22‑nucleotide RNAs as targeting of Dicer- the piRNA pathway as a transposon nism in animals was unknown. guides. However, piRNAs seemed independent surveillance mechanism. Although Then, Gvozdev and colleagues distinct. For example, there was little a plethora of later studies provided demonstrated homology-dependent evidence for overlapping comple- small silencing additional exciting insights into the silencing of testis-specific Stellate mentary RNAs or potential fold-back RNAs piRNA pathway and its function as genes mediated by small RNAs structures, suggesting that piRNAs a safeguard of genome integrity and generated from both strands of the might not be derived from dsRNA fertility, many questions regarding Suppressor of Stellate repeat locus in precursors. Zamore and colleagues the precise molecular mechanisms the Drosophila melanogaster male then provided evidence that Dicer of piRNA generation and their germline. Interestingly, relief of Stellate endonuclease activity — which is diverse silencing functions are still silencing also led to de‑repression of essential for microRNA and short unanswered and the subject remains retrotransposons and other genomic interfering RNA biogenesis — was an active area of research. tandem repeats. This work marked dispensable for piRNA generation Anke Sparmann, Senior Editor, the discovery of piRNAs, although it in D. melanogaster. This finding Nature Structural & Molecular Biology would take another five years until led to the realization that piRNAs they gained this name. represented a novel class of Dicer- MILESTONE STUDIES Aravin, A. A. et al. Double-stranded RNA-mediated silencing of genomic tandem repeats and transposable elements in the D. melanogaster germline. In 2006, four studies used RNA independent small silencing RNAs. Curr. Biol. 11, 1017–1027 (2001) | Girard, A. et al. A germline-specific class of small sequencing to identify a class of Yet, the mechanism governing RNAs binds mammalian Piwi proteins. Nature 442, 199–202 (2006) | Aravin, A. et al. A novel class of small RNAs bind to MILI protein in mouse testes. Nature 442, 203–207 26–30‑nucleotide-long RNAs that piRNA biogenesis remained elusive (2006) | Lau, N. C. et al. Characterization of the piRNA complex from rat testes. Science specifically associated with mamma- until 2007, when two groups inde- 313, 363–367 (2006) | Grivna, S. T. et al. A novel class of small RNAs in mouse lian PIWI-clade Argonaute proteins pendently described an intricate spermatogenic cells. Genes Dev. 20, 1709–1714 (2006) | Vagin, V. V. et al. A distinct small RNA pathway silences selfish genetic elements in the germline. Science 313, 320–324 in mouse, rat and human male germ piRNA amplification loop, the (2006) | Gunawardane, L. S. et al. A slicer-mediated mechanism for repeat-associated cells — hence the name ‘piRNAs’, so‑called ‘piRNA ping-pong cycle’. siRNA 5ʹ end formation in Drosophila. Science 315, 1587–1590 (2007) | Brennecke, J. for PIWI-interacting RNAs. PIWI Sequencing of small RNAs associ- et al. Discrete small RNA-generating loci as master regulators of transposon activity in Drosophila. Cell 128, 1089–1103 (2007). proteins had been genetically linked ated with all three D. melanogaster

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MILESTONE 10 pSUPER start to large-scale RNA interference screening Credit: Cultura Creative (RF) / Alamy Stock Photo Stock Alamy / (RF) Creative Cultura Credit:

In 2002, Reuven Agami, Thijn KRAS-targeted siRNA inhibited the large-scale screening. Berns et al. Brummelkamp and René Bernards expression of mutant (oncogenic) constructed a set of retroviral vectors published two papers (in Science KRAS mRNA without reducing the These vector encoding 23,742 distinct shRNAs, and in Cancer Cell) describing expression of the native mRNA. This systems… which targeted 7,914 human genes different versions of a plasmid-based specificity is essential for studying enabled (that is, three different shRNAs expression system capable of cancer, in which oncogenic and targeting each gene) implicated in targeted driving continuous synthesis of non-oncogenic alleles of the same the promotion or suppression of can- a small interfering RNA (siRNA) gene (which might differ from each knockdown of cer. The researchers then used this in mammalian cells. These vector other by only a single nucleotide) are gene expression RNAi library to screen human cells systems — pSUPER (suppression often co‑expressed. with an efficiency and identified one known and five of endogenous RNA) and the The pSUPER plasmid includes matching that previously unknown components of retroviral version, pRETRO-SUPER the promoter of H1 RNA poly- of synthetic the p53 tumour suppressor pathway, — enabled targeted knockdown of merase III, placed upstream of a which induces cell cycle arrest, cell gene expression with an efficiency 19‑nucleotide sequence derived from siRNAs, yet senescence or apoptosis. This study matching that of synthetic siRNAs, any gene of interest. This sequence without the was the first to use vector-based yet without the loss of inhibition is separated from its reverse comple- loss of inhibition shRNA libraries for large-scale caused by prompt clearance of such ment 19‑nucleotide sequence by a caused by functional genetic screening in siRNAs from mammalian cells, a 9‑nucleotide spacer, and is followed prompt human cells. disadvantage that had curtailed the by a transcription termination Other scientists were quick to clearance of applications of synthetic siRNAs. sequence. Consequently, the RNA realize the significance of this work. Use of the pSUPER vectors transcripts generated by pSUPER such siRNAs Agami, Brummelkamp and Bernards achieved ~90% inhibition of the and pRETRO-SUPER self-fold into received thousands of requests for expression of the target gene and, in a stem–loop structure resembling a reagents from fellow researchers, the case of pRETRO-SUPER, which hairpin, and were therefore called and the use of siRNA-encoding integrates into the host genome, short hairpin RNAs (shRNAs). (viral) vectors has since become a also enabled stable expression of In their first 2002 paper, major method of gene suppression the siRNA. These advances enabled Brummelkamp et al. concluded that in mammalian cells. the transfected cells to be cultured it should be possible to generate Caroline Barranco, Senior Editor, and the downstream effects of gene large collections of pSUPER shRNA Nature Reviews Cross-Journal Team knockdown to be studied in detail. vectors to carry out high-throughput MILESTONE STUDIES Brummelkamp, T. R. et al. A system for stable expression of short Importantly, Brummelkamp genetic screens for loss‑of-function interfering RNAs in mammalian cells. Science 296, 550–553 (2002) | Brummelkamp, T. R. et al. showed that RNA interference phenotypes. Indeed, only two years et al. Stable expression of tumorigenicity by virus-mediated RNA interference. Cancer (RNAi) was highly sequence-specific: later, the Bernards group demon- Cell 2, 243–247 (2002) | Berns, K. et al. A large-scale RNAi screen in human cells identifies new components of the p53 pathway. Nature 428, 431–437 (2004). use of pRETRO-SUPER to deliver a strated the utility of this system for

S14 | DECEMBER 2019 www.nature.com/collections/antisense Credit: Feodora Chiosea / Alamy Stock Vector Targeted siRNA delivery in vivo NATURE MILESTONES (MILESTONE 8) by Judy Lieberman’s group in2003 potential of siRNAs was performed demonstration of therapeutic the tool. The first proof approachthis as atherapeutic interestincreased the inusing interferingsmall RNAs (siRNAs) of gene selective silencing through Advances characterisation inthe body, authors the an antibody used 2005. To target cells inthe specific a subsequent study published in by and in colleagues Lieberman immune cells. to target other cells, for example, it was unclear used ifit be could also worked method the for liver cells but portion ofvolume. blood the Second, pro large a of exchange the involves potential clinical application. First, it which has two major challenges for hydrodynamic tail-vein injection, group administered siRNA by expression inamouse model, the Both challenges wereBoth addressed

MILESTONE 11 . To silence gene ‑ of |

ANTISENSE RNA ‑ principle -

gene gene siRNAs targeting HIV-1 the capsid with F105–Ploaded methods. to transfect with conventional T cells, which are challenging how F105–Pworks inprimary HIV mRNA levels only incells expressing reductiondose-dependent of target of siRNA, and and selective the that F105–Penabled uptake the to transport DNA into cells. F105–P had previously shown been fragment itself not does bind them. siRNAs, antibody the because protamine for was necessary binding TheF105–P molecules. fusion to F105 with protamine, generating are infected with HIV. fused They expressed on of surface the cells that HIV fragment (F105)that recognised viral particles into particles viral culture cell the but reduced also release the of replication in HIV-infected T cells, The researchers first confirmed ‑ ‑ gag gag 1 envelope. tested Then, they 1 envelope protein, which is not only HIV decreased applications… for clinical siRNAs developing step towards important cells was an by selected siRNA uptake …targeted F105–P mRNA expression. proteinssurface to silence or reduce on cell based types cell of selected approachthe enables targeting the medium. This demonstrated that side effects. cations minimising while adverse developing siRNAs for clinical appli- cells was an important step towards targeted siRNA uptake by selected investigatedto be inmore detail, and toxicity assessment, remained macokinetics and safety adetailed some important issues, like phar the siRNA in vivo. delivery Although laid foundation the for targeted less immunogenic. methods Both ies, which are easier to produce and aptamersbased instead of antibod- published ayear later, RNA- used in vivo.delivery Asimilar approach, demonstration of targeted siRNA in ERBB2 to silence Ku70 expression antibody against receptor the of approach their by using an strated broader the applicability F105–P in vivoeffective gene silencing by growth, thereby demonstrating related genes reduced tumour siRNAs against tumour-growth- bindingmolecules amixture of Furthermore, injection of F105–P through intravenous injection. tumourthe cells or administered F105–Pwaswhen injected into surrounding cells, took up siRNA into mice. cells, but Only these not envelope protein were implanted tumour cells expressing an HIV application of F105–P. For this, worksalso in vivo after systemic work was proof the Associate Editor, Communications Nature chimeras. specific delivery of siRNAs with aptamer-siRNA 709–717 (2005) | McNamara II, J. et al. Cell type– via cell-surfacereceptors. Nat.Biotechnol. mediated in vivo delivery of small interfering RNAs MILESTONE STUDIES Song, E. et al. Antibody ERBB2 The next step was to show that The researchers demon also - The major achievement of this MILESTONES ‑ ‑ Nat. Biotechnol. mediated siRNA delivery. mediated gene silencing ‑ expressing cells. DECEMBER 2019 24, 1005–1015(2006). ‑ of Christian Schnell, ‑ principle 23, |

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Credit: Vicky Summersby / Springer Nature Limited

MILESTONE 12 Gene editing by CRISPR–Cas

One of the most revolutionary developments Foundational work was performed in 2012 in biology has its origins in the RNA-based by groups studying the function of CRISPR defence system of bacteria, which encodes and the associated protein Cas9 in bacteria. It clustered regularly interspaced short was known that these CRISPR–Cas systems palindromic repeats (CRISPR) along with function as a bacterial immune system against CRISPR-associated (Cas) proteins. CRISPR– viruses. Jinek et al., and complementary work Cas has been adapted to function as a pro- from Gasiunas et al., demonstrated that two grammable genome-engineering tool that has small RNAs — the CRISPR RNA (crRNA) in a enabled easy targeting and manipulation of complex with a trans-activating CRISPR RNA precise genomic sequences in bacteria, plants, (tracrRNA) — function together in targeting fungi and mammals, including humans. the nuclease Cas9 to specific DNA sequences, Prior work with programmable where it can generate a double-stranded DNA genome-engineering tools focused on the break (DSB). Jinek et al. had the insight that a mammalian context, they demonstrated use of various nucleases for gene targeting. the two RNAs could be joined together to form that Cas9 could be guided to specific target Though precise, the reliance on protein-based a single guide RNA (sgRNA). The groups spec- sequences in the larger and more complex recognition of a target DNA sequence meant ulated that RNA could also be used to guide mammalian genomes where, as in bacteria, that each new target required redesigning the Cas9 to specific genomic sequences and thus it cleaved the DNA. tool, which is often a laborious task. CRISPR– enable gene editing and genome engineering. Mammalian cells attempt to rapidly repair Cas has the advantage of being precise and The concept was demonstrated in a set of these DSBs and this process can be co‑opted highly adaptable. This precision is derived papers published in early 2013, when Jinek for tailored genome engineering. The non- from base-pairing between the complemen- et al., Mali et al. and Cong et al. adapted the homologous end-joining DSB repair pathway tary CRISPR–Cas guiding RNA and target bacterial CRISPR–Cas system to function attempts to stitch the DNA ends back together, DNA, which creates a straightforward and in mouse and human cells. Using sgRNAs often resulting in the introduction of delete- easy‑to‑adapt targeting system. and optimising the system for expression in rious mutations in genes. Furthermore, if a

MILESTONE 13 Chemical optimization improves oligonucleotide delivery

Forty years after their initial discovery, (GalNAc), a ligand of the asialoglycoprotein oligonucleotide therapeutics had begun to receptor (ASGPR). This receptor is expressed on show potential to reach clinical maturity. By the surface of liver cells and is responsible for the 2014, several drugs had been approved for uptake of circulating glycoproteins with exposed disease treatment, and many others were GalNAc glycans. This design enabled targeted undergoing clinical trials at various stages. siRNA–GalNAc delivery to the liver owing to However, being able to deliver effective doses of the high-affinity binding between receptor and oligonucleotides to a specific set of diseased cells ligand. The first tests were performed in cultured or organs was still challenging. mouse liver cells and showed that receptor Small interfering RNAs (siRNAs) had been binding-affinity correlated with siRNA uptake successfully used to inhibit the expression of efficiency. Encouraged by these results, the disease-causing genes, and the implementation researchers tested the ability of siRNA–GalNAc of antibody-mediated and lipid-nanoparticle- conjugates to silence gene expression in vivo. They mediated delivery had significantly improved observed a robust and durable silencing of the their efficiency. However, these treatments often targeted gene, transthyretin, in the liver of mice required high doses and repeated intravenous after a single or multiple low-volume subcutaneous injections to be therapeutically effective, which administrations. The extent of silencing was limited their clinical applicability in cases where higher following subcutaneous administration intravenous drug administration was not feasible. compared with intravenous administration. Therefore, researchers worked to develop In a study published a year later, the same chemically modified oligonucleotides to enable research group optimised the design of the efficient delivery to target cells by subcutaneous conjugates to improve therapeutic effectiveness. administration. The researchers tested various attachment sites How such chemical optimisation could improve for the GalNAc ligand on the RNA molecule, and siRNA delivery was demonstrated in 2014 by evaluated silencing activity both in vitro and Manoharan and colleagues, who covalently in vivo. They found that placing three monovalent GalNAc units in close proximity to each other

conjugated siRNA to N‑acetylgalactosamine Photo Stock Alamy / Kaulitzki Sebastian Credit:

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DNA template is available the cell will MILESTONE 14 attempt to copy genetic information …CRISPR–Cas from it during repair, thereby insert- has opened up ing new genetic information into the An antisense new avenues in our genome, as demonstrated by Mali understanding of et al. and Cong et al. how cells repair From its origins as a bacterial oligonucleotide splicing DNA damage, immune system, CRISPR–Cas has in our ability to been developed into an all-purpose modulator to treat spinal tool for tailored engineering of engineer cells and genomes in a range of species. In the in the possibility of span of less than a decade, CRISPR– muscular atrophy developing new, Cas has opened up new avenues RNA-dependent in our understanding of how cells therapies for repair DNA damage, in our ability to Antisense previously oligonucleotide engineer cells and in the possibility Nucleus intractable genetic of developing new, RNA-dependent diseases therapies for previously intractable hnRNP genetic diseases. Ross Cloney, Senior Editor, Nature Communications

8 3ʹ 199 , 21–25 (2012) Intron 7 MILESTONE STUDIES Jinek, M. et al. Programmable dual-RNA-guided DNA pre-mRNA endonuclease in adaptive bacterial immunity. Science 337, 816–821 (2012) | Gasiunas, G. 6 et al. Cas9–crRNA ribonucleoprotein complex mediates specific DNA cleavage for 5ʹ Biol . J. Cell adaptive immunity in bacteria. Proc. Natl. Acad. Sci. USA 109, E2579–E2586 (2012) | Jinek, M. et al. RNA-programmed genome editing in human cells. eLife 2, e00471 (2013) 6 7 8 | Mali, P. et al. RNA-guided human genome engineering via Cas9. Science 339, 823–826 (2013) | Cong, L. et al. Multiplex genome engineering using CRISPR/Cas systems. from permission with adapted Figure et al. F. Rigo, Science 339, 819–823 (2013). FURTHER READING Pickar-Oliver, A. & Gersbach, C. A. The next generation of On 23 December 2016, the Some individuals with SMA carry CRISPR–Cas technologies and applications. Nat. Rev. Mol. Cell Biol. 20, 490–507 (2019). United States Food and Drug multiple copies of SMN2 and can Administration (FDA) approved thus produce higher levels of full- the antisense oligonucleotide (ASO) length SMN protein, which reduces on the RNA sense strand resulted in a drug nusinersen (Spinraza) to treat the severity and delays the onset of higher-affinity binding to ASGPR on This seminal spinal muscular atrophy (SMA), a the disease. liver cells, and a more robust silencing fatal genetic disease that can affect The molecular basis of SMN2 work in vivo. children and adults. The approval exon 7 skipping was elucidated by established This seminal work established siRNA− was the culmination of a successful several groups, including those of GalNAc as a promising therapeutic siRNA–GalNAc collaboration between researchers Ravendra Singh at University of delivery approach to treat diseases in academia and industry, with Massachusetts Medical School and as a promising involving liver-expressed genes. support and assistance from patient Adrian Krainer at the Cold Spring therapeutic Despite halting of the development advocacy groups and regulatory Harbor Laboratory, in the late 1990s delivery of the first siRNA−GalNAc-based drug agencies. to early 2000s. SMN2 contains a approach to (Revusiran) during clinical trials in 2016, the impressive silencing efficiency, SMA is a devastating neuro- synonymous C‑to‑T substitution in treat diseases good safety profile and encouraging muscular disease that affects 1 in exon 7 that weakens the binding of involving liver- results from more recent clinical trials 10,000 people and is caused by splicing activators, thereby reducing expressed of drugs for acute hepatic porphyria mutations in the gene survival of the efficiency of the 3ʹ splice site. genes (Givosiran) and cardiovascular motor neuron 1 (SMN1). Without In 2003, Cartegni and Krainer disease with elevated LDL cholesterol functional SMN protein, the motor engineered bifunctional ASOs that (Inclisiran), established GalNAc neurons in the spinal cord and operate as synthetic splicing activa- conjugation as a promising solution for brain stem degenerate, resulting in tors: a peptide mimicking a splicing therapeutic siRNA delivery to the liver. muscle weakness and atrophy. Of activator was covalently linked to Alfredo Sansone, the infants born with SMA, 60% an ASO that hybridized to exon 7. Senior Editor, Nature Communications show symptoms before six months This chimeric effector was able to of age, with median life expectancy promote exon 7 inclusion in cell MILESTONE STUDIES Nair, J. K. et al. Multivalent N‑acetylgalactosamine- conjugated siRNA localizes in hepatocytes and elicits robust RNAi-mediated gene of less than two years. A paralog extracts. Those findings prompted silencing. J. Am. Chem. Soc. 136, 16958–16961 (2014) | Matsuda, S. et al. siRNA of SMN1 in the human genome, C. Frank Bennett, from Isis (later conjugates carrying sequentially assembled trivalent N‑acetylgalactosamine SMN2, encodes an identical SMN Ionis) Pharmaceuticals, to contact linked through nucleosides elicit robust gene silencing in vivo in hepatocytes. ACS Chem. Biol. 10, 1181–1187 (2015). protein. However, its pre-mRNA Krainer and initiate a collaboration, FURTHER READING Sardh, E. et al. Phase 1 trial of an RNA interference therapy for undergoes aberrant splicing, with as recounted by Rigo et al. in 2012. acute intermittent porphyria. N. Engl. J. Med. 380, 549–558 (2019) | Ray, K. K. et al. 90% of mature SMN2 transcripts Over the next years, the strategy Inclisiran in patients at high cardiovascular risk with elevated LDL cholesterol. N. Engl. J. Med. 376, 1430–1440 (2017). lacking exon 7 and producing a to control exon 7 inclusion was truncated, unstable polypeptide. optimized for use in cells and

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animal models. ASOs targeting a (skin prick). An interim analysis The FDA approved nusinersen site near the 5ʹ splice site in SMN2 conducted with 82 patients showed only three months after the new intron 7 could efficiently promote …a successful that 40% of those treated with drug application (NDA) was filed exon 7 inclusion without the need of collaboration nusinersen showed improvements by Biogen. This occurred with an appended peptide moiety. They between in motor function milestones, such fast-track designation and priority acted by preventing binding of the as head control, sitting, rolling, review, and without an advisory researchers splicing repressors HNRNPA1 and crawling, standing and walking, committee, as outside expertise was HNRNPA2. In addition, chemical in academia compared to none in the control not deemed necessary given the modifications in the backbone and industry, group. These results led to early lack of controversial issues, as noted (phosphorothioate) and nucleotides with support termination of the trial in August in the agency’s summary report. (2ʹ‑O‑methoxyethyl, or 2ʹ‑MOE) and assistance 2016, so that infants in the control Nusinersen was approved by the of the ASOs improved their from patient group could start receiving the European Medicines Agency in May pharmacological properties. drug. Beneficial effects observed 2017, and it is currently available With promising results in pre- advocacy groups in another trial, with children aged for treating SMA in more than clinical studies, the ASO nusinersen and regulatory 2–12 with later-onset SMA, also 40 countries. entered clinical trial phase I and agencies prompted its early termination in Inês Chen, Chief Editor, II studies in 2011 and 2013–2014, November 2016. Nature Structural & Molecular Biology respectively. A multi-centre, randomized, double-blind phase III study took place in 2014–2016 FURTHER READING Lefebvre, S. L. et al. Identification and characterization of a spinal muscular atrophy-determining gene. Cell 80, 155–165 (1995) | Hofmann, Y. et al. Htra2‑β1 stimulates an exonic splicing enhancer and can restore full-length SMN expression to survival motor neuron 2 and included 121 infants up to (SMN2). Proc. Natl. Acad. Sci. USA 97, 9618–9623 (2000) | Cartegni, L. & Krainer, A. R. Disruption of an SF2/ASF-dependent exonic splicing enhancer seven months of age who had been in SMN2 causes spinal muscular atrophy in the absence of SMN1. Nat. Genet. 30, 377–394 (2002) | Cartegni, L & Krainer, A. R. Correction of disease- associated exon skipping by synthetic exon-specific activators. Nat. Struct. Biol. 10, 120–125 (2003) | Singh, N. K. et al. Splicing of a critical exon of diagnosed with SMA before they human Survival Motor Neuron is regulated by a unique silencer element located in the last intron. Mol. Cell. Biol. 26, 1333–1346 (2006) | Hua, Y. et al. were six months old (infantile Antisense masking of an hnRNP A1/A2 intronic splicing silencer corrects SMN2 splicing in transgenic mice. Am. J. Hum. Genet. 82, 834–848 (2008) | onset). Participants received the Singh, N. K. et al. A short antisense oligonucleotide masking a unique intronic motif prevents skipping of a critical exon in spinal muscular atrophy. RNA Biol. 6, 341–350 (2009) | Hua, Y. et al. Peripheral SMN restoration is essential for long-term rescue of a severe SMA mouse model. Nature 478, drug injected intrathecally (that 123–126 (2011) | Rigo, F. et al. Antisense-based therapy for the treatment of spinal muscular atrophy. J. Cell Biol. 199, 21–25 (2012) | Chiriboga, C. A. et al. Results from a phase 1 study of nurinersen (ISIS-SMN ) in children with spinal muscular atrophy. Neurology , 890–897 (2016) | Wadman, M. is, through a lumbar puncture for Rx 86 Updated: FDA approves drug that rescues babies with fatal neurodegenerative disease. Science https://doi.org/dbg2 (2016) | Finkel, R. S. et al. delivery into the cerebrospinal Treatment of infantile-onset spinal muscular atrophy with nusinersen: a phase 2, open-label, dose-escalation study. The Lancet 388, 3017–3026 fluid, to reach targets in the central (2017) | Finkel, R. S. et al. Nusinersen versus sham control in infantile-onset spinal muscular atrophy. N. Engl. J. Med. 377, 1723–1732 (2017) | nervous system). A control group Mercuri, E. et al. Nusinersen versus sham control in later-onset spinal muscular atrophy. N. Engl. J. Med. 378, 625–635 (2018) | Bastings, E. Division Director Summary Review (FDA, 2016); https://go.nature.com/2lP28zc was treated with a mock procedure

MILESTONE 15 A new dawn for RNAi drugs

The 10th of August, 2018 marked a new era for the field of RNA therapeutics, with the first approval of an RNA interference (RNAi)-based drug by the United States Food and Drug Administration. The drug — patisiran (Onpattro) — is approved for the treatment of polyneuropathy in people with hereditary transthyretin-mediated amyloidosis (hATTR). This rare and devastating neurodegenerative disease is caused by deposition of amyloid fibrils formed by misfolded transthyretin protein. A double-stranded small interfering RNA composed of two modified 21‑mer oligonucleotides and encapsulated in a lipid nanoparticle formulated for hepatocyte uptake, patisiran silences transthyretin mRNAs in the liver to reduce serum levels of the protein. The approval of patisiran brings new hope to patients with hATTR who previously had no effective treatment options.

FURTHER READING Garber, K. Alnylam launches era of RNAi drugs. Nat. Biotechnol. 36, 777–778 (2018) | Setten, R. L. et al. The current state and future directions of RNAi-based therapeutics. Nat. Rev. Drug Discov. 18, 421–446 (2019) Credit: GiroScience / Alamy Stock Photo Stock Alamy / GiroScience Credit:

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