Abstract
Oligonucleotides can be used to modulate gene expression via a range of processes including RNAi, target degradation by RNase H-mediated cleavage, splicing modulation, non-coding RNA inhibition, gene activation and programmed gene editing. As such, these molecules have potential therapeutic applications for myriad indications, with several oligonucleotide drugs recently gaining approval. However, despite recent technological advances, achieving efficient oligonucleotide delivery, particularly to extrahepatic tissues, remains a major translational limitation. Here, we provide an overview of oligonucleotide-based drug platforms, focusing on key approaches — including chemical modification, bioconjugation and the use of nanocarriers — which aim to address the delivery challenge.
This is a preview of subscription content, access via your institution
Access options
Access Nature and 54 other Nature Portfolio journals
Get Nature+, our best-value online-access subscription
£14.99 / 30 days
cancel any time
Subscribe to this journal
Receive 12 print issues and online access
£139.00 per year
only £11.58 per issue
Buy this article
- Purchase on SpringerLink
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Kim, J. et al. Patient-customized oligonucleotide therapy for a rare genetic disease. N. Engl. J. Med. 381, 1644–1652 (2019). This is the first study to utilize an oligonucleotide therapy tailored to a single patient.
Giorgio, E. et al. Allele-specific silencing as treatment for gene duplication disorders: proof-of-principle in autosomal dominant leukodystrophy. Brain 142, 1905–1920 (2019).
Southwell, A. L. et al. In vivo evaluation of candidate allele-specific mutant huntingtin gene silencing antisense oligonucleotides. Mol. Ther. 22, 2093–2106 (2014).
Miller, V. M. et al. Allele-specific silencing of dominant disease genes. Proc. Natl Acad. Sci. USA 100, 7195–7200 (2003).
Klein, A. F. et al. Peptide-conjugated oligonucleotides evoke long-lasting myotonic dystrophy correction in patient-derived cells and mice. J. Clin. Invest. 129, 4739–4744 (2019).
Crnković-Mertens, I., Semzow, J., Hoppe-Seyler, F. & Butz, K. Isoform-specific silencing of the Livin gene by RNA interference defines Livin β as key mediator of apoptosis inhibition in HeLa cells. J. Mol. Med. 84, 232–240 (2006).
Valencia-Serna, J. et al. siRNA-mediated BCR–ABL silencing in primary chronic myeloid leukemia cells using lipopolymers. J. Controlled Rel. 310, 141–154 (2019).
Wu, S. Y., Lopez-Berestein, G., Calin, G. A. & Sood, A. K. RNAi therapies: drugging the undruggable. Sci. Transl. Med. 6, 240ps7 (2014).
Tushir-Singh, J. Antibody–siRNA conjugates: drugging the undruggable for anti-leukemic therapy. Expert Opin. Biol. Ther. 17, 325–338 (2017).
Bobbin, M. L., Burnett, J. C. & Rossi, J. J. RNA interference approaches for treatment of HIV-1 infection. Genome Med. 7, 50 (2015).
Hermann, T. & Patel, D. J. Adaptive recognition by nucleic acid aptamers. Science 287, 820–825 (2000).
Knott, G. J. & Doudna, J. A. CRISPR–Cas guides the future of genetic engineering. Science 361, 866–869 (2018).
Jackson, A. L. et al. Expression profiling reveals off-target gene regulation by RNAi. Nat. Biotechnol. 21, 635–637 (2003).
Jackson, A. L. et al. Widespread siRNA ‘off-target’ transcript silencing mediated by seed region sequence complementarity. RNA 12, 1179–1187 (2006).
Scacheri, P. C. et al. Short interfering RNAs can induce unexpected and divergent changes in the levels of untargeted proteins in mammalian cells. Proc. Natl Acad. Sci. USA 101, 1892–1897 (2004).
Persengiev, S. P., Zhu, X. & Green, M. R. Nonspecific, concentration-dependent stimulation and repression of mammalian gene expression by small interfering RNAs (siRNAs). RNA 10, 12–18 (2004).
Doench, J. G., Petersen, C. P. & Sharp, P. A. siRNAs can function as miRNAs. Genes Dev. 17, 438–442 (2003).
Grimm, D. et al. Fatality in mice due to oversaturation of cellular microRNA/short hairpin RNA pathways. Nature 441, 537–541 (2006).
Wu, H. et al. Determination of the role of the human RNase H1 in the pharmacology of DNA-like antisense drugs. J. Biol. Chem. 279, 17181–17189 (2004).
Crooke, S. T. Molecular mechanisms of antisense oligonucleotides. Nucleic Acid. Ther. 27, 70–77 (2017).
Monia, B. P. et al. Evaluation of 2′-modified oligonucleotides containing 2′-deoxy gaps as antisense inhibitors of gene expression. J. Biol. Chem. 268, 14514–14522 (1993).
Liang, 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. 25, 2075–2092 (2017).
Lennox, K. A. & Behlke, M. A. Cellular localization of long non-coding RNAs affects silencing by RNAi more than by antisense oligonucleotides. Nucleic Acids Res. 44, 863–877 (2016).
Dominski, Z. & Kole, R. Restoration of correct splicing in thalassemic pre-mRNA by antisense oligonucleotides. Proc. Natl Acad. Sci. USA 90, 8673–8677 (1993).
Singh, R. N. & Singh, N. N. Mechanism of splicing regulation of spinal muscular atrophy genes. Adv. Neurobiol. 20, 31–61 (2018).
Aartsma-Rus, A. et al. Development of exon skipping therapies for Duchenne muscular dystrophy: a critical review and a perspective on the outstanding issues. Nucleic Acid. Ther. 27, 251–259 (2017).
Wan, L. & Dreyfuss, G. Splicing-correcting therapy for SMA. Cell 170, 5 (2017).
Ward, A. J., Norrbom, M., Chun, S., Bennett, C. F. & Rigo, F. Nonsense-mediated decay as a terminating mechanism for antisense oligonucleotides. Nucleic Acids Res. 42, 5871–5879 (2014).
Boiziau, C. et al. Inhibition of translation initiation by antisense oligonucleotides via an RNase-H independent mechanism. Nucleic Acids Res. 19, 1113–1119 (1991).
Baker, B. F. et al. 2′-O-(2-methoxy)ethyl-modified anti-intercellular adhesion molecule 1 (ICAM-1) oligonucleotides selectively increase the ICAM-1 mRNA level and inhibit formation of the ICAM-1 translation initiation complex in human umbilical vein endothelial cells. J. Biol. Chem. 272, 11994–12000 (1997).
Calvo, S. E., Pagliarini, D. J. & Mootha, V. K. Upstream open reading frames cause widespread reduction of protein expression and are polymorphic among humans. Proc. Natl Acad. Sci. USA 106, 7507–7512 (2009).
Liang, X.-H. et al. Translation efficiency of mRNAs is increased by antisense oligonucleotides targeting upstream open reading frames. Nat. Biotechnol. 34, 875–880 (2016).
Nomakuchi, T. T., Rigo, F., Aznarez, I. & Krainer, A. R. Antisense oligonucleotide-directed inhibition of nonsense-mediated mRNA decay. Nat. Biotechnol. 34, 164–166 (2016).
Vickers, T. A., Wyatt, J. R., Burckin, T., Bennett, C. F. & Freier, S. M. Fully modified 2′ MOE oligonucleotides redirect polyadenylation. Nucleic Acids Res. 29, 1293–1299 (2001).
Elbashir, S. M. et al. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411, 494–498 (2001).
Liu, J. et al. Argonaute2 is the catalytic engine of mammalian RNAi. Science 305, 1437–1441 (2004).
Roberts, T. C. The microRNA machinery. Adv. Exp. Med. Biol. 887, 15–30 (2015).
Schürmann, N., Trabuco, L. G., Bender, C., Russell, R. B. & Grimm, D. Molecular dissection of human Argonaute proteins by DNA shuffling. Nat. Struct. Mol. Biol. 20, 818–826 (2013).
Kim, D.-H. et al. Synthetic dsRNA Dicer substrates enhance RNAi potency and efficacy. Nat. Biotechnol. 23, 222–226 (2005).
Bramsen, J. B. et al. Improved silencing properties using small internally segmented interfering RNAs. Nucleic Acids Res. 35, 5886–5897 (2007).
Byrne, M. et al. Novel hydrophobically modified asymmetric RNAi compounds (sd-rxRNA) demonstrate robust efficacy in the eye. J. Ocul. Pharmacol. Ther. 29, 855–864 (2013).
Yu, D. et al. Single-stranded RNAs use RNAi to potently and allele-selectively inhibit mutant huntingtin expression. Cell 150, 895–908 (2012).
Lima, W. F. et al. Single-stranded siRNAs activate RNAi in animals. Cell 150, 883–894 (2012).
Alterman, J. F. et al. A divalent siRNA chemical scaffold for potent and sustained modulation of gene expression throughout the central nervous system. Nat. Biotechnol. 37, 884–894 (2019).
Svoronos, A. A., Engelman, D. M. & Slack, F. J. OncomiR or tumor suppressor? The duplicity of microRNAs in cancer. Cancer Res. 76, 3666–3670 (2016).
Okada, N. et al. A positive feedback between p53 and miR-34 miRNAs mediates tumor suppression. Genes Dev. 28, 438–450 (2014).
Bueno, M. J. & Malumbres, M. MicroRNAs and the cell cycle. Biochim. Biophys. Acta 1812, 592–601 (2011).
Shimakami, T. et al. Stabilization of hepatitis C virus RNA by an Ago2–miR-122 complex. Proc. Natl Acad. Sci. USA 109, 941–946 (2012).
Balasubramaniam, M., Pandhare, J. & Dash, C. Are microRNAs important players in HIV-1 infection? An update. Viruses 10, 110 (2018).
Xu, S. J., Hu, H. T., Li, H. L. & Chang, S. The role of miRNAs in immune cell development, immune cell activation, and tumor immunity: with a focus on macrophages and natural killer cells. Cells 8, 1140 (2019).
Wendt, A., Esguerra, J. L. & Eliasson, L. Islet microRNAs in health and type-2 diabetes. Curr. Opin. Pharmacol. 43, 46–52 (2018).
Vienberg, S., Geiger, J., Madsen, S. & Dalgaard, L. T. MicroRNAs in metabolism. Acta Physiol. 219, 346–361 (2017).
van Rooij, E., Liu, N. & Olson, E. N. MicroRNAs flex their muscles. Trends Genet. 24, 159–166 (2008).
Chen, J.-F. et al. microRNA-1 and microRNA-206 regulate skeletal muscle satellite cell proliferation and differentiation by repressing Pax7. J. Cell Biol. 190, 867–879 (2010).
Roberts, T. C. et al. Expression analysis in multiple muscle groups and serum reveals complexity in the microRNA transcriptome of the MDX mouse with implications for therapy. Mol. Ther. Nucleic Acids 1, e39 (2012).
Cacchiarelli, D. et al. MicroRNAs involved in molecular circuitries relevant for the Duchenne muscular dystrophy pathogenesis are controlled by the dystrophin/nNOS pathway. Cell Metab. 12, 341–351 (2010).
Provost, P. et al. Ribonuclease activity and RNA binding of recombinant human Dicer. EMBO J. 21, 5864–5874 (2002).
Lee, Y. et al. The nuclear RNase III Drosha initiates microRNA processing. Nature 425, 415–419 (2003).
Guo, H., Ingolia, N. T., Weissman, J. S. & Bartel, D. P. Mammalian microRNAs predominantly act to decrease target mRNA levels. Nature 466, 835–840 (2010).
Filipowicz, W., Bhattacharyya, S. N. & Sonenberg, N. Mechanisms of post-transcriptional regulation by microRNAs: are the answers in sight? Nat. Rev. Genet. 9, 102–114 (2008).
Roberts, T. C. & Wood, M. J. A. Therapeutic targeting of non-coding RNAs. Essays Biochem. 54, 127–145 (2013).
Krützfeldt, J. et al. Silencing of microRNAs in vivo with ‘antagomirs’. Nature 438, 685–689 (2005).
Lindow, M. & Kauppinen, S. Discovering the first microRNA-targeted drug. J. Cell Biol. 199, 407–412 (2012).
Jopling, C. L., Yi, M., Lancaster, A. M., Lemon, S. M. & Sarnow, P. Modulation of hepatitis C virus RNA abundance by a liver-specific microRNA. Science 309, 1577–1581 (2005).
Jopling, C. L., Schütz, S. & Sarnow, P. Position-dependent function for a tandem microRNA miR-122-binding site located in the hepatitis C virus RNA genome. Cell Host Microbe 4, 77–85 (2008).
Jopling, C. L. Targeting microRNA-122 to treat hepatitis C virus infection. Viruses 2, 1382–1393 (2010).
Janssen, H. L. A. et al. Treatment of HCV infection by targeting microRNA. N. Engl. J. Med. 368, 1685–1694 (2013).
Machlin, E. S., Sarnow, P. & Sagan, S. M. Masking the 5′ terminal nucleotides of the hepatitis C virus genome by an unconventional microRNA–target RNA complex. Proc. Natl Acad. Sci. USA 108, 3193–3198 (2011).
Ottosen, S. et al. In vitro antiviral activity and preclinical and clinical resistance profile of miravirsen, a novel anti-hepatitis C virus therapeutic targeting the human factor miR-122. Antimicrob. Agents Chemother. 59, 599–608 (2015).
Gomez, I. G. et al. Anti-microRNA-21 oligonucleotides prevent Alport nephropathy progression by stimulating metabolic pathways. J. Clin. Invest. 125, 141–156 (2015).
Lee, E. C. et al. Discovery and preclinical evaluation of anti-miR-17 oligonucleotide RGLS4326 for the treatment of polycystic kidney disease. Nat. Commun. 10, 1–14 (2019).
Seto, A. G. et al. Cobomarsen, an oligonucleotide inhibitor of miR-155, co-ordinately regulates multiple survival pathways to reduce cellular proliferation and survival in cutaneous T-cell lymphoma. Br. J. Haematol. 183, 428–444 (2018).
Gallant-Behm, C. L. et al. A microRNA-29 mimic (remlarsen) represses extracellular matrix expression and fibroplasia in the skin. J. Invest. Dermatol. 139, 1073–1081 (2019).
Choi, W.-Y., Giraldez, A. J. & Schier, A. F. Target protectors reveal dampening and balancing of Nodal agonist and antagonist by miR-430. Science 318, 271–274 (2007).
Wang, Z. The principles of miRNA-masking antisense oligonucleotides technology. Methods Mol. Biol. 676, 43–49 (2011).
Roberts, T. C., Morris, K. V. & Wood, M. J. A. The role of long non-coding RNAs in neurodevelopment, brain function and neurological disease. Philos. Trans. R. Soc. Lond. B Biol. Sci. 369, (2014).
Wahlestedt, C. Targeting long non-coding RNA to therapeutically upregulate gene expression. Nat. Rev. Drug Discov. 12, 433–446 (2013).
Yoon, S. & Rossi, J. J. Therapeutic potential of small activating RNAs (saRNAs) in human cancers. Curr. Pharm. Biotechnol. 19, 604–610 (2018).
Faghihi, M. A. et al. Expression of a noncoding RNA is elevated in Alzheimer’s disease and drives rapid feed-forward regulation of β-secretase. Nat. Med. 14, 723–730 (2008).
Modarresi, F. et al. Inhibition of natural antisense transcripts in vivo results in gene-specific transcriptional upregulation. Nat. Biotechnol. 30, 453–459 (2012).
Meng, L. et al. Towards a therapy for Angelman syndrome by targeting a long non-coding RNA. Nature 518, 409–412 (2015).
Hsiao, J. et al. Upregulation of haploinsufficient gene expression in the brain by targeting a long non-coding RNA improves seizure phenotype in a model of Dravet syndrome. EBioMedicine 9, 257–277 (2016).
Schwartz, J. C. et al. Antisense transcripts are targets for activating small RNAs. Nat. Struct. Mol. Biol. 15, 842–848 (2008).
Matsui, M. et al. Promoter RNA links transcriptional regulation of inflammatory pathway genes. Nucleic Acids Res. 41, 10086–10109 (2013).
Turunen, T. A. et al. Changes in nuclear and cytoplasmic microRNA distribution in response to hypoxic stress. Sci. Rep. 9, 10332 (2019).
Gagnon, K. T., Li, L., Chu, Y., Janowski, B. A. & Corey, D. R. RNAi factors are present and active in human cell nuclei. Cell Rep. 6, 211–221 (2014).
Voutila, J. et al. Development and mechanism of small activating RNA targeting CEBPA, a novel therapeutic in clinical trials for liver cancer. Mol. Ther. 25, 2705–2714 (2017).
Reebye, V. et al. Gene activation of CEBPA using saRNA: preclinical studies of the first in human saRNA drug candidate for liver cancer. Oncogene 37, 3216–3228 (2018).
Sarker, D. et al. MTL-CEBPA, a small activating RNA therapeutic up-regulating C/EBP-α, in patients with advanced liver cancer: a first-in-human, multi-centre, open-label, phase I trial. Clin. Cancer Res. https://doi.org/10.1158/1078-0432.CCR-20-0414 (2020). This paper presents the currently most advanced oligonucleotide therapy aimed at upregulating a target gene.
Tsui, N. B. Y., Ng, E. K. O. & Lo, Y. M. D. Stability of endogenous and added RNA in blood specimens, serum, and plasma. Clin. Chem. 48, 1647–1653 (2002).
Iversen, F. et al. Optimized siRNA–PEG conjugates for extended blood circulation and reduced urine excretion in mice. Theranostics 3, 201–209 (2013).
Geary, R. S., Norris, D., Yu, R. & Bennett, C. F. Pharmacokinetics, biodistribution and cell uptake of antisense oligonucleotides. Adv. Drug Deliv. Rev. 87, 46–51 (2015).
Shemesh, C. S. et al. Pharmacokinetic and pharmacodynamic investigations of ION-353382, a model antisense oligonucleotide: using α2-macroglobulin and murinoglobulin double-knockout mice. Nucleic Acid. Ther. 26, 223–235 (2016).
Allen, T. M. The use of glycolipids and hydrophilic polymers in avoiding rapid uptake of liposomes by the mononuclear phagocyte system. Adv. Drug Delivery Rev. 13, 285–309 (1994).
Sahay, G. et al. Efficiency of siRNA delivery by lipid nanoparticles is limited by endocytic recycling. Nat. Biotechnol. 31, 653–658 (2013).
Finkel, R. S. et al. Treatment of infantile-onset spinal muscular atrophy with nusinersen: a phase 2, open-label, dose-escalation study. Lancet 388, 3017–3026 (2016). This landmark study shows efficacy of an oligonucleotide therapy for spinal muscular atrophy.
Wan, W. B. & Seth, P. P. The medicinal chemistry of therapeutic oligonucleotides. J. Med. Chem. 59, 9645–9667 (2016).
Hung, G. et al. Characterization of target mRNA reduction through in situ RNA hybridization in multiple organ systems following systemic antisense treatment in animals. Nucleic Acid. Ther. 23, 369–378 (2013).
Tabrizi, S. J. et al. Targeting huntingtin expression in patients with Huntington’s disease. N. Engl. J. Med. 380, 2307–2316 (2019).
Eckstein, F. Phosphorothioates, essential components of therapeutic oligonucleotides. Nucleic Acid. Ther. 24, 374–387 (2014).
Hall, A. H. S., Wan, J., Shaughnessy, E. E., Ramsay Shaw, B. & Alexander, K. A. RNA interference using boranophosphate siRNAs: structure–activity relationships. Nucleic Acids Res. 32, 5991–6000 (2004).
Braasch, D. A. et al. RNA interference in mammalian cells by chemically-modified RNA. Biochemistry 42, 7967–7975 (2003).
Bijsterbosch, M. K. et al. In vivo fate of phosphorothioate antisense oligodeoxynucleotides: predominant uptake by scavenger receptors on endothelial liver cells. Nucleic Acids Res. 25, 3290–3296 (1997).
Ezzat, K. et al. Self-assembly into nanoparticles is essential for receptor mediated uptake of therapeutic antisense oligonucleotides. Nano Lett. 15, 4364–4373 (2015).
Miller, C. M. et al. Stabilin-1 and Stabilin-2 are specific receptors for the cellular internalization of phosphorothioate-modified antisense oligonucleotides (ASOs) in the liver. Nucleic Acids Res. 44, 2782–2794 (2016).
Gaus, H. J. et al. Characterization of the interactions of chemically-modified therapeutic nucleic acids with plasma proteins using a fluorescence polarization assay. Nucleic Acids Res. 47, 1110–1122 (2019).
Brown, D. A. et al. Effect of phosphorothioate modification of oligodeoxynucleotides on specific protein binding. J. Biol. Chem. 269, 26801–26805 (1994).
Liang, X., Sun, H., Shen, W. & Crooke, S. T. Identification and characterization of intracellular proteins that bind oligonucleotides with phosphorothioate linkages. Nucleic Acids Res. 43, 2927–2945 (2015).
Weidner, D. A., Valdez, B. C., Henning, D., Greenberg, S. & Busch, H. Phosphorothioate oligonucleotides bind in a non sequence-specific manner to the nucleolar protein C23/nucleolin. FEBS Lett. 366, 146–150 (1995).
Shen, W., Liang, X. & Crooke, S. T. Phosphorothioate oligonucleotides can displace NEAT1 RNA and form nuclear paraspeckle-like structures. Nucleic Acids Res. 42, 8648–8662 (2014).
Liang, X., Shen, W., Sun, H., Prakash, T. P. & Crooke, S. T. TCP1 complex proteins interact with phosphorothioate oligonucleotides and can co-localize in oligonucleotide-induced nuclear bodies in mammalian cells. Nucleic Acids Res. 42, 7819–7832 (2014).
Monia, B. P., Johnston, J. F., Sasmor, H. & Cummins, L. L. Nuclease resistance and antisense activity of modified oligonucleotides targeted to Ha-ras. J. Biol. Chem. 271, 14533–14540 (1996).
Iwamoto, N. et al. Control of phosphorothioate stereochemistry substantially increases the efficacy of antisense oligonucleotides. Nat. Biotechnol. 35, 845–851 (2017). This study demonstrates the influence of phosphorothioate stereochemistry on the properties of gapmer ASOs.
Wexler, M. M. S. Wave Life Sciences discontinues development of suvodirsen for DMD. Muscular Dystrophy News https://musculardystrophynews.com/2019/12/17/wave-life-sciences-discontinues-suvodirsen-development-for-dmd/ (2019).
Wan, W. B. et al. Synthesis, biophysical properties and biological activity of second generation antisense oligonucleotides containing chiral phosphorothioate linkages. Nucleic Acids Res. 42, 13456–13468 (2014).
Lee, H.-S. et al. Abasic pivot substitution harnesses target specificity of RNA interference. Nat. Commun. 6, 10154 (2015).
Liu, J. et al. RNA duplexes with abasic substitutions are potent and allele-selective inhibitors of huntingtin and ataxin-3 expression. Nucleic Acids Res. 41, 8788–8801 (2013).
Schirle, N. T. & MacRae, I. J. The crystal structure of human Argonaute2. Science 336, 1037–1040 (2012).
Frank, F., Sonenberg, N. & Nagar, B. Structural basis for 5′-nucleotide base-specific recognition of guide RNA by human AGO2. Nature 465, 818–822 (2010).
Haraszti, R. A. et al. 5′-Vinylphosphonate improves tissue accumulation and efficacy of conjugated siRNAs in vivo. Nucleic Acids Res. 45, 7581–7592 (2017).
Behlke, M. A. Chemical modification of siRNAs for in vivo use. Oligonucleotides 18, 305–319 (2008).
Southwell, A. L., Skotte, N. H., Bennett, C. F. & Hayden, M. R. Antisense oligonucleotide therapeutics for inherited neurodegenerative diseases. Trends Mol. Med. 18, 634–643 (2012).
Manoharan, M. 2′-Carbohydrate modifications in antisense oligonucleotide therapy: importance of conformation, configuration and conjugation. Biochim. Biophys. Acta 1489, 117–130 (1999).
Roberts, T. C., Ezzat, K., El Andaloussi, S. & Weinberg, M. S. Synthetic siRNA delivery: progress and prospects. Methods Mol. Biol. 1364, 291–310 (2016).
Prakash, T. P. et al. Positional effect of chemical modifications on short interference RNA activity in mammalian cells. J. Med. Chem. 48, 4247–4253 (2005).
Allerson, C. R. et al. Fully 2′-modified oligonucleotide duplexes with improved in vitro potency and stability compared to unmodified small interfering RNA. J. Med. Chem. 48, 901–904 (2005).
Hassler, M. R. et al. Comparison of partially and fully chemically-modified siRNA in conjugate-mediated delivery in vivo. Nucleic Acids Res. 46, 2185–2196 (2018).
Jackson, A. L. et al. Position-specific chemical modification of siRNAs reduces ‘off-target’ transcript silencing. RNA 12, 1197–1205 (2006).
Springer, A. D. & Dowdy, S. F. GalNAc–siRNA conjugates: leading the way for delivery of RNAi therapeutics. Nucleic Acid. Ther. 28, 109–118 (2018).
Garber, K. Alnylam terminates revusiran program, stock plunges. Nat. Biotechnol. 34, 1213–1214 (2016).
Nair, J. K. et al. Impact of enhanced metabolic stability on pharmacokinetics and pharmacodynamics of GalNAc–siRNA conjugates. Nucleic Acids Res. 45, 10969–10977 (2017).
Judge, A. D. et al. Sequence-dependent stimulation of the mammalian innate immune response by synthetic siRNA. Nat. Biotechnol. 23, 457–462 (2005).
Shen, W., Liang, X.-H., Sun, H. & Crooke, S. T. 2′-Fluoro-modified phosphorothioate oligonucleotide can cause rapid degradation of P54nrb and PSF. Nucleic Acids Res. 43, 4569–4578 (2015).
Iwasaki, A. & Medzhitov, R. Toll-like receptor control of the adaptive immune responses. Nat. Immunol. 5, 987–995 (2004).
Hornung, V. et al. Sequence-specific potent induction of IFN-α by short interfering RNA in plasmacytoid dendritic cells through TLR7. Nat. Med. 11, 263–270 (2005).
Hartmann, G. Nucleic acid immunity. Adv. Immunol. 133, 121–169 (2017).
Morrissey, D. V. et al. Potent and persistent in vivo anti-HBV activity of chemically modified siRNAs. Nat. Biotechnol. 23, 1002–1007 (2005). This landmark study demonstrates enhanced RNAi-mediated gene silencing using chemically modified siRNA in vivo.
Judge, A. D., Bola, G., Lee, A. C. H. & MacLachlan, I. Design of noninflammatory synthetic siRNA mediating potent gene silencing in vivo. Mol. Ther. 13, 494–505 (2006).
Poeck, H. et al. 5′-Triphosphate-siRNA: turning gene silencing and Rig-I activation against melanoma. Nat. Med. 14, 1256–1263 (2008).
Kortylewski, M. et al. In vivo delivery of siRNA to immune cells by conjugation to a TLR9 agonist enhances antitumor immune responses. Nat. Biotechnol. 27, 925–932 (2009).
Kanzler, H., Barrat, F. J., Hessel, E. M. & Coffman, R. L. Therapeutic targeting of innate immunity with Toll-like receptor agonists and antagonists. Nat. Med. 13, 552–559 (2007).
Kandimalla, E. R. et al. Design, synthesis and biological evaluation of novel antagonist compounds of Toll-like receptors 7, 8 and 9. Nucleic Acids Res. 41, 3947–3961 (2013).
Veedu, R. N. & Wengel, J. Locked nucleic acid as a novel class of therapeutic agents. RNA Biol. 6, 321–323 (2009).
Vester, B. & Wengel, J. LNA (locked nucleic acid): high-affinity targeting of complementary RNA and DNA. Biochemistry 43, 13233–13241 (2004).
Morita, K. et al. 2′-O,4′-C-ethylene-bridged nucleic acids (ENA): highly nuclease-resistant and thermodynamically stable oligonucleotides for antisense drug. Bioorg. Med. Chem. Lett. 12, 73–76 (2002).
Hong, D. et al. AZD9150, a next-generation antisense oligonucleotide inhibitor of STAT3 with early evidence of clinical activity in lymphoma and lung cancer. Sci. Transl Med. 7, 314ra185 (2015).
Koshkin, A. A. et al. LNA (locked nucleic acids): synthesis of the adenine, cytosine, guanine, 5-methylcytosine, thymine and uracil bicyclonucleoside monomers, oligomerisation, and unprecedented nucleic acid recognition. Tetrahedron 54, 3607–3630 (1998).
Obad, S. et al. Silencing of microRNA families by seed-targeting tiny LNAs. Nat. Genet. 43, 371–378 (2011).
Moulton, J. D. Using morpholinos to control gene expression. Curr. Protoc. Nucleic Acid. Chem. 68, 4.30.1–4.30.29 (2017).
Iversen, P. L. Phosphorodiamidate morpholino oligomers: favorable properties for sequence-specific gene inactivation. Curr. Opin. Mol. Ther. 3, 235–238 (2001).
Komaki, H. et al. Systemic administration of the antisense oligonucleotide NS-065/NCNP-01 for skipping of exon 53 in patients with Duchenne muscular dystrophy. Sci. Transl Med. 10, eaan0713 (2018).
Larsen, H. J., Bentin, T. & Nielsen, P. E. Antisense properties of peptide nucleic acid. Biochim. Biophys. Acta 1489, 159–166 (1999).
Saarbach, J., Sabale, P. M. & Winssinger, N. Peptide nucleic acid (PNA) and its applications in chemical biology, diagnostics, and therapeutics. Curr. Opin. Chem. Biol. 52, 112–124 (2019).
Renneberg, D. & Leumann, C. J. Watson–Crick base-pairing properties of tricyclo-DNA. J. Am. Chem. Soc. 124, 5993–6002 (2002).
Goyenvalle, A. et al. Functional correction in mouse models of muscular dystrophy using exon-skipping tricyclo-DNA oligomers. Nat. Med. 21, 270–275 (2015).
Dowling, J. J. Eteplirsen therapy for Duchenne muscular dystrophy: skipping to the front of the line. Nat. Rev. Neurol. 12, 675–676 (2016).
Yin, H. et al. Optimization of peptide nucleic acid antisense oligonucleotides for local and systemic dystrophin splice correction in the MDX mouse. Mol. Ther. 18, 819–827 (2010).
Imbert, M., Blandel, F., Leumann, C., Garcia, L. & Goyenvalle, A. Lowering mutant huntingtin using tricyclo-DNA antisense oligonucleotides as a therapeutic approach for Huntington’s disease. Nucleic Acid. Ther. 29, 256–265 (2019).
Meade, B. R. et al. Efficient delivery of RNAi prodrugs containing reversible charge-neutralizing phosphotriester backbone modifications. Nat. Biotechnol. 32, 1256–1261 (2014).
Wolfrum, C. et al. Mechanisms and optimization of in vivo delivery of lipophilic siRNAs. Nat. Biotechnol. 25, 1149–1157 (2007).
Soutschek, J. et al. Therapeutic silencing of an endogenous gene by systemic administration of modified siRNAs. Nature 432, 173–178 (2004).
Lorenz, C., Hadwiger, P., John, M., Vornlocher, H.-P. & Unverzagt, C. Steroid and lipid conjugates of siRNAs to enhance cellular uptake and gene silencing in liver cells. Bioorg. Med. Chem. Lett. 14, 4975–4977 (2004).
Eguchi, A. et al. Efficient siRNA delivery into primary cells by a peptide transduction domain–dsRNA binding domain fusion protein. Nat. Biotechnol. 27, 567–571 (2009).
Betts, C. et al. Pip6–PMO, a new generation of peptide–oligonucleotide conjugates with improved cardiac exon skipping activity for DMD treatment. Mol. Ther. Nucleic Acids 1, e38 (2012).
Alam, Md. R. et al. Multivalent cyclic RGD conjugates for targeted delivery of small interfering RNA. Bioconjugate Chem. 22, 1673–1681 (2011).
Ämmälä, C. et al. Targeted deliivery of antisense oligonucleotides to pancreatic β-cells. Sci. Adv. 4, eaat3386 (2018).
Liu, X. et al. Tumor-targeted in vivo gene silencing via systemic delivery of cRGD-conjugated siRNA. Nucleic Acids Res. 42, 11805–11817 (2014).
McNamara, J. O. et al. Cell type-specific delivery of siRNAs with aptamer–siRNA chimeras. Nat. Biotechnol. 24, 1005–1015 (2006).
Song, E. et al. Antibody mediated in vivo delivery of small interfering RNAs via cell-surface receptors. Nat. Biotechnol. 23, 709–717 (2005). This paper demonstrates antibody–siRNA conjugates for gene silencing in mice.
Nair, J. K. et al. Multivalent N-acetylgalactosamine-conjugated siRNA localizes in hepatocytes and elicits robust RNAi-mediated gene silencing. J. Am. Chem. Soc. 136, 16958–16961 (2014). This paper demonstrates long-term gene silencing after weekly dosing of an optimized GalNAc–siRNA conjugate in mice.
Matsuda, S. et al. siRNA conjugates carrying sequentially assembled trivalent N-acetylgalactosamine linked through nucleosides elicit robust gene silencing in vivo in hepatocytes. ACS Chem. Biol. 10, 1181–1187 (2015).
Tai, W. Current aspects of siRNA bioconjugate for in vitro and in vivo delivery. Molecules 24, 2211 (2019).
Juliano, R. L. The delivery of therapeutic oligonucleotides. Nucleic Acids Res. 44, 6518–6548 (2016).
Khan, T. et al. Silencing myostatin using cholesterol-conjugated siRNAs induces muscle growth. Mol. Ther. Nucleic Acids 5, e342 (2016).
Nishina, K. et al. Efficient in vivo delivery of siRNA to the liver by conjugation of α-tocopherol. Mol. Ther. 16, 734–740 (2008).
Osborn, M. F. et al. Hydrophobicity drives the systemic distribution of lipid-conjugated siRNAs via lipid transport pathways. Nucleic Acids Res. 47, 1070–1081 (2019).
Spiess, M. The asialoglycoprotein receptor: a model for endocytic transport receptors. Biochemistry 29, 10009–10018 (1990).
Tanowitz, M. et al. Asialoglycoprotein receptor 1 mediates productive uptake of N-acetylgalactosamine-conjugated and unconjugated phosphorothioate antisense oligonucleotides into liver hepatocytes. Nucleic Acids Res. 45, 12388–12400 (2017).
Prakash, T. P. et al. Targeted delivery of antisense oligonucleotides to hepatocytes using triantennary N-acetyl galactosamine improves potency 10-fold in mice. Nucleic Acids Res. 42, 8796–8807 (2014).
Ray, K. K. et al. Inclisiran in patients at high cardiovascular risk with elevated LDL cholesterol. N. Engl. J. Med. 376, 1430–1440 (2017).
Zimmermann, T. S. et al. Clinical proof of concept for a novel hepatocyte-targeting GalNAc–siRNA conjugate. Mol. Ther. 25, 71–78 (2017).
Viney, N. J. et al. Antisense oligonucleotides targeting apolipoprotein(a) in people with raised lipoprotein(a): two randomised, double-blind, placebo-controlled, dose-ranging trials. Lancet 388, 2239–2253 (2016).
Sardh, E. et al. Phase 1 trial of an RNA interference therapy for acute intermittent porphyria. N. Engl. J. Med. 380, 549–558 (2019).
No authors listed. Novartis to acquire the medicines company for USD 9.7 bn, adding inclisiran, a potentially transformational investigational cholesterol-lowering therapy to address leading global cause of death. Novartis https://www.novartis.com/news/media-releases/novartis-acquire-medicines-company-usd-97-bn-adding-inclisiran-potentially-transformational-investigational-cholesterol-lowering-therapy-address-leading-global (2019).
Hooper, A. J. & Burnett, J. R. Anti-PCSK9 therapies for the treatment of hypercholesterolemia. Expert Opin. Biol. Ther. 13, 429–435 (2013).
Mousavi, S. A., Berge, K. E. & Leren, T. P. The unique role of proprotein convertase subtilisin/kexin 9 in cholesterol homeostasis. J. Intern. Med. 266, 507–519 (2009).
Fitzgerald, K. et al. A highly durable RNAi therapeutic inhibitor of PCSK9. N. Engl. J. Med. 376, 41–51 (2017).
Sievers, E. L. & Senter, P. D. Antibody–drug conjugates in cancer therapy. Annu. Rev. Med. 64, 15–29 (2013).
Yao, Y. et al. Targeted delivery of PLK1–siRNA by ScFv suppresses Her2+ breast cancer growth and metastasis. Sci. Transl Med. 4, 130ra48 (2012).
Kumar, P. et al. T cell-specific siRNA delivery suppresses HIV-1 infection in humanized mice. Cell 134, 577–586 (2008).
Sugo, T. et al. Development of antibody–siRNA conjugate targeted to cardiac and skeletal muscles. J. Control. Rel. 237, 1–13 (2016).
Cuellar, T. L. et al. Systematic evaluation of antibody-mediated siRNA delivery using an industrial platform of THIOMAB–siRNA conjugates. Nucleic Acids Res. 43, 1189–1203 (2015).
Arnold, A. E. et al. Antibody–antisense oligonucleotide conjugate downregulates a key gene in glioblastoma stem cells. Mol. Ther. Nucleic Acids 11, 518–527 (2018).
Astriab-Fisher, A., Fisher, M. H., Juliano, R. & Herdewijn, P. Increased uptake of antisense oligonucleotides by delivery as double stranded complexes. Biochem. Pharmacol. 68, 403–407 (2004).
Nuzzo, S. et al. Potential and challenges of aptamers as specific carriers of therapeutic oligonucleotides for precision medicine in cancer. Cancers 11, 1521 (2019).
Hong, S., Sun, N., Liu, M., Wang, J. & Pei, R. Building a chimera of aptamer–antisense oligonucleotide for silencing galectin-1 gene. RSC Adv. 6, 112445–112450 (2016).
White, R. R., Sullenger, B. A. & Rusconi, C. P. Developing aptamers into therapeutics. J. Clin. Invest. 106, 929–934 (2000).
McClorey, G. & Banerjee, S. Cell-penetrating peptides to enhance delivery of oligonucleotide-based therapeutics. Biomedicines 6, 51 (2018).
Yin H. et al. Cell-penetrating peptide-conjugated antisense oligonucleotides restore systemic muscle and cardiac dystrophin expression and function. Hum. Mol. Genet. 17, 3909–3918 (2008).
McClorey, G., Moulton, H. M., Iversen, P. L., Fletcher, S. & Wilton, S. D. Antisense oligonucleotide-induced exon skipping restores dystrophin expression in vitro in a canine model of DMD. Gene Ther. 13, 1373–1381 (2006).
Wu, B. et al. Effective rescue of dystrophin improves cardiac function in dystrophin-deficient mice by a modified morpholino oligomer. Proc. Natl Acad. Sci. USA 105, 14814–14819 (2008).
Abes, R. et al. Delivery of steric block morpholino oligomers by (R-X-R)4 peptides: structure–activity studies. Nucleic Acids Res. 36, 6343–6354 (2008).
Wender, P. A. et al. The design, synthesis, and evaluation of molecules that enable or enhance cellular uptake: peptoid molecular transporters. Proc. Natl Acad. Sci. USA 97, 13003–13008 (2000).
Yin, H. et al. A fusion peptide directs enhanced systemic dystrophin exon skipping and functional restoration in dystrophin-deficient mdx mice. Hum. Mol. Genet. 18, 4405–4414 (2009).
Yin, H. et al. Context dependent effects of chimeric peptide morpholino conjugates contribute to dystrophin exon-skipping efficiency. Mol. Ther. Nucleic Acids 2, e124 (2013).
Gao, X. et al. Effective dystrophin restoration by a novel muscle-homing peptide–morpholino conjugate in dystrophin-deficient MDX mice. Mol. Ther. 22, 1333–1341 (2014).
Yin, H. et al. Pip5 transduction peptides direct high efficiency oligonucleotide-mediated dystrophin exon skipping in heart and phenotypic correction in MDX mice. Mol. Ther. 19, 1295–1303 (2011).
van Westering, T. L. E. et al. Uniform sarcolemmal dystrophin expression is required to prevent extracellular microRNA release and improve dystrophic pathology. J. Cachexia Sarcopenia Muscle 11, 578–593 (2019).
Gait, M. J. et al. Cell-penetrating peptide conjugates of steric blocking oligonucleotides as therapeutics for neuromuscular diseases from a historical perspective to current prospects of treatment. Nucleic Acid. Ther. 29, 1–12 (2019).
Betts, C. A. et al. Prevention of exercised induced cardiomyopathy following Pip–PMO treatment in dystrophic mdx mice. Sci. Rep. 5, 8986 (2015).
Hammond, S. M. et al. Systemic peptide-mediated oligonucleotide therapy improves long-term survival in spinal muscular atrophy. Proc. Natl Acad. Sci. USA 113, 10962–10967 (2016).
Amantana, A. et al. Pharmacokinetics, biodistribution, stability and toxicity of a cell-penetrating peptide–morpholino oligomer conjugate. Bioconjug. Chem. 18, 1325–1331 (2007).
Moulton, H. M. & Moulton, J. D. Morpholinos and their peptide conjugates: therapeutic promise and challenge for Duchenne muscular dystrophy. Biochim. Biophys. Acta 1798, 2296–2303 (2010).
Lehto, T. et al. Cellular trafficking determines the exon skipping activity of Pip6a–PMO in MDX skeletal and cardiac muscle cells. Nucleic Acids Res. 42, 3207–3217 (2014).
Abes, S. et al. Vectorization of morpholino oligomers by the (R-Ahx-R)4 peptide allows efficient splicing correction in the absence of endosomolytic agents. J. Control. Rel. 116, 304–313 (2006).
Bestas, B. et al. Splice-correcting oligonucleotides restore BTK function in X-linked agammaglobulinemia model. J. Clin. Invest. 124, 4067–4081 (2014).
Du, L. et al. Arginine-rich cell-penetrating peptide dramatically enhances AMO-mediated ATM aberrant splicing correction and enables delivery to brain and cerebellum. Hum. Mol. Genet. 20, 3151–3160 (2011).
Geller, B. L. et al. Gene-silencing antisense oligomers inhibit acinetobacter growth in vitro and in vivo. J. Infect. Dis. 208, 1553–1560 (2013).
Burrer, R. et al. Antiviral effects of antisense morpholino oligomers in murine coronavirus infection models. J. Virol. 81, 5637–5648 (2007).
Neuman, B. W. et al. Inhibition and escape of SARS-CoV treated with antisense morpholino oligomers. Adv. Exp. Med. Biol. 581, 567–571 (2006).
Enterlein, S. et al. VP35 knockdown inhibits Ebola virus amplification and protects against lethal infection in mice. Antimicrob. Agents Chemother. 50, 984–993 (2006).
Moschos, S. A. et al. Lung delivery studies using siRNA conjugated to TAT(48–60) and penetratin reveal peptide induced reduction in gene expression and induction of innate immunity. Bioconjug. Chem. 18, 1450–1459 (2007).
Cavallaro, G., Sardo, C., Craparo, E. F., Porsio, B. & Giammona, G. Polymeric nanoparticles for siRNA delivery: production and applications. Int. J. Pharm. 525, 313–333 (2017).
Dzmitruk, V. et al. Dendrimers show promise for siRNA and microRNA therapeutics. Pharmaceutics 10, 126 (2018).
Mignani, S., Shi, X., Zablocka, M. & Majoral, J.-P. Dendrimer-enabled therapeutic antisense delivery systems as innovation in medicine. Bioconjug. Chem. 30, 1938–1950 (2019).
Crombez, L. et al. Targeting cyclin B1 through peptide-based delivery of siRNA prevents tumour growth. Nucleic Acids Res. 37, 4559–4569 (2009).
El Andaloussi, S. et al. Design of a peptide-based vector, PepFect6, for efficient delivery of siRNA in cell culture and systemically in vivo. Nucleic Acids Res. 39, 3972–3987 (2011).
Kumar, P. et al. Transvascular delivery of small interfering RNA to the central nervous system. Nature 448, 39–43 (2007). This study shows the utility of RVG peptide derivatives for delivering siRNA to the brain after intravenous injection in mice.
Montrose, K., Yang, Y., Sun, X., Wiles, S. & Krissansen, G. W. Xentry, a new class of cell-penetrating peptide uniquely equipped for delivery of drugs. Sci. Rep. 3, 1–7 (2013).
Tabaković, A., Kester, M. & Adair, J. H. Calcium phosphate-based composite nanoparticles in bioimaging and therapeutic delivery applications. Wiley Interdiscip. Rev. Nanomed. Nanobiotechnol. 4, 96–112 (2012).
Semple, S. C. et al. Rational design of cationic lipids for siRNA delivery. Nat. Biotechnol. 28, 172–176 (2010).
de Fougerolles, A. R. Delivery vehicles for small interfering RNA in vivo. Hum. Gene Ther. 19, 125–132 (2008).
Ambegia, E. et al. Stabilized plasmid–lipid particles containing PEG–diacylglycerols exhibit extended circulation lifetimes and tumor selective gene expression. Biochim. Biophys. Acta 1669, 155–163 (2005).
Tam, Y. Y. C., Chen, S. & Cullis, P. R. Advances in lipid nanoparticles for siRNA delivery. Pharmaceutics 5, 498–507 (2013).
Zimmermann, T. S. et al. RNAi-mediated gene silencing in non-human primates. Nature 441, 111–114 (2006).
Hoy, S. M. Patisiran: first global approval. Drugs 78, 1625–1631 (2018).
Akinc, A. et al. Targeted delivery of RNAi therapeutics with endogenous and exogenous ligand-based mechanisms. Mol. Ther. 18, 1357–1364 (2010).
Akinc, A. et al. A combinatorial library of lipid-like materials for delivery of RNAi therapeutics. Nat. Biotechnol. 26, 561–569 (2008).
Akinc, A. et al. Development of lipidoid–siRNA formulations for systemic delivery to the liver. Mol. Ther. 17, 872–879 (2009).
Love, K. T. et al. Lipid-like materials for low-dose, in vivo gene silencing. Proc. Natl Acad. Sci. USA 107, 1864–1869 (2010).
Juliano, R. L. & Akhtar, S. Liposomes as a drug delivery system for antisense oligonucleotides. Antisense Res. Dev. 2, 165–176 (1992).
Wisse, E., Jacobs, F., Topal, B., Frederik, P. & De Geest, B. The size of endothelial fenestrae in human liver sinusoids: implications for hepatocyte-directed gene transfer. Gene Ther. 15, 1193–1199 (2008).
Rungta, R. L. et al. Lipid nanoparticle delivery of siRNA to silence neuronal gene expression in the brain. Mol. Ther. Nucleic Acids 2, e136 (2013).
Seth, P. P., Tanowitz, M. & Bennett, C. F. Selective tissue targeting of synthetic nucleic acid drugs. J. Clin. Invest. 129, 915–925 (2019).
Endsley, A. N. & Ho, R. J. Y. Design and characterization of novel peptide-coated lipid nanoparticles for targeting anti-HIV drug to CD4 expressing cells. AAPS J. 14, 225–235 (2012).
Suk, J. S., Xu, Q., Kim, N., Hanes, J. & Ensign, L. M. PEGylation as a strategy for improving nanoparticle-based drug and gene delivery. Adv. Drug Delivery Rev. 99, 28–51 (2016).
Li, S.-D. & Huang, L. Targeted delivery of antisense oligodeoxynucleotide and small interference RNA into lung cancer cells. Mol. Pharm. 3, 579–588 (2006).
Tam, Y. Y. C. et al. Small molecule ligands for enhanced intracellular delivery of lipid nanoparticle formulations of siRNA. Nanomedicine 9, 665–674 (2013).
Sato, Y. et al. Resolution of liver cirrhosis using vitamin A-coupled liposomes to deliver siRNA against a collagen-specific chaperone. Nat. Biotechnol. 26, 431–442 (2008).
Coelho, T. et al. Safety and efficacy of RNAi therapy for transthyretin amyloidosis. N. Engl. J. Med. 369, 819–829 (2013).
Wiklander, O. P. B., Brennan, M. Á., Lötvall, J., Breakefield, X. O. & El Andaloussi, S. Advances in therapeutic applications of extracellular vesicles. Sci. Transl Med. 11, eaav8521 (2019).
Kalluri, R. & LeBleu, V. S. The biology, function, and biomedical applications of exosomes. Science 367, eaau6977 (2020).
Valadi, H. et al. Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells. Nat. Cell Biol. 9, 654–659 (2007).
Skog, J. et al. Glioblastoma microvesicles transport RNA and proteins that promote tumour growth and provide diagnostic biomarkers. Nat. Cell Biol. 10, 1470–1476 (2008).
Alvarez-Erviti, L. et al. Delivery of siRNA to the mouse brain by systemic injection of targeted exosomes. Nat. Biotechnol. 29, 341–345 (2011). This study is the first to demonstrate exosome-mediated siRNA delivery to the mouse brain.
Kamerkar, S. et al. Exosomes facilitate therapeutic targeting of oncogenic KRAS in pancreatic cancer. Nature 546, 498–503 (2017).
Kordelas, L. et al. MSC-derived exosomes: a novel tool to treat therapy-refractory graft-versus-host disease. Leukemia 28, 970–973 (2014).
Lai, R. C. et al. Exosome secreted by MSC reduces myocardial ischemia/reperfusion injury. Stem Cell Res. 4, 214–222 (2010).
Giebel, B., Kordelas, L. & Börger, V. Clinical potential of mesenchymal stem/stromal cell-derived extracellular vesicles. Stem Cell Investig. 4, 84 (2017).
Katakowski, M. et al. Exosomes from marrow stromal cells expressing miR-146b inhibit glioma growth. Cancer Lett. 335, 201–204 (2013).
Mendt, M. et al. Generation and testing of clinical-grade exosomes for pancreatic cancer. JCI Insight 3, e99263 (2018).
Lamichhane, T. N. et al. Oncogene knockdown via active loading of small RNAs into extracellular vesicles by sonication. Cell Mol. Bioeng. 9, 315–324 (2016).
Haraszti, R. A. et al. Optimized cholesterol–siRNA chemistry improves productive loading onto extracellular vesicles. Mol. Ther. 26, 1973–1982 (2018).
Didiot, M.-C. et al. Exosome-mediated delivery of hydrophobically modified siRNA for huntingtin mRNA silencing. Mol. Ther. 24, 1836–1847 (2016).
Gao, X. et al. Anchor peptide captures, targets, and loads exosomes of diverse origins for diagnostics and therapy. Sci. Transl Med. 10, eaat0195 (2018).
Cooper, J. M. et al. Systemic exosomal siRNA delivery reduced α-synuclein aggregates in brains of transgenic mice. Mov. Disord. 29, 1476–1485 (2014).
Yang, J., Zhang, X., Chen, X., Wang, L. & Yang, G. Exosome mediated delivery of miR-124 promotes neurogenesis after ischemia. Mol. Ther. Nucleic Acids 7, 278–287 (2017).
Ohno, S. et al. Systemically injected exosomes targeted to EGFR deliver antitumor microRNA to breast cancer cells. Mol. Ther. 21, 185–191 (2013).
Pi, F. et al. Nanoparticle orientation to control RNA loading and ligand display on extracellular vesicles for cancer regression. Nat. Nanotechnol. 13, 82–89 (2018).
Nordin, J. Z. et al. Ultrafiltration with size-exclusion liquid chromatography for high yield isolation of extracellular vesicles preserving intact biophysical and functional properties. Nanomedicine 11, 879–883 (2015).
Kapadia, C. H., Melamed, J. R. & Day, E. S. Spherical nucleic acid nanoparticles: therapeutic potential. BioDrugs 32, 297–309 (2018).
Jensen, S. A. et al. Spherical nucleic acid nanoparticle conjugates as an RNAi-based therapy for glioblastoma. Sci. Transl Med. 5, 209ra152 (2013).
Randeria, P. S. et al. siRNA-based spherical nucleic acids reverse impaired wound healing in diabetic mice by ganglioside GM3 synthase knockdown. Proc. Natl Acad. Sci. USA 112, 5573–5578 (2015).
Nemati, H. et al. Using siRNA-based spherical nucleic acid nanoparticle conjugates for gene regulation in psoriasis. J. Control. Rel. 268, 259–268 (2017).
Lee, H. et al. Molecularly self-assembled nucleic acid nanoparticles for targeted in vivo siRNA delivery. Nat. Nanotechnol. 7, 389–393 (2012).
Mohri, K. et al. Design and development of nanosized DNA assemblies in polypod-like structures as efficient vehicles for immunostimulatory CpG motifs to immune cells. ACS Nano 6, 5931–5940 (2012).
Jiang, D. et al. Efficient renal clearance of DNA tetrahedron nanoparticles enables quantitative evaluation of kidney function. Nano Res. 12, 637–642 (2019).
Li, H. et al. siRNA suppression of hTERT using activatable cell-penetrating peptides in hepatoma cells. Biosci Rep 35, e00181 (2015).
Jiang, T. et al. Tumor imaging by means of proteolytic activation of cell-penetrating peptides. Proc. Natl Acad. Sci. USA 101, 17867–17872 (2004).
Rozema, D. B. et al. Dynamic polyconjugates for targeted in vivo delivery of siRNA to hepatocytes. Proc. Natl Acad. Sci. USA 104, 12982–12987 (2007).
Douglas, S. M., Bachelet, I. & Church, G. M. A logic-gated nanorobot for targeted transport of molecular payloads. Science 335, 831–834 (2012).
Torchilin, V. P. Multifunctional, stimuli-sensitive nanoparticulate systems for drug delivery. Nat. Rev. Drug Disco. 13, 813–827 (2014).
Prasad, V. Nusinersen for spinal muscular atrophy: are we paying too much for too little? JAMA Pediatr. 172, 123–125 (2018).
Ng, E. W. M. et al. Pegaptanib, a targeted anti-VEGF aptamer for ocular vascular disease. Nat. Rev. Drug Discov. 5, 123–132 (2006).
Zhao, X. et al. Mechanisms involved in the activation of C/EBPα by small activating RNA in hepatocellular carcinoma. Oncogene 38, 3446–3457 (2019).
Tuerk, C. & Gold, L. Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. Science 249, 505–510 (1990).
Robertson, D. L. & Joyce, G. F. Selection in vitro of an RNA enzyme that specifically cleaves single-stranded DNA. Nature 344, 467–468 (1990).
Ellington, A. D. & Szostak, J. W. In vitro selection of RNA molecules that bind specific ligands. Nature 346, 818–822 (1990).
Burmeister, P. E. et al. Direct in vitro selection of a 2′-O-methyl aptamer to VEGF. Chem. Biol. 12, 25–33 (2005).
Ruckman, J. et al. 2′-Fluoropyrimidine RNA-based aptamers to the 165-amino acid form of vascular endothelial growth factor (VEGF165). Inhibition of receptor binding and VEGF-induced vascular permeability through interactions requiring the exon 7-encoded domain. J. Biol. Chem. 273, 20556–20567 (1998).
Eulberg, D. & Klussmann, S. Spiegelmers: biostable aptamers. Chembiochem 4, 979–983 (2003).
Jinek, M. et al. A programmable dual-RNA–guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816–821 (2012).
Cong, L. et al. Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819–823 (2013).
Mali, P. et al. RNA-guided human genome engineering via Cas9. Science 339, 823–826 (2013).
Chavez, A. et al. Highly-efficient Cas9-mediated transcriptional programming. Nat. Methods 12, 326–328 (2015).
Kiani, S. et al. CRISPR transcriptional repression devices and layered circuits in mammalian cells. Nat. Methods 11, 723–726 (2014).
Ran, F. A. et al. In vivo genome editing using Staphylococcus aureus Cas9. Nature 520, 186–191 (2015).
Xu, X. et al. Delivery of CRISPR/Cas9 for therapeutic genome editing. J. Gene Med. 21, e3107 (2019).
Lee, K. et al. Nanoparticle delivery of Cas9 ribonucleoprotein and donor DNA in vivo induces homology-directed DNA repair. Nat. Biomed. Eng. 1, 889–901 (2017).
Zuris, J. A. et al. Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editing in vitro and in vivo. Nat. Biotechnol. 33, 73–80 (2015).
Gao, X. et al. Treatment of autosomal dominant hearing loss by in vivo delivery of genome editing agents. Nature 553, 217–221 (2018).
Ramakrishna, S. et al. Gene disruption by cell-penetrating peptide-mediated delivery of Cas9 protein and guide RNA. Genome Res. 24, 1020–1027 (2014).
Acknowledgements
The authors thank R. Raz for assistance with chemical structures.
Author information
Authors and Affiliations
Contributions
T.C.R. and M.J.A.W. discussed content and wrote the article; T.C.R., R.L. and M.J.A.W. revised the manuscript before submission; and T.C.R. developed all of the figures.
Corresponding authors
Ethics declarations
Competing interests
Complete details of relationships, compensated and uncompensated, for R.L. can be found in the Supplementary information. M.J.A.W. is a founder and shareholder of Evox Therapeutics and PepGen Ltd, companies dedicated to the commercialization of extracellular vesicle therapeutics and peptide-enhanced therapeutic oligonucleotide delivery, respectively. T.C.R. declares no competing financial interests.
Additional information
Peer review information
Nature Reviews Drug Discovery thanks the anonymous reviewers for their contribution to the peer review of this work.
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Related links
n-Lorem Foundation: www.nlorem.org
Regulatory approval for viltolarsen in Japan: https://www.nippon-shinyaku.co.jp/file/download.php?file_id=2965
Sarepta Therapeutics’ pipeline: https://www.sarepta.com/products-pipeline/pipeline
Stoke Therapeutics’ science: https://www.stoketherapeutics.com/our-science/
Supplementary information
Glossary
- Spliceosome
-
A large riboprotein complex that mediates the splicing of mRNA transcripts.
- Nonsense-mediated decay
-
A cellular pathway through which mRNA transcripts containing premature termination codons are eliminated.
- RNAi
-
A cellular pathway through which small interfering RNAs mediate gene silencing via the slicing of target mRNA transcripts. Much of the RNAi machinery is shared with the miRNA processing pathway.
- Exocytosis
-
A cellular mechanism in which molecules are exported from the cell in an energy-dependent manner. This is achieved through the fusion of intracellular vesicles with the plasma membrane, thereby secreting their contents into the extracellular space. Vesicles released in this manner are called exosomes.
- Blood–brain barrier
-
(BBB). A physical barrier that selectively prevents molecules and pathogens from crossing from the blood and into the extracellular space in the brain and spinal cord. The blood–brain barrier is composed of blood capillary endothelial cells, pericytes and astrocyte end-feet.
- Endocytosis
-
The process of internalization of material (for example, nanoparticles) into the cell. There are multiple distinct mechanisms of endocytosis, including clathrin-mediated endocytosis, caveolae-mediated endocytosis and micropinocytosis.
- Multivesicular bodies
-
Membrane-bound compartments within cells that contain intraluminal vesicles that form as a consequence of inward budding of the multivesicular body membrane. When multivesicular bodies fuse with the plasma membrane, their intraluminal vesicles are released into the extracellular space and are now considered exosomes.
Rights and permissions
About this article
Cite this article
Roberts, T.C., Langer, R. & Wood, M.J.A. Advances in oligonucleotide drug delivery. Nat Rev Drug Discov 19, 673–694 (2020). https://doi.org/10.1038/s41573-020-0075-7
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/s41573-020-0075-7
This article is cited by
-
MolGraph: a Python package for the implementation of molecular graphs and graph neural networks with TensorFlow and Keras
Journal of Computer-Aided Molecular Design (2025)
-
Regulation of human microglial gene expression and function via RNAase-H active antisense oligonucleotides in vivo in Alzheimer’s disease
Molecular Neurodegeneration (2024)
-
miR-NPs-RVG promote spinal cord injury repair: implications from spinal cord-derived microvascular endothelial cells
Journal of Nanobiotechnology (2024)
-
Recent advances in gene delivery nanoplatforms based on spherical nucleic acids
Journal of Nanobiotechnology (2024)
-
CRISPR-dCas13d-based deep screening of proximal and distal splicing-regulatory elements
Nature Communications (2024)