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The promises and pitfalls of RNA-interference-based therapeutics

Abstract

The discovery that gene expression can be controlled by the Watson–Crick base-pairing of small RNAs with messenger RNAs containing complementary sequence — a process known as RNA interference — has markedly advanced our understanding of eukaryotic gene regulation and function. The ability of short RNA sequences to modulate gene expression has provided a powerful tool with which to study gene function and is set to revolutionize the treatment of disease. Remarkably, despite being just one decade from its discovery, the phenomenon is already being used therapeutically in human clinical trials, and biotechnology companies that focus on RNA-interference-based therapeutics are already publicly traded.

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Figure 1: Mechanisms of cellular gene silencing.
Figure 2: In vivo delivery strategies for therapeutic siRNAs.

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References

  1. Zamore, P. D. RNA interference: big applause for silencing in Stockholm. Cell 127, 1083–1086 (2006).

    CAS  PubMed  Google Scholar 

  2. McCaffrey, A. P. et al. RNA interference in adult mice. Nature 418, 38–39 (2002). This study was the first to show siRNA activity in vivo in mammals.

    ADS  CAS  PubMed  Google Scholar 

  3. Song, E. et al. RNA interference targeting Fas protects mice from fulminant hepatitis. Nature Med. 9, 347–351 (2003). This paper provided the first therapeutic RNAi demonstration in animals.

    Article  CAS  PubMed  Google Scholar 

  4. Grimm, D. et al. Fatality in mice due to oversaturation of cellular microRNA/short hairpin RNA pathways. Nature 441, 537–541 (2006). This article raised cautionary concerns about the danger of high-level shRNA expression in animals.

    Article  ADS  CAS  PubMed  Google Scholar 

  5. Castanotto, D. et al. Combinatorial delivery of small interfering RNAs reduces RNAi efficacy by selective incorporation into RISC. Nucleic Acids Res. 35, 5154–5164 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  6. Hornung, V. et al. Sequence-specific potent induction of IFN-α by short interfering RNA in plasmacytoid dendritic cells through TLR7. Nature Med. 11, 263–270 (2005).

    CAS  PubMed  Google Scholar 

  7. Judge, A. D. et al. Sequence-dependent stimulation of the mammalian innate immune response by synthetic siRNA. Nature Biotechnol. 23, 457–462 (2005).

    CAS  Google Scholar 

  8. Gantier, M. P. & Williams, B. R. The response of mammalian cells to double-stranded RNA. Cytokine Growth Factor Rev. 18, 363–371 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  9. Elbashir, S. M. et al. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411, 494–498 (2001). This study was the first to show that RNAi triggers can work in mammalian cells without stimulating interferon pathways.

    ADS  CAS  PubMed  Google Scholar 

  10. Tolia, N. H. & Joshua-Tor, L. Slicer and the argonautes. Nature Chem. Biol. 3, 36–43 (2007).

    ADS  CAS  Google Scholar 

  11. Zamore, P. D., Tuschl, T., Sharp, P. A. & Bartel, D. P. RNAi: double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals. Cell 101, 25–33 (2000).

    CAS  PubMed  Google Scholar 

  12. Matzke, M. A. & Birchler, J. A. RNAi-mediated pathways in the nucleus. Nature Rev. Genet. 6, 24–35 (2005).

    CAS  PubMed  Google Scholar 

  13. Wassenegger, M. The role of the RNAi machinery in heterochromatin formation. Cell 122, 13–16 (2005).

    CAS  PubMed  Google Scholar 

  14. Wassenegger, M., Heimes, S., Riedel, L. & Sanger, H. L. RNA-directed de novo methylation of genomic sequences in plants. Cell 76, 567–576 (1994).

    CAS  PubMed  Google Scholar 

  15. Morris, K. V., Chan, S. W., Jacobsen, S. E. & Looney, D. J. Small interfering RNA-induced transcriptional gene silencing in human cells. Science 305, 1289–1292 (2004).

    ADS  CAS  PubMed  Google Scholar 

  16. Castanotto, D. et al. Short hairpin RNA-directed cytosine (CpG) methylation of the RASSF1A gene promoter in HeLa cells. Mol. Ther. 12, 179–183 (2005).

    CAS  PubMed  Google Scholar 

  17. Ting, A. H., Schuebel, K. E., Herman, J. G. & Baylin, S. B. Short double-stranded RNA induces transcriptional gene silencing in human cancer cells in the absence of DNA methylation. Nature Genet. 37, 906–910 (2005).

    CAS  PubMed  Google Scholar 

  18. Verdel, A. et al. RNAi-mediated targeting of heterochromatin by the RITS complex. Science 303, 672–676 (2004).

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  19. Janowski, B. A. et al. Involvement of AGO1 and AGO2 in mammalian transcriptional silencing. Nature Struct. Mol. Biol. 13, 787–792 (2006).

    CAS  Google Scholar 

  20. Kim, D. H., Villeneuve, L. M., Morris, K. V. & Rossi, J. J. Argonaute-1 directs siRNA-mediated transcriptional gene silencing in human cells. Nature Struct. Mol. Biol. 13, 793–797 (2006).

    CAS  Google Scholar 

  21. Kim, D. H., Saetrom, P., Snove, O. Jr & Rossi, J. J. MicroRNA-directed transcriptional gene silencing in mammalian cells. Proc. Natl Acad. Sci. USA 105, 16230–16235 (2008).

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  22. Aravin, A. A., Hannon, G. J. & Brennecke, J. The Piwi–piRNA pathway provides an adaptive defense in the transposon arms race. Science 318, 761–764 (2007).

    ADS  CAS  PubMed  Google Scholar 

  23. Klattenhoff, C. & Theurkauf, W. Biogenesis and germline functions of piRNAs. Development 135, 3–9 (2008).

    CAS  PubMed  Google Scholar 

  24. Batista, P. J. et al. PRG-1 and 21U-RNAs interact to form the piRNA complex required for fertility in C. elegans . Mol. Cell 31, 67–78 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Das, P. P. et al. Piwi and piRNAs act upstream of an endogenous siRNA pathway to suppress Tc3 transposon mobility in the Caenorhabditis elegans germline. Mol. Cell 31, 79–90 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. Ghildiyal, M. et al. Endogenous siRNAs derived from transposons and mRNAs in Drosophila somatic cells. Science 320, 1077–1081 (2008).

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  27. Czech, B. et al. An endogenous small interfering RNA pathway in Drosophila . Nature 453, 798–802 (2008).

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  28. Kawamura, Y. et al. Drosophila endogenous small RNAs bind to Argonaute 2 in somatic cells. Nature 453, 793–797 (2008).

    ADS  CAS  PubMed  Google Scholar 

  29. Okamura, K. et al. The Drosophila hairpin RNA pathway generates endogenous short interfering RNAs. Nature 453, 803–806 (2008).

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  30. Tam, O. H. et al. Pseudogene-derived small interfering RNAs regulate gene expression in mouse oocytes. Nature 453, 534–538 (2008).

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  31. Watanabe, T. et al. Endogenous siRNAs from naturally formed dsRNAs regulate transcripts in mouse oocytes. Nature 453, 539–543 (2008).

    ADS  CAS  PubMed  Google Scholar 

  32. Matranga, C., Tomari, Y., Shin, C., Bartel, D. P. & Zamore, P. D. Passenger-strand cleavage facilitates assembly of siRNA into Ago2-containing RNAi enzyme complexes. Cell 123, 607–620 (2005).

    CAS  PubMed  Google Scholar 

  33. Rand, T. A., Petersen, S., Du, F. & Wang, X. Argonaute2 cleaves the anti-guide strand of siRNA during RISC activation. Cell 123, 621–629 (2005). References 32 and 33 were the first studies to demonstrate the mechanism of guide-strand selection for siRNAs.

    CAS  PubMed  Google Scholar 

  34. Li, W. & Cha, L. Predicting siRNA efficiency. Cell. Mol. Life Sci. 64, 1785–1792 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. Tafer, H. et al. The impact of target site accessibility on the design of effective siRNAs. Nature Biotechnol. 26, 578–583 (2008).

    CAS  Google Scholar 

  36. Huesken, D. et al. Design of a genome-wide siRNA library using an artificial neural network. Nature Biotechnol. 23, 995–1001 (2005).

    CAS  Google Scholar 

  37. Morrissey, D. V. et al. Potent and persistent in vivo anti-HBV activity of chemically modified siRNAs. Nature Biotechnol. 23, 1002–1007 (2005).

    CAS  Google Scholar 

  38. Robbins, M. et al. 2′-O-Methyl-modified RNAs act as TLR7 antagonists. Mol. Ther. 15, 1663–1669 (2007).

    CAS  PubMed  Google Scholar 

  39. Dowler, T. et al. Improvements in siRNA properties mediated by 2′-deoxy-2′-fluoro-β-d-arabinonucleic acid (FANA). Nucleic Acids Res. 34, 1669–1675 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  40. Watts, J. K. et al. 2′-Fluoro-4′-thioarabino-modified oligonucleotides: conformational switches linked to siRNA activity. Nucleic Acids Res. 35, 1441–1451 (2007).

    MathSciNet  CAS  PubMed  PubMed Central  Google Scholar 

  41. Fisher, M. et al. Inhibition of MDR1 expression with altritol-modified siRNAs. Nucleic Acids Res. 35, 1064–1074 (2007).

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  42. 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).

    CAS  PubMed  Google Scholar 

  43. Howard, K. A. et al. RNA interference in vitro and in vivo using a novel chitosan/siRNA nanoparticle system. Mol. Ther. 14, 476–484 (2006).

    CAS  PubMed  Google Scholar 

  44. Bitko, V., Musiyenko, A., Shulyayeva, O. & Barik, S. Inhibition of respiratory viruses by nasally administered siRNA. Nature Med. 11, 50–55 (2005).

    CAS  PubMed  Google Scholar 

  45. Li, B. J. et al. Using siRNA in prophylactic and therapeutic regimens against SARS coronavirus in Rhesus macaque . Nature Med. 11, 944–951 (2005).

    ADS  CAS  PubMed  Google Scholar 

  46. Palliser, D. et al. An siRNA-based microbicide protects mice from lethal herpes simplex virus 2 infection. Nature 439, 89–94 (2006).

    ADS  CAS  PubMed  Google Scholar 

  47. 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).

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  48. Bartlett, D. W., Su, H., Hildebrandt, I. J., Weber, W. A. & Davis, M. E. Impact of tumor-specific targeting on the biodistribution and efficacy of siRNA nanoparticles measured by multimodality in vivo imaging. Proc. Natl Acad. Sci. USA 104, 15549–15554 (2007).

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  49. Soutschek, J. et al. Therapeutic silencing of an endogenous gene by systemic administration of modified siRNAs. Nature 432, 173–178 (2004).

    ADS  CAS  PubMed  Google Scholar 

  50. Zimmermann, T. S. et al. RNAi-mediated gene silencing in non-human primates. Nature 441, 111–114 (2006).

    ADS  CAS  PubMed  Google Scholar 

  51. Peer, D., Park, E. J., Morishita, Y., Carman, C. V. & Shimaoka, M. Systemic leukocyte-directed siRNA delivery revealing cyclin D1 as an anti-inflammatory target. Science 319, 627–630 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  52. Dorn, G. et al. siRNA relieves chronic neuropathic pain. Nucleic Acids Res. 32, e49 (2004).

    PubMed  PubMed Central  Google Scholar 

  53. Kawasaki, Y. et al. Distinct roles of matrix metalloproteases in the early- and late-phase development of neuropathic pain. Nature Med. 14, 331–336 (2008).

    CAS  PubMed  Google Scholar 

  54. Dore-Savard, L. et al. Central delivery of Dicer-substrate siRNA: a direct application for pain research. Mol. Ther. 16, 1331–1339 (2008).

    CAS  PubMed  Google Scholar 

  55. Shishkina, G. T., Kalinina, T. S. & Dygalo, N. N. Attenuation of α2A-adrenergic receptor expression in neonatal rat brain by RNA interference or antisense oligonucleotide reduced anxiety in adulthood. Neuroscience 129, 521–528 (2004).

    CAS  PubMed  Google Scholar 

  56. Pardridge, W. M. shRNA and siRNA delivery to the brain. Adv. Drug Deliv. Rev. 59, 141–152 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  57. Kumar, P. et al. Transvascular delivery of small interfering RNA to the central nervous system. Nature 448, 39–43 (2007). This paper demonstrated the important concept that an acetylcholine-receptor-binding peptide–polyarginine conjugate can deliver siRNAs across the blood–brain barrier.

    ADS  CAS  PubMed  Google Scholar 

  58. Song, E. et al. Antibody mediated in vivo delivery of small interfering RNAs via cell-surface receptors. Nature Biotechnol. 23, 709–717 (2005).

    CAS  Google Scholar 

  59. McNamara, J. O. et al. Cell type-specific delivery of siRNAs with aptamer–siRNA chimeras. Nature Biotechnol. 24, 1005–1015 (2006).

    CAS  Google Scholar 

  60. Chu, T. C., Twu, K. Y., Ellington, A. D. & Levy, M. Aptamer mediated siRNA delivery. Nucleic Acids Res. 34, e73 (2006). References 59 and 60 were the first to show aptamer-mediated delivery of siRNAs to a specific cellular receptor.

    PubMed  PubMed Central  Google Scholar 

  61. Sato, Y. et al. Resolution of liver cirrhosis using vitamin A-coupled liposomes to deliver siRNA against a collagen-specific chaperone. Nature Biotechnol. 26, 431–442 (2008).

    CAS  Google Scholar 

  62. Akinc, A. et al. A combinatorial library of lipid-like materials for delivery of RNAi therapeutics. Nature Biotechnol. 26, 561–569 (2008).

    CAS  Google Scholar 

  63. Brummelkamp, T. R., Bernards, R. & Agami, R. Stable suppression of tumorigenicity by virus-mediated RNA interference. Cancer Cell 2, 243–247 (2002).

    CAS  PubMed  Google Scholar 

  64. Raoul, C. et al. Lentiviral-mediated silencing of SOD1 through RNA interference retards disease onset and progression in a mouse model of ALS. Nature Med. 11, 423–428 (2005).

    CAS  PubMed  Google Scholar 

  65. Ralph, G. S. et al. Silencing mutant SOD1 using RNAi protects against neurodegeneration and extends survival in an ALS model. Nature Med. 11, 429–433 (2005).

    CAS  PubMed  Google Scholar 

  66. Carlson, M. E., Hsu, M. & Conboy, I. M. Imbalance between pSmad3 and Notch induces CDK inhibitors in old muscle stem cells. Nature 454, 528–532 (2008).

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  67. Farah, M. H. RNAi silencing in mouse models of neurodegenerative diseases. Curr. Drug Deliv. 4, 161–167 (2007).

    CAS  PubMed  Google Scholar 

  68. Xia, H. et al. RNAi suppresses polyglutamine-induced neurodegeneration in a model of spinocerebellar ataxia. Nature Med. 10, 816–820 (2004).

    CAS  PubMed  Google Scholar 

  69. Shen, J. et al. Suppression of ocular neovascularization with siRNA targeting VEGF receptor 1. Gene Ther. 13, 225–234 (2006).

    CAS  PubMed  Google Scholar 

  70. Kleinman, M. E. et al. Sequence- and target-independent angiogenesis suppression by siRNA via TLR3. Nature 452, 591–597 (2008). This study found that macular vascularization could be inhibited in a non-sequence-specific manner by siRNA-mediated activation of TLR3.

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  71. Li, M. J. et al. Inhibition of HIV-1 infection by lentiviral vectors expressing Pol III-promoted anti-HIV RNAs. Mol. Ther. 8, 196–206 (2003).

    CAS  PubMed  Google Scholar 

  72. Chang, J. et al. Liver-specific microRNA miR-122 enhances the replication of hepatitis C virus in nonhepatic cells. J. Virol. 82, 8215–8223 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  73. Randall, G. et al. Cellular cofactors affecting hepatitis C virus infection and replication. Proc. Natl Acad. Sci. USA 104, 12884–12889 (2007).

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  74. van Rooij, E. et al. Control of stress-dependent cardiac growth and gene expression by a microRNA. Science 316, 575–579 (2007).

    ADS  CAS  PubMed  Google Scholar 

  75. Calin, G. A. & Croce, C. M. MicroRNA–cancer connection: the beginning of a new tale. Cancer Res. 66, 7390–7394 (2006).

    CAS  PubMed  Google Scholar 

  76. Esau, C. C. & Monia, B. P. Therapeutic potential for microRNAs. Adv. Drug Deliv. Rev. 59, 101–114 (2007).

    CAS  PubMed  Google Scholar 

  77. Soifer, H. S., Rossi, J. J. & Saetrom, P. MicroRNAs in disease and potential therapeutic applications. Mol. Ther. 15, 2070–2079 (2007).

    CAS  PubMed  Google Scholar 

  78. Baek, D. et al. The impact of microRNAs on protein output. Nature 455, 64–71 (2008).

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  79. Selbach, M. et al. Widespread changes in protein synthesis induced by microRNAs. Nature 455, 58–63 (2008).

    ADS  CAS  PubMed  Google Scholar 

  80. Amarzguioui, M. et al. Rational design and in vitro and in vivo delivery of Dicer substrate siRNA. Nature Protoc. 1, 508–517 (2006).

    CAS  Google Scholar 

  81. Rose, S. D. et al. Functional polarity is introduced by Dicer processing of short substrate RNAs. Nucleic Acids Res. 33, 4140–4156 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  82. Kim, D. H. et al. Synthetic dsRNA Dicer substrates enhance RNAi potency and efficacy. Nature Biotechnol. 23, 222–226 (2005).

    CAS  Google Scholar 

  83. Siolas, D. et al. Synthetic shRNAs as potent RNAi triggers. Nature Biotechnol. 23, 227–231 (2005).

    CAS  Google Scholar 

  84. 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).

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  85. Jackson, A. L. et al. Expression profiling reveals off-target gene regulation by RNAi. Nature Biotechnol. 21, 635–637 (2003).

    CAS  Google Scholar 

  86. Birmingham, A. et al. 3′ UTR seed matches, but not overall identity, are associated with RNAi off-targets. Nature Methods 3, 199–204 (2006).

    CAS  PubMed  Google Scholar 

  87. Jackson, A. L. et al. Position-specific chemical modification of siRNAs reduces 'off-target' transcript silencing. RNA 12, 1197–1205 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  88. Ui-Tei, K. et al. Functional dissection of siRNA sequence by systematic DNA substitution: modified siRNA with a DNA seed arm is a powerful tool for mammalian gene silencing with significantly reduced off-target effect. Nucleic Acids Res. 36, 2136–2151 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  89. Chiu, Y. L. & Rana, T. M. RNAi in human cells: basic structural and functional features of small interfering RNA. Mol. Cell 10, 549–561 (2002).

    CAS  PubMed  Google Scholar 

  90. Agrawal, S. & Kandimalla, E. R. Role of Toll-like receptors in antisense and siRNA. Nature Biotechnol. 22, 1533–1537 (2004).

    CAS  Google Scholar 

  91. Judge, A. D., Bola, G., Lee, A. C. & MacLachlan, I. Design of noninflammatory synthetic siRNA mediating potent gene silencing in vivo . Mol. Ther. 13, 494–505 (2006).

    CAS  PubMed  Google Scholar 

  92. Schlee, M., Hornung, V. & Hartmann, G. siRNA and isRNA: two edges of one sword. Mol. Ther. 14, 463–470 (2006).

    CAS  PubMed  Google Scholar 

  93. Armstrong, M. E. et al. Small interfering RNAs induce macrophage migration inhibitory factor production and proliferation in breast cancer cells via a double-stranded RNA-dependent protein kinase-dependent mechanism. J. Immunol. 180, 7125–7133 (2008).

    CAS  PubMed  Google Scholar 

  94. Sioud, M. Does the understanding of immune activation by RNA predict the design of safe siRNAs? Front. Biosci. 13, 4379–4392 (2008).

    CAS  PubMed  Google Scholar 

  95. Medarova, Z., Pham, W., Farrar, C., Petkova, V. & Moore, A. In vivo imaging of siRNA delivery and silencing in tumors. Nature Med. 13, 372–377 (2007).

    CAS  PubMed  Google Scholar 

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Acknowledgements

I thank the National Institutes of Health for grant assistance.

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J.J.R. is a cofounder, and chairman of the scientific advisory board, of Dicerna Pharmaceuticals (Watertown, Massachusetts), which is developing RNAi-based therapeutics. He is also a cofounder of Calando Pharmaceuticals (Pasadena, California), which is developing delivery vehicles for siRNAs, and a scientific adviser for Benitec (Melbourne, Australia), which is developing methodologies for expressing small RNAs.

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Correspondence should be addressed to J.J.R. (jrossi@coh.org).

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Castanotto, D., Rossi, J. The promises and pitfalls of RNA-interference-based therapeutics. Nature 457, 426–433 (2009). https://doi.org/10.1038/nature07758

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