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Therapeutic Applications of RNAi for Silencing Virus Replication

  • Ralph A. Tripp
  • Stephen Mark Tompkins
Protocol
Part of the Methods in Molecular Biology™ book series (MIMB, volume 555)

Abstract

RNA interference (RNAi) is an evolutionarily conserved gene-silencing mechanism in which small 19–23-nucleotide double-stranded RNA molecules, or small interfering RNAs (siRNAs), target cognate RNA for destruction with exquisite potency and selectivity. The RNAi machinery is believed to be expressed in all eukaryotic cells and has been shown to regulate host gene expression. Given this ability, RNAi silencing strategies have been developed to inhibit viral genes and replication in host cells. One area of growing interest is the development of synthetic siRNA drugs to target acute viral infections in which long-term gene silencing is not required or desirable. To achieve synthetic siRNA drug efficacy, these anti-viral agents need to be delivered to the appropriate host cells, as they do not readily cross the cell membrane. Varied delivery and siRNA chemical stabilization strategies are being investigated for siRNA drug delivery; however, several studies have shown that naked, unmodified siRNA drugs can be effective in silencing replication of some viruses in animal models of infection. These findings suggest that RNAi-based drugs may offer breakthrough technology to protect and treat humans and animals from viral infection. However, there are four major considerations for evaluating successful RNAi efficacy: the siRNAs must have high efficiency, show low cytotoxicity, result in minimal off-target effects, and lead to results that are reproducible between experiments. The methods and caveats to achieve these goals are discussed.

Key words

RNA interference RNAi small interfering RNA siRNA transfection drug delivery virus prophylactic therapeutic 

References

  1. 1.
    Elbashir, S. M., Harborth, J., Lendeckel, W., Yalcin, A., Weber, K., and Tuschl, T. (2001) Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells Nature 411, 494.PubMedCrossRefGoogle Scholar
  2. 2.
    Amarzguioui, M., Rossi, J. J., and Kim, D. (2005) Approaches for chemically synthesized siRNA and vector-mediated RNAi FEBS Lett 579, 5974–5981.PubMedCrossRefGoogle Scholar
  3. 3.
    de Fougerolles, A., Vornlocher, H.-P., Maraganore, J., and Lieberman, J. (2007) Interfering with disease: a progress report on siRNA-based therapeutics Nat Rev Drug Discov 6, 443–453.PubMedCrossRefGoogle Scholar
  4. 4.
    Fewell, G. D., and Schmitt, K. (2006) Vector-based RNAi approaches for stable, inducible and genome-wide screens Drug Discov Today 11, 975–982.PubMedCrossRefGoogle Scholar
  5. 5.
    Hamasaki, K., Kogure, K., and Ohwada, K. (1996) A biological method for the quantitative measurement of tetrodotoxin (TTX): tissue culture bioassay in combination with a water-soluble tetrazolium salt. Toxicon 34, 490–495.PubMedCrossRefGoogle Scholar
  6. 6.
    Elbashir, S. M., Lendeckel, W., and Tuschl, T. (2001) RNA interference is mediated by 21- and 22-nucleotide RNAs Genes Dev. 15, 188–200.PubMedCrossRefGoogle Scholar
  7. 7.
    Birmingham, A., Anderson, E., Sullivan, K., Reynolds, A., Boese, Q., Leake, D., Karpilow, J., and Khvorova, A. (2007) A protocol for designing siRNAs with high functionality and specificity Nat Protocols 2, 2068–2078.CrossRefGoogle Scholar
  8. 8.
    Gonzalez-Alegre, P., Bode, N., Davidson, B. L., and Paulson, H. L. (2005) Silencing primary dystonia: lentiviral-mediated RNA interference therapy for DYT1 dystonia. J Neurosci 25, 10502–10509.PubMedCrossRefGoogle Scholar
  9. 9.
    Dann, C. (2007) New technology for an old favorite: lentiviral transgenesis and RNAi in rats. Transgenic Res 16, 571–580.PubMedCrossRefGoogle Scholar
  10. 10.
    Ge, Q., McManus, M. T., Nguyen, T., Shen, C. H., Sharp, P. A., Eisen, H. N., and Chen, J. (2003) RNA interference of influenza virus production by directly targeting mRNA for degradation and indirectly inhibiting all viral RNA transcription Proc Natl Acad Sci USA 100, 2718–2723.PubMedCrossRefGoogle Scholar
  11. 11.
    Sledz, C. A., Holko, M., de Veer, M. J., Silverman, R. H., and Williams, B. R. (2003) Activation of the interferon system by short-interfering RNAs. Nat Cell Biol 5, 834–839.PubMedCrossRefGoogle Scholar
  12. 12.
    Sledz, C. A., and Williams, B. R. G. (2004) RNA interference and double-stranded-RNA-activated pathways Biochem Soc Trans 32, 952–956.PubMedCrossRefGoogle Scholar
  13. 13.
    Perrimon, N., Friedman, A., Mathey-Prevot, B., and Eggert, U. S. (2007) Drug–target identification in Drosophila cells: combining high-throughout RNAi and small-molecule screens. Drug Discov Today 12, 28–33.PubMedCrossRefGoogle Scholar
  14. 14.
    Birmingham, A., Anderson, E. M., Reynolds, A., Ilsley-Tyree, D., Leake, D., Fedorov, Y., Baskerville, S., Maksimova, E., Robinson, K., Karpilow, J., Marshall, W. S., and Khvorova, A. (2006) 3' UTR seed matches, but not overall identity, are associated with RNAi off-targets. Nat Meth 3, 199–204.CrossRefGoogle Scholar
  15. 15.
    Cullen, L. M., and Arndt, G. M. (2005) Genome-wide screening for gene function using RNAi in mammalian cells. Immunol Cell Biol 83, 217–223.PubMedCrossRefGoogle Scholar
  16. 16.
    Masters, J. R., Thomson, J. A., Daly-Burns, B., Reid, Y. A., Dirks, W. G., Packer, P., Toji, L. H., Ohno, T., Tanabe, H., Arlett, C. F., Kelland, L. R., Harrison, M., Virmani, A., Ward, T. H., Ayres, K. L., and Debenham, P. G. (2001) Short tandem repeat profiling provides an international reference standard for human cell lines Proc Natl Acad Sci USA 98, 8012–8017.PubMedCrossRefGoogle Scholar
  17. 17.
    Lingel, A., and Izaurralde, E. (2004) RNAi: finding the elusive endonuclease. RNA 10, 1675–1679.PubMedCrossRefGoogle Scholar
  18. 18.
    Koller, E., Propp, S., Murray, H., Lima, W., Bhat, B., Prakash, T. P., Allerson, C. R., Swayze, E. E., Marcusson, E. G., and Dean, N. M. (2006) Competition for RISC binding predicts in vitro potency of siRNA. Nucl Acids Res 34, 4467–4476.PubMedCrossRefGoogle Scholar
  19. 19.
    Vickers, T. A., Lima, W. F., Nichols, J. G., and Crooke, S. T. (2007) Reduced levels of Ago2 expression result in increased siRNA competition in mammalian cells Nucl Acids Res. 35, 6598–6610.PubMedCrossRefGoogle Scholar
  20. 20.
    Reynolds, A., Anderson, E. M., Vermeulen, A., Fedorov, Y., Robinson, K., Leake, D., Karpilow, J., Marshall, W. S., and Khvorova, A. (2006) Induction of the interferon response by siRNA is cell type- and duplex length-dependent. RNA 12, 988–993.PubMedCrossRefGoogle Scholar
  21. 21.
    Lin, X., Ruan, X., Anderson, M. G., McDowell, J. A., Kroeger, P. E., Fesik, S. W., and Shen, Y. (2005) siRNA-mediated off-target gene silencing triggered by a 7 nt complementation. Nucl Acids Res 33, 4527–4535.PubMedCrossRefGoogle Scholar
  22. 22.
    Jackson, A. L., Burchard, J., Schelter, J., Chau, B. N., Cleary, M., Lim, L., and Linsley, P. S. (2006) Widespread siRNA “off-target” transcript silencing mediated by seed region sequence complementarity. RNA 12, 1179–1187.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2009

Authors and Affiliations

  • Ralph A. Tripp
    • 1
  • Stephen Mark Tompkins
    • 1
  1. 1.Department of Infectious Diseases, College of Veterinary Medicine, Center for Disease InterventionUniversity of GeorgiaAthensUSA

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