Skip to main content
SpringerLink
Log in

Promises and Challenges in Developing RNAi as a Research Tool and Therapy

  • Protocol
  • First Online:
RNA

Part of the book series: Methods in Molecular Biology ((MIMB,volume 703))

Abstract

Small interfering RNA (siRNAs), the main effector of RNA interference (RNAi), are now routinely used to assess gene function, both in vitro and in vivo, and many innovative screens have been reported on the use of RNAi to identify potential drug targets. Despite several technical advances, however, there are still many challenges in determining the ideal design of siRNA sequence, the activation of the immune system, off-target effects, and competition with endogenous microRNAs for cellular miRNA-processing machinery. Therefore, the translation of RNAi technology into the clinic depends on resolving these challenges. This chapter summarizes recent progress in siRNA design, sensing by the immune system, and discusses some of the promising approaches that are currently being explored in separating siRNA unwanted effects from gene silencing.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 129.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 169.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Fire, A., Xu, S., Montgomery, M. K., Kostas, S. A., Driver, S.E, Mello, C. C. (1998) Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391, 806–811.

    Article  CAS  PubMed  Google Scholar 

  2. Jorgensen, R. (1990) Altered gene expression in plants due to trans interactions between homologous genes. Trends Biotechnol 8, 340–344.

    Article  CAS  PubMed  Google Scholar 

  3. Hamilton, A.J, Baucombe, D. C. (1999) A species of small antisense RNA in posttranscriptional gene silencing in plants. Science 286, 950–952.

    Article  CAS  PubMed  Google Scholar 

  4. Sen, G. C. (2001) Viruses and interferons. Annu Rev Microbiol 55, 255–281.

    Article  CAS  PubMed  Google Scholar 

  5. Elbashir, S. M., Harborth, J., Lendeckel, W., Yalcin, A., Weber, K., Tuschl, T. (2001) Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411, 494–498.

    Article  CAS  PubMed  Google Scholar 

  6. Sioud, M. (2004) Therapeutic siRNAs. Trends Pharmacol Sci 25, 22–28.

    Article  CAS  PubMed  Google Scholar 

  7. Hannon, G. J., Rossi, J. J. (2004) Unlocking the potential of the human genome with RNA interference. Nature 431, 371–378.

    Article  CAS  PubMed  Google Scholar 

  8. Liu, J., Carmell, M. A., Rivas, F. V., Marsden, C. G., Thomson, J. M., Song, J. J., Hammond, S. M., Joshua-Tor, L., Hannon, G. J. (2004) Argonaute2 is the catalytic engine of mammalian RNAi. Science 305, 1437–1441.

    Article  CAS  PubMed  Google Scholar 

  9. Song, J. J., Smith, S. K., Hannon, G. J., Joshua-Tor, L. (2004) Crystal structure of Argonaute and its implications for RISC slicer activity. Science 305, 1434–1437.

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  11. Rand, T. A., Petersen, S., Du, F., Wang, X. (2005) Argonaute2 cleaves the anti-guide strand of siRNA during RISC activation. Cell 123, 621–629.

    Article  CAS  PubMed  Google Scholar 

  12. Ma, J. B., Yuan, Y. R., Meister, G., Pei, Y., Tuschl, T., Partel, D. J. (2005) Structural basis for 5′-end-specific recognition of guide RNA by the A. fulgidus Piwi protein. Nature 434, 666–670.

    Article  CAS  PubMed  Google Scholar 

  13. Lee, Y. S., et al. (2004) Distinct roles for Drosophila Dicer-1 and Dicer-2 in the siRNA/miRNA silencing pathway. Cell 117, 69–81.

    Article  CAS  PubMed  Google Scholar 

  14. Sioud, M., Sørensen, D. R. (2003) Cationic liposome-mediated delivery of siRNAs in adult mice. Biochem Biophys Res Commun 312, 1220–1225.

    Article  CAS  PubMed  Google Scholar 

  15. Jackson, A. L., Bartz, S. R., Schelter, J., Kobayashi, S. V., Burchard, J., Mao, M., Li, B., Cavet, G., Linsley, P. S. (2003) Expression profiling reveals off-target gene regulation by RNAi. Nat Biotechnol 21, 635–637.

    Article  CAS  PubMed  Google Scholar 

  16. Semizarov, D., Frost, L., Sarthy, A., Kroeger, P., Halbert, D. N., Fesik, S. W. (2003) Specificity of short interfering RNA determined through gene expression signatures. Proc Natl Acad Sci USA 100, 6347–6352.

    Article  CAS  PubMed  Google Scholar 

  17. Sledz, C. A., Holko, M., de Veer, M. J., Silverman, R. H., Williams, B. R. (2003) Activation of the interferon system by short-interfering RNAs. Nat Cell Biol 5, 834–839.

    Article  CAS  PubMed  Google Scholar 

  18. Holen, T., Amrzguioui, M., Wiiger, M. T., Babaie, E., Prydz, H. (2002) Positional effects of short interfering RNAs targeting the human coagulation tissue factor. Nucleic Acids Res 30, 1757–1766.

    Article  CAS  PubMed  Google Scholar 

  19. Schwarz, D. S, et al. (2003) Asymmetry in the assembly of the RNAi enzyme complex. Cell 115, 199–208.

    Article  CAS  PubMed  Google Scholar 

  20. Khvorova, A., Reynolds, A., Jayasena, S. D. (2003) Functional siRNAs and miRNAs exhibit strand bias. Cell 115, 209–216.

    Article  CAS  PubMed  Google Scholar 

  21. Ui-Tei, K., Naito, Y., Saigo, K. (2007) Guidelines for the selection of effectives short-interferin RNA sequences for functional genomics. Methods Mol Biol 361, 201–216.

    CAS  PubMed  Google Scholar 

  22. Li, S., Peters, G. A., Ding, K., Zhang, X., Qin. J., Sen, G. C. (2006) Molecular basis for PKR activation by PACT or dsRNA. Proc Natl Acad Sci USA 103, 1005–1010.

    Google Scholar 

  23. Kato, H., Takeuchi, O., Sato, S., Yoneyama, M., Yamamoto, M., Matsui, K., Uematsu, S., Jung, A., Kawai, T., Ishii, K. J., Yamaguchi, O., Otsu, K., Tsujimura, T., Koh, C. S., Reis e Sousa, C., Matsuura, Y., Fujita, T., Akira, S. (2006) Differential roles of MDA5 and RIG-I helicases in the recognition of RNA viruses. Nature 441, 101–105.

    Article  CAS  PubMed  Google Scholar 

  24. Meylan, E., Tschopp, T., Karin, M. (2006) Intracellular pattern recognition receptors in the host response. Nature 442, 39–44.

    Article  CAS  PubMed  Google Scholar 

  25. Takeda, K., Akira, S. (2005) Toll-like receptors in innate immunity. Int Immunol 17, 1–14.

    Article  CAS  PubMed  Google Scholar 

  26. Sioud, M. (2005) Innate sensing of self and non-self RNAs by Toll-like receptors. Trends Mol Med 12, 167–176.

    Article  Google Scholar 

  27. Alexopoulou, L., Holt, A. C., Medzhitov, R., Flavell, R. A. (2001) Recognition of double-stranded RNA and activation of NF-kappaB by Toll-like receptor 3. Nature 413, 732–738.

    Article  CAS  PubMed  Google Scholar 

  28. Heil, F., Hemmi, H., Hochrein, H., Ampenberger, A., Kirschning, C., Akira, S., Lipford, G., Wagner, H., Bauer, S. (2004) Species-specific recognition of single-stranded RNA via toll-like receptor 7 and 8. Science 303, 1526–1529.

    Article  CAS  PubMed  Google Scholar 

  29. Krieg, A. M. (2002) CpG motifs in bacterial DNA and their immune effects. Annu Rev Immunol 20, 709–760.

    Article  CAS  PubMed  Google Scholar 

  30. Cao, W., Liu, Y. J. (2007) Innate immune functions of plasmacytoid dendritic cells. Curr Opin Immunol 19, 24–30.

    Article  PubMed  Google Scholar 

  31. Kariko, K., Bhuyan, P., Capodici, J., Weissman, D. (2004) Small interfering RNAs mediate sequence-independent gene suppression and induce immune activation by signaling through toll-like receptor 3. J. Immunol 172, 6545–6549.

    CAS  PubMed  Google Scholar 

  32. Sioud, M. (2005) Induction of inflammatory cytokines and interferon responses by double-stranded and single-stranded siRNAs is sequence-dependent and requires endosomal localization. J Mol Biol 348, 1079–1090.

    Article  CAS  PubMed  Google Scholar 

  33. Hornung, V., Guenthner-Biller, M., Bourquin, C., Ablasser, A., Schlee, M., Uematsu, S., Noronha, a., Manoharan, M., Akira, S., de Fougerolles, A., Endres, S., Hartmann, G. (2005) Sequence-specific potent induction of IFN-alpha by short interfering RNA in plasmacytoid dendritic cells through TLR7. Nat Med 11, 263–270.

    Article  CAS  PubMed  Google Scholar 

  34. Judge, A. D., Sood, V., Shaw, J. R., Fang, D., McClintock, K., MacLachlan, I. (2005) Sequence-dependent stimulation of the mammalian innate immune response by synthetic siRNA. Nat Biotechnol 23, 457–462.

    Article  CAS  PubMed  Google Scholar 

  35. Sioud, M. (2006) Single-stranded small interfering RNA are more immunostimulatory than their double-stranded counterparts: a central role for 2′-hydroxyl uridines in immune responses. Eur J Immunol 36, 1222–1230.

    Article  CAS  PubMed  Google Scholar 

  36. Cekaite, L., Furset, G., Hovig, E., Sioud, M. (2007) Gene expression analysis in blood cells in response to unmodified and 2′-modified siRNAs reveals TLR-dependent and independent effects. J Mol Biol 365, 90–108.

    Article  CAS  PubMed  Google Scholar 

  37. Goodchild, A., Nopper, N., King, A., Doan, T., Tanudji, M., Arndt, G. M., Poidinger, M., Rivory, L. P., Passioura, T. (2009) Sequence determinants of innate immune activation by short interfering RNAs. BMC Immunol 10, 40–48.

    Article  PubMed  Google Scholar 

  38. Marques, J. T., Devosse, T., Wang, D., Zamanian-Daryoush, M., Serbinowski, P., Hartmann, R., Fujita, T., Behlke, M. A., Williams, B. R. (2006) A structural basis for discriminating between self and nonself double-stranded RNAs in mammalian cells. Nat Biotechnol 24, 559–565.

    Article  CAS  PubMed  Google Scholar 

  39. Bartel, D. P. (2004) MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116, 281–294.

    Article  CAS  PubMed  Google Scholar 

  40. Han, J., Lee, Y., Yeom, K. H., Nam, J. W., Heo, I., Rhee, J. K., Sohn, S. Y., Cho, Y., Zhang, B. T., Kim, V. N. (2006) Molecular basis for the recognition of primary microRNAs by the Drosha-DGCR8 complex. Cell 125, 887–901.

    Article  CAS  PubMed  Google Scholar 

  41. Furset, G., Sioud, M. (2007) Design of bifunctional siRNAs: combining immunostimulation and gene-silencing in one single siRNA molecule. Biochem Biophys Res Commun 352, 642–649.

    Article  CAS  PubMed  Google Scholar 

  42. Hornung, V., Ellegast, J., Kim, S., Brzozka, K., Jung, A., Kato, H., Poeck, H., Akira, S., Conzelmann, K. K., Schlee, M., Endres. S., Hartmann, G. (2006) 5′-Triphosphate RNA is the ligand for RIG-I. Science 314, 994–997.

    Article  PubMed  Google Scholar 

  43. Morrissey, D. V., Lockridge, J. A., Shaw, L., Blanchard, K., Jensen, K., Breen, W., Hartsough, K., Machemer, L., Radka, S., Jadhav, V., Vaish, N., Zinnen, S., Vargeese, C., Bowman, K., Shaffer, C. S., Jeffs, L. B., Judge, A., MacLachlan, I., Polisky, B. (2005) Potent and persistent in vivo anti-HBV activity of chemically modified siRNAs. Nat Biotechnol 23, 1002–1007.

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  45. Kariko, K., Buckstein, M., Ni, H., Weissman, D. (2005) Suppression of RNA recognition by Toll-like receptors: the impact of nucleoside modification and the evolutionary origin of RNA. Immunity 23, 165–175.

    Article  CAS  PubMed  Google Scholar 

  46. Sioud, M. (2007) RNA interference and innate immunity. Adv Drug Deliv Rev 59, 153–163.

    Article  CAS  PubMed  Google Scholar 

  47. Sioud, M., Furset, G., Cekaite, L. (2007) Suppression of immunostimulatory siRNA-driven innate immune activation by 2′-modified RNAs. Biochem Biophys Res Commun 361, 122–126.

    Article  CAS  PubMed  Google Scholar 

  48. Furset, G., Floisand, Y., Sioud, M. (2007) Impaired expression of indoleamine 2, 3-dioxygenase in monocyte-derived dendritic cells in response to Toll-like receptor-7/8 ligands. Immunology 123, 263–271.

    PubMed  Google Scholar 

  49. Robbins, M., Judge, A., Liang, L., McClintock, K., Yaworski, E., MacLachlan, I. (2007) 2′-O-methyl-modified RNAs act as TLR7 antagonists. Mol Ther 15, 1663–1669.

    Article  CAS  PubMed  Google Scholar 

  50. Zhu, F. G., Reich, C. F., Pisetsky, D. S. (2002) Inhibition of murine dendritic cell activation by synthetic phosphorothioate oligodeoxynucleotides. J Leukoc Biol 72, 1154–1163.

    CAS  PubMed  Google Scholar 

  51. Jackson, A. L., Burchard, J., Leake, D., Reynolds, A., Schelter, J., Guo, J., Johnson, J. M., Lim, L., Karpilow, J., Nichols, K., Marshall, W., Khvorova, A., Linsley, P. S. (2006) Position-specific chemical modification of siRNAs reduces “off-target” transcript silencing. RNA 12, 1197–1205.

    Article  CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Molecular Medicine Group, Department of Immunology, Institute for Cancer Research, The Norwegian Radium Hospital, Montebello, Oslo, Norway

    Mouldy Sioud

Authors
  1. Mouldy Sioud
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Mouldy Sioud .

Editor information

Editors and Affiliations

  1. The Panum Institute, Dept. of Cellular & Molecular Medicine, University of Copenhagen, Blegdamsvej 3, Copenhagen, 2200N, Denmark

    Henrik Nielsen

Rights and permissions

Reprints and permissions

Copyright information

© 2011 Springer Science+Business Media, LLC

About this protocol

Cite this protocol

Sioud, M. (2011). Promises and Challenges in Developing RNAi as a Research Tool and Therapy. In: Nielsen, H. (eds) RNA. Methods in Molecular Biology, vol 703. Humana Press. https://doi.org/10.1007/978-1-59745-248-9_12

Download citation

  • DOI: https://doi.org/10.1007/978-1-59745-248-9_12

  • Published:

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-58829-913-0

  • Online ISBN: 978-1-59745-248-9

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation