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Modulation of Dendritic Cell Maturation and Function by siRNA-Bearing 5ʹ-Triphosphate

  • Mouldy Sioud
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 629)

Abstract

Small interfering RNAs (siRNAs) have emerged as a powerful technique for sequence-specific gene silencing in a wide variety of organisms. However, it has been shown that in vitro transcribed siRNA can activate innate immunity through RIG-1 helicase, a cytosolic sensor of viral RNA. We recently demonstrated that the activation of RIG-1 by siRNA-bearing 5ʹ-triphosphate can be used to enhance dendritic cell maturation and function. Indeed, immature DCs that had been transfected with siRNA-bearing 5ʹ-triphosphate activated T cells, indicating that, even in the absence of external stimuli such as TNF-α, those DCs were sufficiently mature to initiate T cell activation. Also, a recent study has shown that 5ʹ-bearing siRNAs targeting Bcl-2 can induce apoptosis in tumour cells, further supporting the use of this strategy to combat cancer. This chapter describes the in vitro synthesis of siRNA targeting indoleamine 2,3-dioxygenase (IDO), an enzyme that plays a critical role in hindering immunity against tumours.

Key words

Indoleamine 2 3-dioxygenase siRNA bifunctional siRNAs dendritic cells immunosuppression 

References

  1. 1.
    Sioud, M. (2001) Nucleic acid enzymes as a novel generation of anti-gene agents. Curr Mol Med, 1, 575–588.PubMedCrossRefGoogle Scholar
  2. 2.
    Sharp, P.A. (2001) RNA interference. Genes Dev, 15, 188–200.CrossRefGoogle Scholar
  3. 3.
    Fire, A., Xu, S., Montgomery, M.K., Kostas, S.A., Driver, S.E., and Mello, C.C. (1998) Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature, 391, 806–811.PubMedCrossRefGoogle Scholar
  4. 4.
    Sioud, M. (2004) Therapeutic siRNAs. Trends Pharmacol Sci, 25, 22–28.PubMedCrossRefGoogle Scholar
  5. 5.
    Meister, G. and Tuschl, T. (2004) Mechanisms of gene silencing by double-stranded RNA. Nature, 431, 343–349.PubMedCrossRefGoogle Scholar
  6. 6.
    Corey, D.R. (2007) Chemical modification: the key to clinical application of RNA interference? J Clin Invest, 117, 3615–3622.PubMedCrossRefGoogle Scholar
  7. 7.
    Leirdal, M. and Sioud, M. (2002) Gene silencing by preformed small RNA duplexes. Biochem Biophys Res Commun, 295, 744–748.PubMedCrossRefGoogle Scholar
  8. 8.
    Pichlmair, A., Schulz, O., Tan, C.P., Näslund, T.I., Liljeström, P., Weber, F., and Reisesousa, C. (2006) RIG-1-mediated antiviral responses to single-stranded RNA-bearing 5ʹ-phosphates. Science, 314, 997–1001.PubMedCrossRefGoogle Scholar
  9. 9.
    Hornung, V., Ellegast, J., Kim, S., Brzózka, K., Jung, A., Kato, H., Poeck, H., Akira, S., Conzelmann, K.K., Schlee, M., Endres, S., and Hartmann, G. (2006) 5ʹ-triphosphate RNA is the ligand for RIG-1. Science, 314, 994–997.PubMedCrossRefGoogle Scholar
  10. 10.
    Furset, G. and Sioud, M. (2007) Design of bifunctional siRNAs: combining immunostimulation and gene-silencing in one single siRNA molecule. Biochem Biophys Res Commun, 352, 642–649.PubMedCrossRefGoogle Scholar
  11. 11.
    Munn, D.H. and Mellor, A.L. (2004) IDO expression by dendritic cells: tolerance and tryptophan catabolism. Nat Rev Immunol, 4, 762–774.PubMedCrossRefGoogle Scholar
  12. 12.
    Furset Flatekval, G. and Sioud, M. (2009) Modulation of dendritic cell function and maturation with mono- and bifunctional small interfering RNAs targeting indoleamine 2,3-dioxygenase (p). Immunology DOI: 10.1111/j.1365–2567.2009.03093.x.Google Scholar
  13. 13.
    Poeck, H., Besch, R., Maihoefer, C., Renn, M., Tormo, D., Morskaya, S.S., Kirschnek, S., Gaffal, E., Landsberg, J., Hellmuth, J., Schmidt, A., Anz, D., Bscheider, M., Schwerd, T., Berking, C., Bourquin, C., Kalinke, U., Kremmer, E., Kato, H., Akira, S., Meyers, R., Häcker, G., Neuenhahn, M., Busch, D., Ruland, J., Rothenfusser, S., Prinz, M., Hornung, V., Endres, S., Tüting, T., and Hartmann, G. (2008) 5ʹ-Triphosphate-siRNA: turning gene silencing and Rig-I activation against melanoma. Nat Med, 14, 1256–1263.PubMedCrossRefGoogle Scholar
  14. 14.
    Hunter, R.L. (2002) Overview of vaccine adjuvants: present and future. Vaccine, 20, S7–S12.PubMedCrossRefGoogle Scholar
  15. 15.
    Banchereau, J. and Steinman, R.M. (1998) Dendritic cells and the control of immunity. Nature, 392, 245–252.PubMedCrossRefGoogle Scholar
  16. 16.
    Kawai, T. and Akira, S. (2008) Toll-like receptor and RIG-1-like receptor signaling. Ann NY Acad Sci, 1143, 1–20.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Mouldy Sioud
    • 1
  1. 1.Department of ImmunologyInstitute for Cancer Research, Rikshospitalet-Radiumhospitalet, University HospitalOsloNorway

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