Molecules and Cells

Korean Society for Molecular and Cellular Biology (한국분자세포생물학회)
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Immune Activation by siRNA/Liposome Complexes in Mice Is Sequence- independent: Lack of a Role for Toll-like Receptor 3 Signaling

  • Kim, Ji Young (Gene2Drug Research Center, Bioneer Corporation) ;
  • Choung, Sorim (Gene2Drug Research Center, Bioneer Corporation) ;
  • Lee, Eun-Ju (Gene2Drug Research Center, Bioneer Corporation) ;
  • Kim, Young Joo (National Genome Information Center, Korea Research Institute of Bioscience and Biotechnology) ;
  • Choi, Young-Chul (Gene2Drug Research Center, Bioneer Corporation)
  • Received : 2007.04.04
  • Accepted : 2007.06.06
  • Published : 2007.10.31
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Abstract

Improvement in the pharmacokinetic properties of short interfering RNAs (siRNAs) is a prerequisite for the therapeutic application of RNA interference technology. When injected into mice as unmodified siRNAs complexed to DOTAP/Chol-based cationic liposomes, all 12 tested siRNA duplexes caused a strong induction of cytokines including interferon ${\alpha}$, indicating that the immune activation by siRNA duplexes is independent of sequence context. When modified by various combinations of 2'-OMe, 2'-F, and phosphorothioate substitutions, introduction of as little as three 2'-OMe substitutions into the sense strand was sufficient to suppress immune activation by siRNA duplexes, whereas the same modifications were much less efficient at inhibiting the immune response of single stranded siRNAs. It is unlikely that Toll-like receptor 3 (TLR3) signaling is involved in immune stimulation by siRNA/liposome complexes since potent immune activation by ds siRNAs was induced in TLR3 knockout mice. Together, our results indicate that chemical modification of siRNA provides an effective means to avoid unwanted immune activation by therapeutic siRNAs. This improvement in the in vivo properties of siRNAs should greatly facilitate successful development of siRNA therapeutics.

Keywords

Acknowledgement

Supported by : Ministry of Commerce, Industry, and Energy in Korea

References

  1. Alexopoulou, L., Holt, A. C., Medzhitov, R., and Flavell, R. A. (2001) Recognition of double-stranded RNA and activation of NF-kappaB by Toll-like receptor 3. Nature 413, 732−738
  2. Cekaite, L., Furset, G., Hovig, E., and 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
  3. Choung, S., Kim, Y. J., Kim, S., Park, H. O., and Choi, Y. C. (2006) Chemical modification of siRNAs to improve serum stability without loss of efficacy. Biochem. Biophys. Res. Commun. 342, 919−927
  4. Diebold, S. S., Kaisho, T., Hemmi, H., Akira, S., and Reis e Sousa, C. (2004) Innate antiviral responses by means of TLR7-mediated recognition of single-stranded RNA. Science 303, 1529−1531
  5. Dorsett, Y. and Tuschl, T. (2004) siRNAs: applications in functional genomics and potential as therapeutics. Nat. Rev. Drug Discov. 3, 318−329
  6. Elbashir, S. M., Harborth, J., Lendeckel, W., Yalcin, A., Weber, K., et al. (2001) Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411, 494−498
  7. Fire, A., Xu, S., Montgomery, M. K., Kostas, S. A., Driver, S. E., et al. (1998) Potent and specific genetic interference by double- stranded RNA in Caenorhabditis elegans. Nature 391, 806−811
  8. 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
  9. Hannon, G. J. and Rossi, J. J. (2004) Unlocking the potential of the human genome with RNA interference. Nature 431, 371−378
  10. Heidel, J. D., Hu, S., Liu, X. F., Triche, T. J., and Davis, M. E. (2004) Lack of interferon response in animals to naked siRNAs. Nat. Biotechnol. 22, 1579−1582
  11. Heil, F., Hemmi, H., Hochrein, H., Ampenberger, F., Kirschning, C., et al. (2004) Species-specific recognition of single-stranded RNA via toll-like receptor 7 and 8. Science 303, 1526−1529
  12. Hornung, V., Guenthner-Biller, M., Bourquin, C., Ablasser, A., Schlee, M., et al. (2005) Sequence-specific potent induction of IFN-alpha by short interfering RNA in plasmacytoid dendritic cells through TLR7. Nat. Med. 11, 263−270
  13. Jackson, A. L., Burchard, J., Leake, D., Reynolds, A., Schelter, J., et al. (2006) Position-specific chemical modification of siRNAs reduces 'off-target' transcript silencing. RNA 12, 1197−1205
  14. Judge, A. D., Sood, V., Shaw, J. R., Fang, D., McClintock, K., et al. (2005) Sequence-dependent stimulation of the mammalian innate immune response by synthetic siRNA. Nat. Biotechnol. 23, 457−462
  15. Judge, A. D., Bola, G., Lee, A. C., and MacLachlan, I. (2006) Design of noninflammatory synthetic siRNA mediating potent gene silencing in vivo. Mol. Ther. 13, 494−505
  16. Lee, M. S. and Kim, Y. J. (2007) Pattern-recognition receptor signaling initiated from extracellular, membrane, and cytoplasmic space. Mol. Cells 23, 1−10
  17. Ma, Z., Li, J., He, F., Wilson, A., Pitt, B., et al. (2005) Cationic lipids enhance siRNA-mediated interferon response in mice. Biochem. Biophys. Res. Commun. 330, 755−759
  18. Marques, J. T., Devosse, T., Wang, D., Zamanian-Daryoush, M., Serbinowski, P., et al. (2006) A structural basis for discriminating between self and nonself double-stranded RNAs in mammalian cells. Nat. Biotechnol. 24, 559−565
  19. Persengiev, S. P., Zhu, X., and Green, M. R. (2004) Nonspecific, concentration-dependent stimulation and repression of mammalian gene expression by small interfering RNAs (siRNAs). RNA 10, 12−18
  20. Reynolds, A., Anderson, E. M., Vermeulen, A., Fedorov, Y., Robinson, K., et al. (2006) Induction of the interferon response by siRNA is cell type- and duplex length-dependent. RNA 12, 988−993
  21. Sen, G. C. (2001) Viruses and interferons. Annu. Rev. Microbiol. 55, 255−281
  22. 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
  23. 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
  24. 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
  25. Song, E., Zhu, P., Lee, S. K., Chowdhury, D., Kussman, S., et al. (2005) Antibody mediated in vivo delivery of small interfering RNAs via cell-surface receptors. Nat. Biotechnol. 23, 709−717
  26. Stark, G. R., Kerr, I. M., Williams, B. R., Silverman, R. H., and Schreiber, R. D. (1998) How cells respond to interferons. Annu. Rev. Biochem. 67, 227−264
  27. Takeda, K. and Akira, S. (2005) Toll-like receptors in innate immunity. Int. Immunol. 17, 1−14
  28. Yoneyama, M., Kikuchi, M., Natsukawa, T., Shinobu, N., Imaizumi, T., et al. (2004) The RNA helicase RIG-I has an essential function in double-stranded RNA-induced innate antiviral responses. Nat. Immunol. 5, 730−737