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Impact of chemical modifications in the structure of isRNA on its antiproliferative and immunostimulatory properties

Abstract

Short double-stranded RNAs, depending on their structure, sequence, and the method of delivery to cells, can activate the system of innate and adaptive immunity. The immunostimulatory activity of nucleic acids can be used in antitumor and antiviral therapy. We have previously identified a biologically active immunostimulatory 19-bp dsRNA (isRNA) with 3′-nucleotide overhangs, which inhibits the proliferation of tumor cells, stimulates interferon synthesis, and suppresses the growth and metastasis of tumors. Here, we have studied the impact of chemical modifications in the isRNA structure and the type of the transfection agent on the antiproliferative and immunostimulatory properties of isRNA. It has been shown that the attachment of an aminohexyl group or a cholesterol residue to the 5′-terminus of the first strand of the isRNA duplex does not impair its antiproliferative and immunostimulatory properties in vitro and in vivo when it is used in complex with a transfection agent, whereas the modification at the 5′-end of the second strand has an adverse effect on the biological activity of isRNA. It has been found that, when used without the transfection agent, isRNA conjugated with a cholesterol residue does not display antiproliferative and immunostimulatory properties. The use of isRNA in complex with low-toxicity liposomes 2Х3-DOPE enhances its activity: the intravenous injection of the isRNA/2Х3-DOPE complex induces a 55-fold increase in the level of interferon α (INF-α) in the murine blood 6 h after the injection, which is significantly higher than the INF-α level after the injection of the isRNA/Lipofectamine 2000 complex. The cytoplasmic localization of isRNA is crucially important for the manifestation of its antiproliferative activity since the selective inhibition of the dsRNA cytoplasmic sensor PKR (dsRNA-dependent protein kinase R) by 2-aminopurine completely blocks the antiproliferative effect of isRNA.

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Abbreviations

isRNA:

immunostimulatory RNA

dsRNA:

double-stranded RNA

PBMC:

peripheral blood mononuclear cells

siRNA:

small interfering RNA

IFN-α:

interferon α

TLR:

toll-like receptor

TNF-α:

tumor necrosis factor α

IL-6:

interleukin 6

References

  1. Bernstein, E., Caudy, A.A., Hammond, S.M., and Hannon, G.J., Nature, 2001, vol. 409, pp. 363–366.

    CAS  Article  PubMed  Google Scholar 

  2. Karikó, K., Bhuyan, P., Capodici, J., and Weissman, D., J. Immunol., 2004, vol. 172, pp. 6545–6549.

    Article  PubMed  Google Scholar 

  3. Kim, D.H., Longo, M., Han, Y., Lundberg, P., Cantin, E., and Rossi, J.J., Nat. Biotechnol., 2004, vol. 22, pp. 321–325.

    CAS  Article  PubMed  Google Scholar 

  4. Hornung, V., Guenthner-Biller, M., Bourquin, C., Ablasser, A., Schlee, M., Uematsu, S., Noronha, A., Manoharan, M., Akira, S., de Fougerolles, A., Endres, S., and Hartmann, G., Nat. Med., 2005, vol. 11, pp. 263–270.

    CAS  Article  PubMed  Google Scholar 

  5. Judge, A.D., Sood, V., Shaw, J.R., Fang, D., McClintock, K., and MacLachlan, I., Nat. Biotechnol., 2005, vol. 23, pp. 457–462.

    CAS  Article  PubMed  Google Scholar 

  6. Sioud, M., J. Mol. Biol., 2005, vol. 348, pp. 1079–1090.

    CAS  Article  PubMed  Google Scholar 

  7. Marques, J.T., Devosse, T., Wang, D., Zamanian-Daryoush, M., Serbinowski, P., Hartmann, R., Fujita, T., Behlke, M.A., and Williams, B.R., Nat. Biotechnol., 2006, vol. 24, pp. 559–565.

    CAS  Article  PubMed  Google Scholar 

  8. Schlee, M., Hartmann, E., Coch, C., Wimmenauer, V., Janke, M., Barchet, W., and Hartmann, G., Immunol. Rev., 2009, vol. 227, pp. 66–74.

    CAS  Article  PubMed  Google Scholar 

  9. Judge, A. and MacLachlan, I., Hum. Gene Ther., 2008, vol. 19, pp. 111–124.

    CAS  Article  PubMed  Google Scholar 

  10. Robbins, M., Judge, A., Ambegia, E., Choi, C., Yaworski, E., Palmer, L., McClintock, K., and MacLachlan, I., Hum. Gene Ther., 2008, vol. 19, pp. 991–999.

    CAS  Article  PubMed  Google Scholar 

  11. Gantier, M.P. and Williams, B.R., Nat. Biotechnol., 2009, vol. 27, pp. 911–912.

    CAS  Article  PubMed  Google Scholar 

  12. Olejniczak, M., Galka-Marciniak, P., Polak, K., Fligier, A., and Krzyzosiak, W.J., RNA, 2012, vol. 18, pp. 930–935.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  13. Desmet, C.J. and Ishii, K.J., Nat. Rev. Immunol., 2012, vol. 12, pp. 479–491.

    CAS  Article  PubMed  Google Scholar 

  14. Chang, C.I., Lee, T.Y., Dua, P., Kim, S., Li, C.J., and Lee, D.K., Nucleic Acid Ther., 2011, vol. 21, pp. 149–155.

    CAS  Article  PubMed  Google Scholar 

  15. Sajeesh, S., Lee, T.Y., Hong, S.W., Dua, P., Choe, J.Y., Kang, A., Yun, W.S., Song, C., Park, S.H., Kim, S., Li, C., and Lee, D.K., Mol. Pharm., 2014, vol. 11, pp. 872–884.

    CAS  Article  PubMed  Google Scholar 

  16. Nechaev, S., Gao, C., Moreira, D., Swiderski, P., Jozwiak, A., Kowolik, C.M., Zhou, J., Armstrong, B., Raubitschek, A., Rossi, J.J., and Kortylewski, M., J. Control Release, 2013, vol. 170, pp. 307–315.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  17. Du, H.Y., Dong, L.H., Zhao, B.J., Fu, J., Wang, Q.Q., Chen, F., Ou, L., Li, N., Sun, X., Tang, Z.M., and Song, H.F., Acta Pharmacol. Sin., 2012, vol. 33, pp. 1047–1054.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  18. Kortylewski, M., Swiderski, P., Herrmann, A., Wang, L., Kowolik, C., Kujawski, M., Lee, H., Scuto, A., Liu, Y., Yang, C., Deng, J., Soifer, H.S., Raubitschek, A., Forman, S., Rossi, J.J., Pardoll, D.M., Jove, R., and Yu, H., Nat. Biotechnol., 2009, vol. 27, pp. 925–932.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  19. Zhang, Q., Hossain, D.M., Nechaev, S., Kozlowska, A., Zhang, W., Liu, Y., Kowolik, C.M., Swiderski, P., Rossi, J.J., Forman, S., Pal, S., Bhatia, R., Raubitschek, A., Yu, H., and Kortylewski, M., Blood, 2013, vol. 121, pp. P. 1304–1315.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  20. Khairuddin, N., Blake, S.J., Firdaus, F., Steptoe, R.J., Behlke, M.A., Hertzog, P.J., and McMillan, N.A., Immunol. Cell Biol., 2014, vol. 92, pp. 156–163.

    CAS  Article  PubMed  Google Scholar 

  21. Khairuddin, N., Gantier, M.P., Blake, S.J., Wu, S.Y., Behlke, M.A., Williams, B.R., and McMillan, N.A., Immunol. Cell Biol., 2012, vol. 90, pp. 187–196.

    CAS  Article  PubMed  Google Scholar 

  22. Stewart, C.R., Karpala, A.J., Lowther, S., Lowenthal, J.W., and Bean, A.G., PLoS One, 2011, vol. 6, p. e21552.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  23. Hashimoto, Y., Abu, Lila A.S., Shimizu, T., Ishida, T., and Kiwada, H., J. Control Release, 2014, vol. 184, pp. 1–8.

    CAS  Article  PubMed  Google Scholar 

  24. Anz, D., Koelzer, V.H., Moder, S., Thaler, R., Schwerd, T., Lahl, K., Sparwasser, T., Besch, R., Poeck, H., Hornung, V., Hartmann, G., Rothenfusser, S., Bourquin, C., and Endres, S., J. Immunol., 2010, vol. 184, pp. 939–946.

    CAS  Article  PubMed  Google Scholar 

  25. Wang, K., Chen, X., Yan, F., Xing, Y., Yang, X., Tu, J., and Chen, Z., J. Immunother., 2013, vol. 36, pp. 294–304.

    CAS  Article  PubMed  Google Scholar 

  26. Chen, X., Qian, Y., Yan, F., Tu, J., Yang, X., Xing, Y., and Chen, Z., Eur J. Pharmacol., 2013, vol. 721, pp. 86–95.

    CAS  Article  PubMed  Google Scholar 

  27. 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., Nat. Med., 2008, vol. 14, pp. 1256–1263.

    CAS  Article  PubMed  Google Scholar 

  28. Prins, R.M., Craft, N., Bruhn, K.W., Khan-Farooqi, H., Koya, R.C., Stripecke, R., Miller, J.F., and Liau, L.M., J. Immunol., 2006, vol. 176, pp. 157–164.

    CAS  Article  PubMed  Google Scholar 

  29. Kabilova, T.O., Chernolovskaya, E.L., Zenkova, M.A., and Vlasov, V.V., Double-stranded RNA fragments exhibiting antiproliferative and interferon-inducing activities, RF Patent no. 2391405, 2010.

    Google Scholar 

  30. Kabilova, T.O., Vladimirova, A.V., Zenkova, M.A., Chernolovskaya, E.L., and Vlassov, V.V., Dokl. Biochem. Biophys., 2011, vol. 436, pp. 8–11.

    CAS  Article  PubMed  Google Scholar 

  31. Kabilova, T.O., Meschaninova, M.I., Venyaminova, A.G., Nikolin, V.P., Zenkova, M.A., Vlassov, V.V., and Chernolovskaya, E.L., Nucleic Acid Ther., 2012, vol. 22, pp. 196–204.

    CAS  PubMed  Google Scholar 

  32. Kabilova, T.O., Kovtonyuk, L.V., Zonov, E.V., Ryabchikova, E.I., Popova, N.A., Nikolin, V.P., Kaledin, V.I., Zenkova, M.A., Vlassov, V.V., and Chernolovskaya, E.L., BMC Cancer, 2014, vol. 14, p. 338.

    Article  PubMed  PubMed Central  Google Scholar 

  33. Kabilova, T.O., Sen’kova, A.V., Nikolin, V.P., Popova, N.A., Zenkova, M.A., Vlassov, V.V., and Chernolovskaya, E.L., PLoS One, 2016, vol. 11, p. e0150751.

    Article  PubMed  PubMed Central  Google Scholar 

  34. Maslov, M.A., Kabilova, T.O., Petukhov, I.A., Morozova, N.G., Serebrennikova, G.A., Vlassov, V.V., and Zenkova, M.A., J. Control Release, 2012, vol. 160, pp. 182–193.

    CAS  Article  PubMed  Google Scholar 

  35. Muzio, M., Bosisio, D., Polentarutti, N., D’amico, G., Stoppacciaro, A., Mancinelli, R., Veer, C., Penton-Rol, G., Ruco, L.P., Allavena, P., and Mantovani, A., J. Immunol., 2000, vol. 164, pp. 5998–6004.

    CAS  Article  PubMed  Google Scholar 

  36. Ge, Q., Dallas, A., Ilves, H., Shorenstein, J., Behlke, M.A., and Johnston, B.H., RNA, 2010, vol. 16, pp. 118–130.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  37. Petrova, N.S., Chernikov, I.V., Meschaninova, M.I., Dovydenko, I.S., Venyaminova, A.G., Zenkova, M.A., Vlassov, V.V., and Chernolovskaya, E.L., Nucleic Acids Res., 2012, vol. 40, pp. 2330–2344.

    CAS  Article  PubMed  Google Scholar 

  38. Castanotto, D. and Rossi, J.J., Nature, 2009, vol. 457, pp. 426–433.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  39. Reynolds, A., Anderson, E.M., Vermeulen, A., Fedorov, Y., Robinson, K., Leake, D., Karpilow, J., Marshall, W.S., and Khvorova, A., RNA, 2006, vol. 12, pp. 988–993.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  40. Puthenveetil, S., Whitby, L., Ren, J., Kelnar, K., Krebs, J.F., and Beal, P.A., Nucleic Acids Res., 2006, vol. 34, pp. 4900–4911.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  41. Hu, Y. and Conway, T.W., J. Interferon Res., 1993, vol. 13, pp. 323–328.

    CAS  Article  PubMed  Google Scholar 

  42. Zhang, Z., Weinschenk, T., Guo, K., and Schluesener, H.J., J. Cell Biochem., 2006, vol. 97, pp. 1217–1229.

    CAS  Article  PubMed  Google Scholar 

  43. Bellon, L., in Curr. Protoc Nucleic Acid Chemistry, Beaucage, S.L., Bergstrom, D.E., Glick, G.D., and Jones, R.A., Eds., New York: John Wiley and Sons, Inc., 2001, pp. 3.6.1–3.6.13.

  44. Carmichael, J., DeGraff, W.G., Gazdar, A.F., Minna, J.D., and Mitchell, J.B., Cancer Res., 1987, vol. 47, pp. 936–942.

    CAS  PubMed  Google Scholar 

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Correspondence to E. L. Chernolovskaya.

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Original Russian Text © T.O. Kabilova, M.I. Meschaninova, A.G. Venyaminova, V.V. Vlassov, M.A. Zenkova, E.L. Chernolovskaya, 2017, published in Bioorganicheskaya Khimiya, 2017, Vol. 43, No. 1, pp. 59–67.

The paper is based on the materials of the “Chemical Biology 2016” conference; Novosibirsk, Russia, July 24–29, 2016.

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Kabilova, T.O., Meschaninova, M.I., Venyaminova, A.G. et al. Impact of chemical modifications in the structure of isRNA on its antiproliferative and immunostimulatory properties. Russ J Bioorg Chem 43, 50–57 (2017). https://doi.org/10.1134/S1068162017010046

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  • DOI: https://doi.org/10.1134/S1068162017010046

Keywords

  • immunostimulatory RNA
  • chemical modifications
  • cholesterol
  • transfection
  • pattern recognition receptors