medigraphic.com
Órgano Oficial de la Asociación Mexicana de Hepatología

2007, Number 3

<< Back Next >>

Ann Hepatol 2007; 6 (3)

Antiviral therapy: Inhibition of hepatitis C Virus expression by RNA interference directed against the NS5B region of the Viral Genome

Trejo-Ávila L, Elizondo-González R, Trujillo-Murillo KC, Zapata-Benavides P, Rodríguez-Padilla C, Rivas-Estilla AM

Language: English
References: 35
Page: 174-180
PDF size: 110.66 Kb.


ABSTRACT

Background: Hepatitis C virus (HCV) is a major public health problem with 170 million chronically infected people throughout the world. Currently, the only treatment available consists of a combination of pegylated interferon (INF-α) and ribavirin, but only half of the patients treated show a sufficient antiviral response. Thus there is a great need for the development of new treatments for HCV infections. RNA interference (RNAi) represents a new promising approach to develop effective antiviral drugs and has been extremely effective against HCV gene expression in short-term cell culture. Our aim was to determine the effect of RNAi directed against the NS5B-HCV region on HCV expression in a human hepatoma cell line that expresses HCV-subgenomic replicon (Huh7 HCV replicon cells). Methods: We transfected Huh7 HCV replicon cells with different concentrations of RNAi (100-200 nM) targeting the NS5B region of the viral genome. 2-6 days post-transfection HCV-RNA was quantified by semiquantitative and real-time RT-PCR, and HCV NS5B protein levels were assayed by western blot. Cell viability was also quantified by MTT assay. Results: Our results indicate that the NS5B-siRNAs used in this study can specifically inhibit HCV-RNA replication and protein expression (more than 90%) compared to control cells. Conclusions: Synthetic siRNA against NS5B-HCV inhibited HCV replication and viral proteins levels and thereby becomes a powerful strategy to combat hepatitis C virus.


REFERENCES

  1. Lieber CS. Alcohol and hepatitis C. Alcohol Res Health 2001; 25: 245-54.

  2. Poynard T, Yuen MF, Ratziu V, Lai CL. Viral hepatitis C. Lancet 2003; 362: 2095-100.

  3. Simmonds P, Bukh J, Combet C, Deleage G, Enomoto N, Feinstone S, Halfon P, Inchauspe G, Kuiken C, Maertens G, Mizokami M, Murphy DG, Okamoto H, Pawlotsky JM, Penin F, Sablon E, Shin IT, Stuyver LJ, Thiel HJ, Viazov S, Weiner AJ, Widell A. Consensus proposals for a unified system of nomenclature of hepatitis C virus genotypes. Hepatology 2005; 42: 962-73.

  4. Penin F, Dubuisson J, Rey FA, Moradpour D, Pawlotsky JM. Structural biology of hepatitis C virus. Hepatology 2004; 39: 5-19.

  5. Chisari FV. Unscrambling hepatitis C virus-host interactions. Nature 2005; 436: 930-2.

  6. Tellinghuisen TL, Rice CM. Interaction between hepatitis C virus proteins and host cell factors. Curr Opin Microbiol 2002; 5: 419-27.

  7. Bartenschlager R, Lohmann V. Novel cell culture systems for the hepatitis C virus. Antiviral Res 2001; 52: 1-17.

  8. Behrens SE, Tomei L, De Francesco R. Identification and properties of the RNA-dependent RNA polymerase of hepatitis C virus. Embo J 1996; 15: 12-22.

  9. Bressanelli S, Tomei L, Rey FA, De Francesco R. Structural analysis of the hepatitis C virus RNA polymerase in complex with ribonucleotides. J Virol 2002; 76: 3482-92.

  10. Sookoian SC. New therapies on the horizon for hepatitis C. Ann Hepatol 2003; 2: 164-70.

  11. Neyts J. Selective inhibitors of hepatitis C virus replication. Antiviral Res 2006; 71: 363-71.

  12. Zeuzem S, Heathcote JE, Martin N, Nieforth K, Modi M. Peginterferon alfa-2a (40 kDa) monotherapy: a novel agent for chronic hepatitis C therapy. Expert Opin Investig Drugs 2001; 10: 2201-13.

  13. Feld JJ, Hoofnagle JH. Mechanism of action of interferon and ribavirin in treatment of hepatitis C. Nature 2005; 436: 967-72.

  14. Barth H, Liang TJ, Baumert TF. Hepatitis C virus entry: molecular biology and clinical implications. Hepatology 2006; 44: 527-35.

  15. Lohmann V, Korner F, Koch J, Herian U, Theilmann L, Bartenschlager R. Replication of subgenomic hepatitis C virus RNAs in a hepatoma cell line. Science 1999; 285: 110-3.

  16. Bartenschlager R. Hepatitis C virus replicons: potential role for drug development. Nat Rev Drug Discov 2002; 1: 911-6.

  17. Bartenschlager R, Pietschmann T. Efficient hepatitis C virus cell culture system: what a difference the host cell makes. Proc Natl Acad Sci USA 2005; 102: 9739-40.

  18. Escuret V, Martin A, Durantel D, Parent R, Hantz O, Trepo C, Menguy T, Bottius E, Dardy J, Maral J, Escary JL, Zoulim F. Novel alpha interferon (IFN-alpha) variant with improved inhibitory activity against hepatitis C virus genotype 1 replication compared to IFN-alpha2b therapy in a subgenomic replicon system. Antimicrob Agents Chemother 2006; 50: 3984-91.

  19. Bumcrot D, Manoharan M, Koteliansky V, Sah DW. RNAi therapeutics: a potential new class of pharmaceutical drugs. Nat Chem Biol 2006; 2: 711-9.

  20. Scherer LJ, Rossi JJ. Approaches for the sequence-specific knockdown of mRNA. Nat Biotechnol 2003; 21: 1457-65.

  21. Schwarz DS, Hutvagner G, Haley B, Zamore PD. Evidence that siRNAs function as guides, not primers, in the Drosophila and human RNAi pathways. Mol Cell 2002; 10: 537-48.

  22. Wilson JA, Jayasena S, Khvorova A, Sabatinos S, Rodrigue-Gervais IG, Arya S, Sarangi F, Harris-Brandts M, Beaulieu S, Richardson CD. RNA interference blocks gene expression and RNA synthesis from hepatitis C replicons propagated in human liver cells. Proc Natl Acad Sci USA 2003; 100: 2783-8.

  23. Takigawa Y, Nagano-Fujii M, Deng L, Hidajat R, Tanaka M, Mizuta H, Hotta H. Suppression of hepatitis C virus replicon by RNA interference directed against the NS3 and NS5B regions of the viral genome. Microbiol Immunol 2004; 48: 591-8.

  24. Hamazaki H, Ujino S, Miyano-Kurosaki N, Shimotohno K, Takaku H. Inhibition of hepatitis C virus RNA replication by short hairpin RNA synthesized by T7 RNA polymerase in hepatitis C virus subgenomic replicons. Biochem Biophys Res Commun 2006; 343: 988-94.

  25. Lindsay KL, Trepo C, Heintges T, Shiffman ML, Gordon SC, Hoefs JC, Schiff ER, Goodman ZD, Laughlin M, Yao R, Albrecht JK. A randomized, double-blind trial comparing pegylated interferon alfa-2b to interferon alfa-2b as initial treatment for chronic hepatitis C. Hepatology 2001; 34: 395-403.

  26. Fried MW, Shiffman ML, Reddy KR, Smith C, Marinos G, Goncales FL, Jr., Haussinger D, Diago M, Carosi G, Dhumeaux D, Craxi A, Lin A, Hoffman J, Yu J. Peginterferon alfa-2a plus ribavirin for chronic hepatitis C virus infection. N Engl J Med 2002; 347: 975-82.

  27. Pause A, Kukolj G, Bailey M, Brault M, Do F, Halmos T, Lagace L, Maurice R, Marquis M, McKercher G, Pellerin C, Pilote L, Thibeault D, Lamarre D. An NS3 serine protease inhibitor abrogates replication of subgenomic hepatitis C virus RNA. J Biol Chem 2003; 278: 20374-80.

  28. Perni RB, Almquist SJ, Byrn RA, Chandorkar G, Chaturvedi PR, Courtney LF, Decker CJ, Dinehart K, Gates CA, Harbeson SL, Heiser A, Kalkeri G, Kolaczkowski E, Lin K, Luong YP, Rao BG, Taylor WP, Thomson JA, Tung RD, Wei Y, Kwong AD, Lin C. Preclinical profile of VX-950, a potent, selective, and orally bioavailable inhibitor of hepatitis C virus NS3-4A serine protease. Antimicrob Agents Chemother 2006; 50: 899-909.

  29. Nakagawa M, Sakamoto N, Enomoto N, Tanabe Y, Kanazawa N, Koyama T, Kurosaki M, Maekawa S, Yamashiro T, Chen CH, Itsui Y, Kakinuma S, Watanabe M. Specific inhibition of hepatitis C virus replication by cyclosporin A. Biochem Biophys Res Commun 2004; 313: 42-7.

  30. Paeshuyse J, Kaul A, De Clercq E, Rosenwirth B, Dumont JM, Scalfaro P, Bartenschlager R, Neyts J. The non-immunosuppressive cyclosporin DEBIO-025 is a potent inhibitor of hepatitis C virus replication in vitro. Hepatology 2006; 43: 761-70.

  31. Xia H, Mao Q, Paulson HL, Davidson BL. siRNA-mediated gene silencing in vitro and in vivo. Nat Biotechnol 2002; 20: 1006-10.

  32. McCaffrey AP, Meuse L, Pham TT, Conklin DS, Hannon GJ, Kay MA. RNA interference in adult mice. Nature 2002; 418: 38-9.

  33. Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl T. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 2001; 411: 494-8.

  34. Gu W, Putral L, Hengst K, Minto K, Saunders NA, Leggatt G, McMillan NA. Inhibition of cervical cancer cell growth in vitro and in vivo with lentiviral-vector delivered short hairpin RNA targeting human papillomavirus E6 and E7 oncogenes. Cancer Gene Ther 2006; 13: 1023-32.

  35. Fuchs U, Borkhardt A. The application of siRNA technology to cancer biology discovery. Adv Cancer Res 2007; 96: 75-102.




2020     |     www.medigraphic.com