Skip to main content
Journal of Virology logoLink to Journal of Virology
. 1996 Dec;70(12):8782–8791. doi: 10.1128/jvi.70.12.8782-8791.1996

Elimination of hepatitis C virus RNA in infected human hepatocytes by adenovirus-mediated expression of ribozymes.

A Lieber 1, C Y He 1, S J Polyak 1, D R Gretch 1, D Barr 1, M A Kay 1
PMCID: PMC190975  PMID: 8971007

Abstract

Hepatitis C virus (HCV), a positive-strand RNA virus, is the major infectious agent responsible for causing chronic hepatitis. Currently, there is no vaccine for HCV infection, and the only therapy for chronic hepatitis C is largely ineffective. To investigate new genetic approaches to the management of HCV infection, six hammerhead ribozymes directed against a conserved region of the plus strand and minus strand of the HCV genome were isolated from a ribozyme library, characterized, and expressed from recombinant adenovirus vectors. The expressed ribozymes individually or in combination were efficient at reducing or eliminating the respective plus- or minus-strand HCV RNAs expressed in cultured cells and from primary human hepatocytes obtained from chronic HCV-infected patients. This study demonstrates the potential utility of ribozyme therapy as a strategy for the treatment of hepatitis C virus infection.

Full Text

The Full Text of this article is available as a PDF (583.9 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Alt M., Renz R., Hofschneider P. H., Paumgartner G., Caselmann W. H. Specific inhibition of hepatitis C viral gene expression by antisense phosphorothioate oligodeoxynucleotides. Hepatology. 1995 Sep;22(3):707–717. [PubMed] [Google Scholar]
  2. Alter M. J., Margolis H. S., Krawczynski K., Judson F. N., Mares A., Alexander W. J., Hu P. Y., Miller J. K., Gerber M. A., Sampliner R. E. The natural history of community-acquired hepatitis C in the United States. The Sentinel Counties Chronic non-A, non-B Hepatitis Study Team. N Engl J Med. 1992 Dec 31;327(27):1899–1905. doi: 10.1056/NEJM199212313272702. [DOI] [PubMed] [Google Scholar]
  3. Brown E. A., Zhang H., Ping L. H., Lemon S. M. Secondary structure of the 5' nontranslated regions of hepatitis C virus and pestivirus genomic RNAs. Nucleic Acids Res. 1992 Oct 11;20(19):5041–5045. doi: 10.1093/nar/20.19.5041. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bukh J., Purcell R. H., Miller R. H. Sequence analysis of the 5' noncoding region of hepatitis C virus. Proc Natl Acad Sci U S A. 1992 Jun 1;89(11):4942–4946. doi: 10.1073/pnas.89.11.4942. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Choo Q. L., Richman K. H., Han J. H., Berger K., Lee C., Dong C., Gallegos C., Coit D., Medina-Selby R., Barr P. J. Genetic organization and diversity of the hepatitis C virus. Proc Natl Acad Sci U S A. 1991 Mar 15;88(6):2451–2455. doi: 10.1073/pnas.88.6.2451. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Davis G. L. Interferon treatment of chronic hepatitis C. Am J Med. 1994 Jan 17;96(1A):41S–46S. doi: 10.1016/0002-9343(94)90189-9. [DOI] [PubMed] [Google Scholar]
  7. Durnam D. M., Palmiter R. D. A practical approach for quantitating specific mRNAs by solution hybridization. Anal Biochem. 1983 Jun;131(2):385–393. doi: 10.1016/0003-2697(83)90188-4. [DOI] [PubMed] [Google Scholar]
  8. Fong T. L., Shindo M., Feinstone S. M., Hoofnagle J. H., Di Bisceglie A. M. Detection of replicative intermediates of hepatitis C viral RNA in liver and serum of patients with chronic hepatitis C. J Clin Invest. 1991 Sep;88(3):1058–1060. doi: 10.1172/JCI115368. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Grakoui A., Wychowski C., Lin C., Feinstone S. M., Rice C. M. Expression and identification of hepatitis C virus polyprotein cleavage products. J Virol. 1993 Mar;67(3):1385–1395. doi: 10.1128/jvi.67.3.1385-1395.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Gretch D. R., Bacchi C. E., Corey L., dela Rosa C., Lesniewski R. R., Kowdley K., Gown A., Frank I., Perkins J. D., Carithers R. L., Jr Persistent hepatitis C virus infection after liver transplantation: clinical and virological features. Hepatology. 1995 Jul;22(1):1–9. [PubMed] [Google Scholar]
  11. Han J. H., Shyamala V., Richman K. H., Brauer M. J., Irvine B., Urdea M. S., Tekamp-Olson P., Kuo G., Choo Q. L., Houghton M. Characterization of the terminal regions of hepatitis C viral RNA: identification of conserved sequences in the 5' untranslated region and poly(A) tails at the 3' end. Proc Natl Acad Sci U S A. 1991 Mar 1;88(5):1711–1715. doi: 10.1073/pnas.88.5.1711. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Houghton M., Weiner A., Han J., Kuo G., Choo Q. L. Molecular biology of the hepatitis C viruses: implications for diagnosis, development and control of viral disease. Hepatology. 1991 Aug;14(2):381–388. [PubMed] [Google Scholar]
  13. Kay M. A., Graham F., Leland F., Woo S. L. Therapeutic serum concentrations of human alpha-1-antitrypsin after adenoviral-mediated gene transfer into mouse hepatocytes. Hepatology. 1995 Mar;21(3):815–819. [PubMed] [Google Scholar]
  14. Koziel M. J., Dudley D., Wong J. T., Dienstag J., Houghton M., Ralston R., Walker B. D. Intrahepatic cytotoxic T lymphocytes specific for hepatitis C virus in persons with chronic hepatitis. J Immunol. 1992 Nov 15;149(10):3339–3344. [PubMed] [Google Scholar]
  15. Lanford R. E., Chavez D., Chisari F. V., Sureau C. Lack of detection of negative-strand hepatitis C virus RNA in peripheral blood mononuclear cells and other extrahepatic tissues by the highly strand-specific rTth reverse transcriptase PCR. J Virol. 1995 Dec;69(12):8079–8083. doi: 10.1128/jvi.69.12.8079-8083.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Lieber A., Kay M. A. Adenovirus-mediated expression of ribozymes in mice. J Virol. 1996 May;70(5):3153–3158. doi: 10.1128/jvi.70.5.3153-3158.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Lieber A., Sandig V., Sommer W., Bähring S., Strauss M. Stable high-level gene expression in mammalian cells by T7 phage RNA polymerase. Methods Enzymol. 1993;217:47–66. doi: 10.1016/0076-6879(93)17055-a. [DOI] [PubMed] [Google Scholar]
  18. Lieber A., Sandig V., Strauss M. A mutant T7 phage promoter is specifically transcribed by T7-RNA polymerase in mammalian cells. Eur J Biochem. 1993 Oct 1;217(1):387–394. doi: 10.1111/j.1432-1033.1993.tb18257.x. [DOI] [PubMed] [Google Scholar]
  19. Lieber A., Strauss M. Selection of efficient cleavage sites in target RNAs by using a ribozyme expression library. Mol Cell Biol. 1995 Jan;15(1):540–551. doi: 10.1128/mcb.15.1.540. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Lieber A., Vrancken Peeters M. J., Kay M. A. Adenovirus-mediated transfer of the amphotropic retrovirus receptor cDNA increases retroviral transduction in cultured cells. Hum Gene Ther. 1995 Jan;6(1):5–11. doi: 10.1089/hum.1995.6.1-5. [DOI] [PubMed] [Google Scholar]
  21. Martinot-Peignoux M., Marcellin P., Pouteau M., Castelnau C., Boyer N., Poliquin M., Degott C., Descombes I., Le Breton V., Milotova V. Pretreatment serum hepatitis C virus RNA levels and hepatitis C virus genotype are the main and independent prognostic factors of sustained response to interferon alfa therapy in chronic hepatitis C. Hepatology. 1995 Oct;22(4 Pt 1):1050–1056. [PubMed] [Google Scholar]
  22. Negro F., Pacchioni D., Shimizu Y., Miller R. H., Bussolati G., Purcell R. H., Bonino F. Detection of intrahepatic replication of hepatitis C virus RNA by in situ hybridization and comparison with histopathology. Proc Natl Acad Sci U S A. 1992 Mar 15;89(6):2247–2251. doi: 10.1073/pnas.89.6.2247. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Nouri Aria K. T., Sallie R., Sangar D., Alexander G. J., Smith H., Byrne J., Portmann B., Eddleston A. L., Williams R. Detection of genomic and intermediate replicative strands of hepatitis C virus in liver tissue by in situ hybridization. J Clin Invest. 1993 May;91(5):2226–2234. doi: 10.1172/JCI116449. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Poeschla E., Wong-Staal F. Antiviral and anticancer ribozymes. Curr Opin Oncol. 1994 Nov;6(6):601–606. doi: 10.1097/00001622-199411000-00012. [DOI] [PubMed] [Google Scholar]
  25. Shimizu Y. K., Purcell R. H., Yoshikura H. Correlation between the infectivity of hepatitis C virus in vivo and its infectivity in vitro. Proc Natl Acad Sci U S A. 1993 Jul 1;90(13):6037–6041. doi: 10.1073/pnas.90.13.6037. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Simmonds P. Variability of hepatitis C virus. Hepatology. 1995 Feb;21(2):570–583. doi: 10.1002/hep.1840210243. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Sullivan S. M. Development of ribozymes for gene therapy. J Invest Dermatol. 1994 Nov;103(5 Suppl):85S–89S. doi: 10.1038/jid.1994.15. [DOI] [PubMed] [Google Scholar]
  28. Tsukiyama-Kohara K., Iizuka N., Kohara M., Nomoto A. Internal ribosome entry site within hepatitis C virus RNA. J Virol. 1992 Mar;66(3):1476–1483. doi: 10.1128/jvi.66.3.1476-1483.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Vrancken Peeters M. J., Perkins A. L., Kay M. A. Method for multiple portal vein infusions in mice: quantitation of adenovirus-mediated hepatic gene transfer. Biotechniques. 1996 Feb;20(2):278–285. doi: 10.2144/96202rr05. [DOI] [PubMed] [Google Scholar]
  30. Wakita T., Wands J. R. Specific inhibition of hepatitis C virus expression by antisense oligodeoxynucleotides. In vitro model for selection of target sequence. J Biol Chem. 1994 May 13;269(19):14205–14210. [PubMed] [Google Scholar]
  31. Wang J. T., Sheu J. C., Lin J. T., Wang T. H., Chen D. S. Detection of replicative form of hepatitis C virus RNA in peripheral blood mononuclear cells. J Infect Dis. 1992 Nov;166(5):1167–1169. doi: 10.1093/infdis/166.5.1167. [DOI] [PubMed] [Google Scholar]
  32. Weiner A., Erickson A. L., Kansopon J., Crawford K., Muchmore E., Hughes A. L., Houghton M., Walker C. M. Persistent hepatitis C virus infection in a chimpanzee is associated with emergence of a cytotoxic T lymphocyte escape variant. Proc Natl Acad Sci U S A. 1995 Mar 28;92(7):2755–2759. doi: 10.1073/pnas.92.7.2755. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Journal of Virology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES