BMB Reports

Korean Society for Biochemistry and Molecular Biology (생화학분자생물학회)
권호 표지
DOI QR코드

DOI QR Code

Inhibition of HBV replication and gene expression in vitro and in vivo with a single AAV vector delivering two shRNA molecules

  • Li, Zhi (Department of Microbiology, The University of Hong Kong) ;
  • He, Ming-Liang (Stanley Ho Center for Emerging Infectious Diseases, and Li Ka Shing Institute of Health Sciences, The Chinese University of Hong Kong) ;
  • Yao, Hong (Stanley Ho Center for Emerging Infectious Diseases, and Li Ka Shing Institute of Health Sciences, The Chinese University of Hong Kong) ;
  • Dong, Qing-Ming (Stanley Ho Center for Emerging Infectious Diseases, and Li Ka Shing Institute of Health Sciences, The Chinese University of Hong Kong) ;
  • Chen, Yang-Chao (Stanley Ho Center for Emerging Infectious Diseases, and Li Ka Shing Institute of Health Sciences, The Chinese University of Hong Kong) ;
  • Chan, Chu-Yan (Stanley Ho Center for Emerging Infectious Diseases, and Li Ka Shing Institute of Health Sciences, The Chinese University of Hong Kong) ;
  • Zheng, Bo-Jian (Department of Microbiology, The University of Hong Kong) ;
  • Yuen, Kwok-Yung (Department of Microbiology, The University of Hong Kong) ;
  • Peng, Ying (The Department of Neurology, The Second Affiliated Hospital, Sun Yat-sen University) ;
  • Sun, Qiang (Genetic Laboratory of Development and Diseases, Institute of Biotechnology) ;
  • Yang, Xiao (Genetic Laboratory of Development and Diseases, Institute of Biotechnology) ;
  • Lin, Marie C. (Department of Chemistry, The University of Hong Kong) ;
  • Sung, Joseph J.Y. (Stanley Ho Center for Emerging Infectious Diseases, and Li Ka Shing Institute of Health Sciences, The Chinese University of Hong Kong) ;
  • Kung, Hsiang-Fu (Stanley Ho Center for Emerging Infectious Diseases, and Li Ka Shing Institute of Health Sciences, The Chinese University of Hong Kong)
  • Published : 2009.01.31
Download PDF
⟨ Previous Next ⟩

Abstract

Hepatitis B virus (HBV) infection is highly prevalent worldwide. The major challenge for current antiviral treatment is the elevated drug resistance that occurs via rapid viral mutagenesis. In this study, we developed AAV vectors to simultaneously deliver two or three shRNAs targeting different HBV-related genes. These vectors showed markedly better antiviral effects than ones that delivered a single shRNA in vitro. A dual shRNA expression vector (AAV-157i/1694i), which simultaneously expressed two shRNAs targeted the S and X genes of HBV, reduced HBsAg, HBeAg and HBV DNA levels by $87{\pm}4$, $80.3{\pm}2.6$ and $86.2{\pm}7%$ respectively, eight days post-transduction. In a mouse model of prophylactic treatment, HBsAg and HBeAg were reduced to undetectable levels and the serum HBV DNA level was reduced by at least 100 fold. These results indicate that AAV-157i/1694i generates potent anti-HBV effects and that the strategy of constructing multi-shRNA expression vectors may lead to enhanced anti-HBV efficacy and overcome the evading mechanism of the virus and thus the development of drug resistance.

Keywords

References

  1. Wright T. L. (2006) Introduction to chronic hepatitis B infection. Am. J. Gastroenterol. 101, 1-6 https://doi.org/10.1111/j.1572-0241.2006.00460.x
  2. Lee, S. and Kim, S. (2007) Gene regulations in HBV-related liver cirrhosis closely correlate with disease severity. J. Biochem. Mol. Biol. 40, 814-824 https://doi.org/10.5483/BMBRep.2007.40.5.814
  3. Chan, H. L., Hui, A. Y., Wong, V. W., Chim, A. M., Wong, M. L. and Sung J. J. (2005) Long-term follow-up of peginterferon and lamivudine combination treatment in HBeAg-positive chronic hepatitis B. Hepatology 41, 1357-1364 https://doi.org/10.1002/hep.20695
  4. Buti, M., Rodriguez-Frias, F., Jardi, R. and Esteban, R. (2005) Hepatitis B virus genome variability and disease progression: the impact of pre-core mutants and HBV genotypes. J. Clin. Virol. 34 (Suppl 1), S79-S82 https://doi.org/10.1016/S1386-6532(05)80015-0
  5. Tillmann H. L. (2007) Antiviral therapy and resistance with hepatitis B virus infection. World J. Gastroenterol. 13, 125-140 https://doi.org/10.3748/wjg.v13.i1.125
  6. McManus, M. T. and Sharp, P. A. (2002) Gene silencing in mammals by small interfering RNAs. Nat. Rev. Genet. 3, 737-747 https://doi.org/10.1038/nrg908
  7. Wilson, J. A. and Richardson, C. D. (2005) Hepatitis C virus replicons escape RNA interference induced by a short interfering RNA directed against the NS5b coding region. J. Virol. 79, 7050-7058 https://doi.org/10.1128/JVI.79.11.7050-7058.2005
  8. Morris, K. V. and Rossi, J. J. (2006) Lentivirus-Mediated RNA Interference Therapy for Human Immunodeficiency Virus Type 1 Infection. Hum. Gene. Ther. 17, 479-486 https://doi.org/10.1089/hum.2006.17.479
  9. Novina, C. D., Murray, M. F., Dykxhoorn, D. M., Beresford, P. J., Riess, J., Lee, S. K., Collman, R. G., Lieberman, J., Shankar, P. and Sharp, P. A. (2002) siRNA-directed inhibition of HIV-1 infection. Nat. Med. 8, 681-686
  10. He, M. L., Zheng, B., Peng, Y., Peiris, J. S., Poon, L. L., Yuen, K. Y., Lin, M. C., Kung, H. F. and Guan, Y. (2003) Inhibition of SARS-associated coronavirus infection and replication by RNA interference. Jama 290, 2665-2666 https://doi.org/10.1001/jama.290.20.2665
  11. Mitchell, D. A., O'Donnell, J., Hare, J. T. and Chapman, M. S. (2006) Serotype-specific detection of adeno- associated virus during laboratory preparation. J. Virol. Methods. 136, 277-282 https://doi.org/10.1016/j.jviromet.2006.05.012
  12. Chen, Y., Du, D., Wu, J., Chan, C. P., Tan, Y., Kung, H. F. and He, M. L. (2003) Inhibition of hepatitis B virus replication by stably expressed shRNA. Biochem. Biophys. Res. Commun. 311, 398-404 https://doi.org/10.1016/j.bbrc.2003.10.009
  13. McCaffrey, A. P., Nakai, H., Pandey, K., Huang, Z., Salazar, F. H., Xu, H., Wieland, S. F., Marion, P. L. and Kay, M. A. (2003) Inhibition of hepatitis B virus in mice by RNA interference. Nat. Biotechnol. 21, 639-644 https://doi.org/10.1038/nbt824
  14. Wu, H. L., Huang, L. R., Huang, C. C., Lai, H. L., Liu, C. J., Huang, Y. T., Hsu, Y. W., Lu, C. Y., Chen, D. S. and Chen, P. J. (2005) RNA interference-mediated control of hepatitis B virus and emergence of resistant mutant. Gastroenterology 128, 708-7169 https://doi.org/10.1053/j.gastro.2004.12.007
  15. Sun, Y., Li, Z., Li, L., Li, J., Liu, X. and Li, W. (2007) Effective inhibition of hepatitis B virus replication by small interfering RNAs expressed from human foamy virus vectors. Int. J. Mol. Med. 19, 705-711
  16. Das, A. T., Brummelkamp, T. R., Westerhout, E. M., Vink, M., Madiredjo, M., Bernards, R. and Berkhout, B. (2004) Human immunodeficiency virus type 1 escapes from RNA interference-mediated inhibition. J. Virol. 78, 2601-2605 https://doi.org/10.1128/JVI.78.5.2601-2605.2004
  17. Westerhout, E. M., Ooms, M., Vink, M., Das, A. T. and Berkhout, B. (2005) HIV-1 can escape from RNA interference by evolving an alternative structure in its RNA genome. Nucleic Acids Res. 33, 796-804 https://doi.org/10.1093/nar/gki220
  18. Merten, O. W., Geny-Fiamma, C. and Douar, A. M. (2005) Current issues in adeno-associated viral vector production. Gene Ther. 12 Suppl 1, S51-S61 https://doi.org/10.1038/sj.gt.3302615
  19. Du, L., Kido, M., Lee, D. V., Rabinowitz, J. E., Samulski, R. J., Jamieson, S. W., Weitzman, M. D. and Thistlethwaite, P. A. (2004) Differential myocardial gene delivery by recombinant serotype-specific adeno-associated viral vectors. Mol. Ther. 10, 604-608 https://doi.org/10.1016/j.ymthe.2004.06.110
  20. Rabinowitz, J. E. and Samulski, J. (1998) Adeno-associated virus expression systems for gene transfer. Curr. Opin. Biotechnol. 9, 470-475 https://doi.org/10.1016/S0958-1669(98)80031-1
  21. Carter B. J. (2005) Adeno-associated virus vectors in clinical trials. Hum. Gene Ther. 16, 541-550 https://doi.org/10.1089/hum.2005.16.541
  22. Xu, R., Sun, X., Tse, L. Y., Li, H., Chan, P. C., Xu, S., Xiao, W., Kung, H. F., Krissansen, G. W. and Fan, S. T. (2003) Long-term expression of angiostatin suppresses metastatic liver cancer in mice. Hepatology 37, 1451-1460 https://doi.org/10.1053/jhep.2003.50244
  23. Li, Z., Yao, H., Ma, Y., Dong, Q., Chen, Y., Peng, Y., Zheng, B.J., Huang, J.D., Chan, C.Y., Lin, M.C., Sung, J.J., Yuen, K.Y., Kung, H.F.and He, M.L. (2008) Inhibition of HBV gene expression and replication by stably expressed interferon-alpha1 via adeno-associated viral vectors. J. Gene Med. 6, 619-627
  24. He, M. L., Wu, J., Chen, Y., Lin, M. C., Lau, G. K. and Kung, H. F. (2002) A new and sensitive method for the quantification of HBV cccDNA by real-time PCR. Biochem. Biophys. Res. Commun. 295, 1102-1107 https://doi.org/10.1016/S0006-291X(02)00813-6
  25. Grieger, J. C. and Samulski, R. J. (2005) Adeno-associated virus as a gene therapy vector: vector development, production and clinical applications. Adv. Biochem. Eng. Biotechnol. 99, 119-145 https://doi.org/10.1007/10_005
  26. Jiang, H., Pierce, G. F., Ozelo, M. C., de Paula, E. V., Vargas, J. A., Smith, P., Sommer, J., Luk, A., Manno, C. S., High, K. A. and Arruda, V. R. (2006) Evidence of multiyear factor IX expression by AAV-mediated gene transfer to skeletal muscle in an individual with severe hemophilia B. Mol. Ther. 14, 452-455 https://doi.org/10.1016/j.ymthe.2006.05.004
  27. Le Bec, C. and Douar, A. M. (2006) Gene therapy progress and prospects--vectorology: design and production of expression cassettes in AAV vectors. Gene Ther. 13, 805-813 https://doi.org/10.1038/sj.gt.3302724
  28. McPhee, S. W., Janson, C. G., Li, C., Samulski, R. J., Camp, A. S., Francis, J., Shera, D., Lioutermann, L., Feely, M., Freese, A. and Leone, P. (2006) Immune responses to AAV in a phase I study for Canavan disease. J. Gene. Med. 8, 577-588 https://doi.org/10.1002/jgm.885
  29. Streck, C. J., Dickson, P. V., Ng, C. Y., Zhou, J., Hall, M. M., Gray, J. T., Nathwani, A. C. and Davidoff, A. M. (2006) Antitumor efficacy of AAV-mediated systemic delivery of interferon-beta. Cancer Gene Ther. 13, 99-106 https://doi.org/10.1038/sj.cgt.7700878
  30. Ohashi, K., Nakai, H., Couto, L.B. and Kay, M.A. (2005) Modified infusion procedures affect recombinant adeno-associated virus vector type 2 transduction in the liver. Hum. Gene Ther. 16, 299-306 https://doi.org/10.1089/hum.2005.16.299
  31. Li, X. P., Li, C. Y., Li, X., Ding, Y., Chan, L. L., Yang, P. H., Li, G., Liu, X., Lin, J. S., Wang, J., He, M., Kung, H. F., Lin, M. C. and Peng, Y. (2006) Inhibition of human nasopharyngeal carcinoma growth and metastasis in mice by adenovirus-associated virus-mediated expression of human endostatin. Mol. Cancer. Ther. 5, 1290-1298 https://doi.org/10.1158/1535-7163.MCT-05-0348
  32. Ladner, S. K., Otto, M. J., Barker, C. S., Zaifert, K., Wang, G. H., Guo, J. T., Seeger, C., King, R. W. (1997) Inducible Expression of Human Hepatitis B Virus ( HBV) in Stably Transfected Hepatoblastoma Cells:A Novel System for Screening Potential Inhibitors of HBV Rep2 lication. Antimicrob. Agents Chemother. 41, 1715-1720
  33. Yang, P. L., Althage, A., Chung, J. and Chisari, F. V. (2002) Hydrodynamic injection of viral DNA: a mouse model of acute hepatitis B virus infection. Proc. Natl. Acad. Sci. U.S.A. 99, 13825-13830 https://doi.org/10.1073/pnas.202398599

Cited by

  1. AAV-mediated gene therapy for liver diseases: the prime candidate for clinical application? vol.11, pp.3, 2011, https://doi.org/10.1517/14712598.2011.548799
  2. Effects of HBV Genetic Variability on RNAi Strategies vol.2011, 2011, https://doi.org/10.1155/2011/367908
  3. Enhanced antiviral and antifibrotic effects of short hairpin RNAs targeting HBV and TGF-β in HBV-persistent mice vol.7, pp.1, 2017, https://doi.org/10.1038/s41598-017-04170-1
  4. Controllable inhibition of hepatitis B virus replication by a DR1-targeting short hairpin RNA (shRNA) expressed from a DOX-inducible lentiviral vector vol.46, pp.3, 2013, https://doi.org/10.1007/s11262-013-0886-2
  5. The life cycle of hepatitis B virus and antiviral targets vol.6, pp.5, 2011, https://doi.org/10.2217/fvl.11.29