Construction And Expression Of Anti-hbv Effects Of Antisense Rna And Dominant-negative Mutant Replication Defect Of Hbv

Chronic infection with the hepatitis B virus (I-IBV) is a major problem of public health and a common disease in our country, and currently available therapies have limited efficacy. New antiviral approaches are needed to reduce the risk of developing chronic liver disease and hepatocellular carcinoma. Gene therapy, which has become one of the most attractive antiviral strategies, is bringing a promising light. But HBV gene therapy poses formidable obstacles to gene delivery. The more intractable problem is the development of methods to target the infected tissues or cell type and express efficiently therein. It is impossible for the present vectors to target so many HBV infected liver cells. HBV has many distinct features that make it attractive candidates as vectors for gene therapy of acquired liver diseases. Viral gene expression is directed by hepatocyte-specific promoter-enhancer elements, and, it establishes a stable episomal transcription template. It can selectively target the liver, and it efficiently infects quiescent hepatocytes. If it may express anti-HBV products and meanwhile be packaged, the antiviral function will be amplified in vivo. The fact that a large number of HBV-infected humans appear continuously to harbor the HBV genome and express HBsAg without pathogenic consequences offers a great advantage in terms of long-term maintenance and expression of a recombinant HBV vector bearing a desired gene. A recombinant HBV genome might be attenuated to the extent that the HBV replication level is reduced. So the limitation of other vectors for HBV gene therapy might be overcome. In this study, the HBV genome was manipulated to express antisense RNA or/and dominant negative mutants of HBV core protein. Transducted by EB virus 111 vector or retrovirus vector, the constructs was tested whether it has anti-HBV effects and can still be packaged in helper cell lines. Methods 1. The HBV genome was modified as follows: (1) partial S gene was reversedly recombined back into HBV genome in order to express antisense RNA complementary to S region; (2) S promoter region was reversedly recombined back into HBV genome in order to express antisense RNA complementary to the S promoter region; (3) two nucleotide acids were inserted at BsrBR I site (in the superposition region of core and the P protein) in order to express a fusion protein of core and partial P protein with a stop codon at EcoRI site; (4) BsrBR Ito EcoRI fragment was removed in order to express a fusion protein of core and partial HBsAg; (5) two nucleotide acids were inserted at BsrBR I site as stated above, and then partial S region was reversedly recombined back, which expressed a fusion protein of core and partial P protein combined with antisense RNA complementary to partial S region. 2. The modified HBV genomes in head-to-tail configuration were inserted into the pMEP4 vector with a hygromycin-resistance gene. Accordingly, five constructs were established as follows: (1) pMEP-Sas, which expressed antisense RNA complementary to partial S region of HBV genome; (2) pMEP-Pas, which expressed antisense RNA complementary to S promoter region; (3) pMEP-CP, which expressed a fusion protein of core and partial P protein; (4) pMEP-CS, which expressed a fusion protein of core and HBsAg; (5) pMEP-CPAS, which expressed a fusion protein of core and p...