Effects Of RNA Interference Mediated By Recombinant Adeno-Associated Virus On HBV Replication And Expression

Hepatitis B virus (hepatitis B virus, HBV) infection is a serious global public health problem.About3.5million of the world’s population are troubled with chronic HBV infection, and about one million people die from HBV infection related diseases every year, Including hepatitis, cirrhosis, liver failure, and hepatocellular carcinoma (HCC). Currently the treatment of HBV infection, includes interferon, nucleoside and nucleotide analogs;however, these drugs can produce only a small number of chronically infected long-term response, does not completely clear the hepatitis B virus;besides,there are problems such as a high rate of recurrence after treatment,drug side effects and high cost, therefore we must find a more effective treatment means.RNA interference, known as the small molecule double-stranded (dsRNA) homologous with the target gene is introduced into cells, leading to purpose mRNA degradation, thereby blocking the expression of the corresponding gene products in eukaryotic cells. It has the characteristics including high inhibition efficiency, specificity, and easier cellular uptake etc, becoming a new molecular biology technique. But at the same time,it also has the defects of short duration.To solve this problem, This experiment used adeno-associated virus as a vector, with the advantages of high stability、high targeting low pathogenicity、low immunogenicity、wide host range、long-term expression, etc,as the vector of shRNA anti-HBV expression box, then transfected into HepG2.2.15cells (HCC cells inserted into HBV gene), observing the effects on HBV replication and expression, trying to solve the problem that RNAi technology anti-HBV action time is short.The expression box of hu6-shRNA was placed between two ITRs of AAV, then connected with basic core promoter (BCP) of HBV and BCP driving Rep Gene of AAV, and thus constituted rAAV. As the vector of shRNA anti-HBV expression box, the rAAV was transfected into HepG2.2.15cells (HCC cells inserted into HBV gene). The expressions of HBsAg and HBeAg and replications of HBV-DNA in the cultured supernate were detected on the1st,2nd,3rd and10th day after transfection and the cell genomes in the AAVS1region were sequenced on the3rd day after transfection. The test showed that the target sequenced vectors of PLRBR322-324, PLRBR522-324, PLRBR322-2424and PLRBR522-2424were successfully constructed. The experiments with plasmid transfected cells in vitro show that all the four vectors have inhibiting effects on expressions of HBsAg and HBeAg, and on HBV-DNA replications with the former two, namely, PLRBR322-324and PLRBR522-324, having more significant inhibitory effect on the HBsAg expression and the latter two on HBeAg expression and the third on HBV-DNA replication. The inhibitory effects of HBsAg, HBeAg expression and HBV-DNA replication were the most obvious on the3rd day after transfection, and the inhibition rate remained high on the10th day. The result of sequencing cell genome in the AAVS1region showed that the site-directed integration of the target sequence was located in this region.These above results indicate that the rAAV constructed by several elements of AAV and HBV, serving shRNA expressing box of anti-HBV as the vector together with the help of site-directed integration mediated by Rep protein, is a good exploration to solve the problem of RNAi short-term effect against HBV.