AAV-mediated Establishment Of HBV Persistent Infection And Fibrosis Mouse Model And Antiviral Drug Evaluation

BackgroundChronic hepatitis B is a major infectious disease caused by Hepatitis B virus. Hepatitis B virus is mainly prevalent in China and some other Asian countries, and 10% of Chinese populations are hepatitis B surface antigen carriers. Hepatitis B virus infection can result in cirrhosis and liver cancer. Low efficacy, undesirable side effect and occurrence of resistance to HBV mutations are the major limitations of current drugs. Therefore, new antiviral strategies for improving the treatment of HBV infection are urgently needed.RNA interference is a process of siRNA directed sequence-specific post-transcriptional silencing of homologous genes. It is a useful strategy for developing specific gene-silencing therapeutics. Synthetic siRNA is expensive, unstable, transient effective, and difficult in transfer. So it is necessary to develop a new method to promote siRNA entry and trafficking, AAV(Adeno-associate virus) vector is a promoting gene delivery vector which can meet the requirements.AAV belongs to human parvovirdae. Since the first AAV infectious clone was established, recombinant AAV (rAAV2) vectors are widely used in gene therapy and preclinical/clinical trials. Recombinant AAV vector has been demonstrated a very promising gene delivery tool due to its no pathogenicity, low immunogenicity, the ability to transduce dividing and non-dividing cells, and the ability to mediate long-term transgene expression.The host range of hepatitis B virus is narrow and there is no suitable small animal model for evaluating the effectiveness of antiviral drugs, which prohibit their development. Therefore, developing a hepatitis B virus persistent infectious mouse model is critical for studying pathological mechanism of HBV chronic infection, screening and evaluation of new antiviral therapeutics.Objects:1.Generate HBV persistent infection mouse model.2.Construct AAV2-shRNA expression vectors and evaluate their antiviral efficiency in vitro.3.Evaluate the candidate AAV2-shRNA antiviral efficiency in HBV persistent infection mouse model.Methods:1. To establish a long-term HBV production mouse model, firstly,1.2 times genome-length HBV genome was cloned into the plasmid vector SSV9(rep-/cap-) for constucting plasmid pSSV9-1.2HBV; then recombinant vector was packaged into AAV8 by three plasmids co-transfection method; thirdly AAV8-1.2HBV was injected C57BL/6 mice by tail vein. Mouse serum and tissue were collected at different time points, and determined by qPCR, RT-qPCR, EL1SA, IHC stain, Masson stain and et al. The time-course of HBV DNA. HBsAg and HBeAg were detected in serum samples. HBV DNA, mRNA, HBsAg and HBcAg in liver were also investigated.2. ShRNA plasmid expression vector were constructed, and packaged into AAV2 vector by using three plasmids co-transfection method. HepG2.2.15 cell was infected with AAV2-shRNA to evaluate their antiviral efficiency. Culture supernatant and cell were collected at indicated time points and were investigated by qPCR, RT-qPCR, and ELISA.3. The best AAV2-shRNA was injected HBV persistent infection mouse model by tail vein. The mouse serum and tissue were collected at different time points, and were investigated as described in method 1. The antiviral efficiency of AAV2-shRNA was evaluated.Results:1. AAV vector effectively mediated HBV genome transfer and replication in mouse liver, and persistent over six months. HBV DNA content in mouse serum was up to 107copy/mL. HBsAg and HBeAg were also persistent in mouse serum, and the expression of HBsAg and HBcAg were observed in hepatocytes by IHC stain. AAV-1.2HBV injection mice did not mediate obvious acute inflammation but induced liver fibrosis.2. All AAV2-shRNA can inhibit HBV replication efficiently in vitro and demonstrated a dose-dependant manner, however there was no significant difference at MOI of higher than104. The antiviral efficiency of AAV2-shRNAl+3 was higher than that of AAV2-shRNA1 and AAV2-shRNAl+3+4.3. AAV2-shRNA1+3 showed higher antiviral efficiency than that of other AAV2-shRNAs in vivo, and the results were consistent with that of in vitro. The extents of liver fibrosis and hepatocyte fatty degeneration of model mouse were improved after the treatment of AAV2-shRNA.Conclusions:1. HBV persistent infection mouse model was successfully established. The novel mouse model did not show obvious acute inflammation but induced liver fibrosis. The novel mouse model holds the potential of screening and evaluating of antiviral and antifibrotic therapeutics.2. AAV2-shRNA1+3 could significantly inhibit HBV replication in vitro.3.AAV2-shRNA1+3 could effectively inhibit HBV replication in the novel mouse model and improved the extent of liver fibrosis and fatty degeneration. It provides an efficient alternative for curing chronic hepatitis B and liver cirrhosis.