Development of AAV-mediated RNAi therapy for chronic hepatitis B infection in transgenic and human liver chimeric mice models

• Main Authors: Cheng-Pu Sun, 孫承溥
• Other Authors: Mi-Hua Tao
• Format: Others
• Language: en_US
• Published: 2013
• Online Access: http://ndltd.ncl.edu.tw/handle/29325436781684656815

博士 === 國立陽明大學 === 生化暨分子生物研究所 === 101 === Hepatitis B virus (HBV) is a major human pathogen that causes acute liver disease and chronic infection. More than 350 million people worldwide are estimated to be chronically infected with HBV and at high risk of developing liver failure, cirrhosis, and hepatocellular carcinoma (HCC). Current anti-HBV therapies, including IFN- and nucleoside and nucleotide analogues, have limited effectiveness in complete elimination of viral DNA templates and their use is usually accompanied by selection of drug-resistant mutations and a high rate of relapse when treatment is discontinued. Thus, the development of new treatment strategies for chronic HBV remains a major medical challenge. The main purpose of the present study is to develop efficient and safe adeno-associated virus (AAV)-mediated RNA interference to treat chronic HBV in transgenic and human liver chimeric mice models. Our lab previously showed that a single dose of double-stranded AAV vector serotype 8 (AAV8) carrying a small hairpin RNA (shRNA) effectively reduced HBV replication and gene expression in HBV transgenic mouse, but the silencing effect gradually decreased with time. In this study, we compared the anti-HBV RNA interference (RNAi) effect of AAV8 with those of AAV7 and AAV9, two other hepatotropic AAV vectors, and examined whether the sequential use of these heterologous AAV vectors could prolong the anti-HBV effect. Our results showed that shRNA delivered by each of the three AAV vectors profoundly reduced the serum HBV titer and liver HBV mRNA and DNA levels in the transgenic mice for up to 22 weeks, with AAV8 having the greatest inhibitory effect, followed by AAV9 and AAV7. The potency of AAV8 correlated with the presence of higher levels of vector DNA and anti-HBV shRNA in the liver. An in vivo cross-administration experiment showed that preexisting anti-AAV8 antibody completely blocked the anti-HBV RNAi effect of AAV8, but had no effect on the potency of AAV7 and AAV9. Moreover, we demonstrated that a longer anti-HBV effect could be achieved by the sequential use of AAV8 and AAV9. These results indicate that effective and persistent HBV suppression might be achieved by a combination of the power of RNAi silencing effect and multiple treatments with different AAV serotypes. One major concern of clinical application of RNAi therapy is its potential side effects. In fact, it has been reported that shRNA-encoding AAV8 vectors targeting HBV and a variety of other genes caused dose-dependent acute hepatitis and lethality in the majority of treated mice, a result different from our findings. To clarify these differences, we generated double-stranded AAV vectors containing different promoters and a panel of HBV-specific shRNAs to investigate factors that contribute to the efficacy and pathogenesis of AAV-mediated RNA interference. HBV transgenic mice injected with high doses of AAV vectors containing the U6 promoter produced abundant shRNAs, transiently inhibited HBV, but induced severe hepatotoxicity. Sustained HBV suppression without liver toxicity can be achieved by lowering the dose of AAV8-U6 vectors. AAVs containing the weaker H1 promoter did not cause liver injury, but their therapeutic efficacy was highly dependent on the sequence of the shRNA. Mice treated with the toxic U6 promoter-driven shRNA showed little change in hepatic microRNA levels, but a dramatic increase in hepatic leukocytes and inflammatory cytokines and chemokines. Hepatotoxicity was completely absent in immunodeficient mice and significantly alleviated in wild-type mice depleted of macrophages and granulocytes, suggesting that host inflammatory responses are the major cause of liver injury induced by the over-expressed shRNAs from AAV8-U6 vectors. From this study, we demonstrate that selection of a highly potent shRNA and control its expression level is critical to achieve sustained HBV suppression without inducing inflammatory side-effects. To further evaluate the clinical applicability of AAV-mediated RNAi, we investigated its therapeutic efficacy in human liver chimeric mice which is susceptible to HBV infection. In the IBMS animal facility, we established Fah-/-/Rag2-/-/IL-2rg-/- (FRG) mice, which are genetically defect of the fumarylacetoacetate hydrolase (Fah) gene, resulting in accumulation of toxic metabolites of tyrosine and chronic liver damage. An optimized transplantation procedure was developed to achieve 74% repopulation rate in FRG mice, with an average human liver chimerism of 52%. HBV infection can be successfully established in these human liver chimeric mice. The average serum HBV titer can reach up to 108 copies /ml at week 6 post HBV infection, and the high viremia could be maintain for at least six months. These HBV-infected FRG mice were then treated with AAV/HBV-S1, which led to a peak reduction of 674-fold reduction of serum HBV at week 4, followed by a slowly rebound to the pretreatment level at week 20. We also demonstrated that AAV8/HBV-S1 had less silencing effect against HBVT318C, which is a previously reported HBV mutant isolated clinically with a silent mutation in the HBV-S1 target region. Interestingly, in mice treated with AAV8/HBV-S1 for 20 weeks and having rebound serum HBV titers, we also identified several other mutations located in the HBV-S1 target region, suggesting drug-resistant HBV mutants might be selected under the pressure of RNAi therapy.