441. Short Hairpin RNA Interference Expressed from a Gene-Deleted Adenoviral Vector Results in Reduction of Hepatits B Surface Antigen Levels In Vitro and in a Small Animal Model for Hepatitis B Infection
Authors: Anja Ehrhardt, Hui Xu, Felix H. Salazar, Patricia L. Marion, Mark A. Kay
Journal: Molecular Therapy (2005)

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There is substantial need for new treatment strategies for chronically hepatitis B virus (HBV) infected patients. This study explores the rational design of novel adenoviral gene therapy vectors for efficient delivery of RNA interference (RNAi) inhibitor molecules against HBV. Over the recent years various viral and non-viral vectors have been studied for delivery of short hairpin RNAi (shRNAi) expression cassettes but the in vitro and in vivo efficacy of significantly safer gene-deleted adenoviral vectors (HD vectors) for delivery of shRNAi remains to be tested. To show in vivo efficacy regular FVB mice (n=3 per group) were co-injected with a HD vector with a shRNAi expression cassette against luciferase and a second adenoviral vector expressing luciferase. We found up to 96% reduction of luciferase expression seven days post-injection.

Our laboratory previously demonstrated that a 25-mer shRNAi against HBV (HBVU6no.2) driven by a polymerase III promoter was sufficient to inhibit HBV in a non-viral gene therapy approach in vivo (McCaffrey et al., Nat Biotechnol 21:639-644, 2003) but was lethal in the context of a novel self-complementery adeno-associated viral vector (AAV) in HBV transgenic and normal mice (Grimm et al., 2004). In this study we created a gene-deleted adenoviral vector with the same shRNAi expression cassette (FTC/HBVU6no.2). In cells stably transfected with the HBV genome we observed up to 60% reduction of secreted surface antigen (sAg) levels, a marker for progression of HBV infection.

In a further step we transfused a plasmid containing the HBV-genome into mouse liver of C57Bl/6 mice (n=4 per group) by high pressure tail vein injection followed by injection of 5|[times]|10e9 transducing units of the HD vector FTC/HBVU6no.2. This viral dose was sufficient to transduce 100% of all hepatocytes. We observed a 70% drop in serum sAg levels. Control mice which received only the HBV containing plasmid or a HD vector with a beta-galactosidase (|[beta]|-Gal) expression cassette showed a 29% and a 73% drop in serum sAg levels, respectively.

HBV transgenic mice (n=4 per group) were either injected with 5|[times]|10e9 transducing units of the HD vector FTC/HBVU6no.2 or a |[beta]|-Gal expressing HD vector. After a transient increase in sAg levels in both groups, we observed an up to 45% and 73% reduction in serum sAg levels compared to the vehicle control group, respectively. Limited toxicity studies were performed and highest liver enzyme levels were measured 20 days post-injection which returned to normal levels 40 days post-injection for the FTC/HBVU6no.2 but not the |[beta]|-Gal group. We speculate that an immune response against the transgene |[beta]|-Gal is responsible for the significant drop in serum sAg levels and the sustained increased liver enzyme levels. In summary, this study demonstrates that gene-deleted adenoviral vectors can be used to deliver small inhibitor molecules in vivo and with further improvements these vectors will play an important role in gene therapy approaches based on shRNAi.