260. BB-HB-331, a DNA-Directed RNA Interference (ddRNAi) Agent Targeting Hepatitis B Virus (HBV), Can Effectively Suppress HBV In Vitro and In Vivo (2016)

Background: BB-HB-331 is a recombinant adeno-associated virus serotype 8 (AAV8) vector designed to treat chronic HBV infection using RNA interference. This self-complementary vector expresses three short hairpin RNAs (shRNAs) that simultaneously target three well-conserved sequences on the viral RNAs that correspond to regions that encode the Core, S-antigen and X-proteins. Using an identical capsid for delivery and an identical set of regulatory elements, BB-HB-331 was engineered as a mimic of TT-034, a ddRNAi therapeutic currently in phase I/IIa clinical studies for the treatment for hepatitis C virus (HCV) infection. The only significant difference between TT-034 and BB-HB-331 is that the anti-HCV shRNAs of TT-034 have been replaced with anti-HBV shRNAs in BB-HB-331. Methods: We first tested the efficacy of BB-HB-331 in an in vitro setting using primary hepatocytes (PHs) isolated from the PXB (Phoenix Bio) mice that are largely comprised of human hepatocytes. PHs were subjected to in vitro infection with HBV genotype C for 12 days prior to the treatment of BB-HB-331. Since AAV does not efficiently transduce PHs in vitro, we utilized adenovirus to deliver the recombinant BB-HB-331 DNA (Ad-BB-HB-331). Increasing doses of Ad-BB-HB-331 were applied to HBV infected primary hepatocytes for 16 days. Hepatocyte cultures treated with adenovirus Ad-TT-034 served as a control. For the in vivo study, PXB mice were first infected with HBV genotype C for 28 days to establish HBV baseline infection. This was followed by a single IV infusion of AAV8-BB-HB-331 at a dose of either 2.00E12 or 2.00E13 vg/kg. Untreated HBV infected PXB mice served as the negative control. Serum samples were collected on a weekly basis to assess HBeAg, HBsAg, and extracellular HBV DNA levels. Results: In vitro treatment of PXB primary hepatocytes with Ad-HB-BB-331 led to significant decreases in HBV parameters. PHs harvested at the conclusion of the experiment demonstrated dose dependent expression of the anti-HBV shRNAs and corresponding inhibition of the HBV viral RNAs. At an MOI of 3, the extracellular levels of HBsAg, HBeAg, and HBcrAg were reduced by 87% or more when compared to the control after 16 days. Although the total cellular DNA did not change, Ad-BB-HB-331 treated cells also demonstrated a 89% reduction of intracellular and extracellular HBV DNA quantities. The levels of cccDNA were correspondingly reduced by 70% in the same time frame. Similarly, effective suppression of HBV infection was observed following in vivo treatment of PXB mice with AAV8-BB-HB-331. Through the first 28 days of an ongoing 56-day experiment, treatment with the high dose resulted in decreases in extracellular levels of HBsAg and HBeAg by 90% and 84%, respectively, when compared to the untreated control. In addition, treatment with the same dose resulted in nearly a log reduction of extracellular HBV DNA at 28 days. Conclusion: Collectively, these data demonstrate suppression of HBV infection by HB-BB-331 in both a primary hepatocyte model and chimeric humanized mouse model.