Inhibition of Hepatitis B virus with adeno-associated virus vectors expressing primary microRNA-122/5

Abstract

Chronic hepatitis B virus (HBV) infection is a global pandemic affecting approximately 240 million people world-wide. The shortcomings with nucleoside/nucleotide-analogues or interferon-α based drug therapy begs for alternative approaches such as gene therapy to treat chronic viral hepatitis. The highly conserved and compact HBV genomic structure makes it vulnerable to disruption therefore viral protein expression interruption. Recently, long-term RNA interference (RNAi) mediated inhibition of HBV replication was demonstrated using adeno-associated virus serotype 8 (AAV8) vector. However, prevalence of anti-AAV8 broadly neutralising antibodies in human population remains a challenge to effective therapeutic gene transfer. This study investigated transfer and liver-specific gene expression of primary-microRNA (pri-miR) mimic (pri-miR 122/5) using an in-silico constructed ancestral viral vector (Anc80L65) to activate RNAi against HBV. Anti-HBV Anc80L65 vector (Anc80L65-122/5) was produced and characterized in liver-derived Huh7 cells and in HBV transgenic mouse model. For assessment of transduction efficiency and long term transgene expression by Anc80L65 in vivo, a nano-luciferase expressing vector (Anc80L65-NLuc) was also produced. Significant expression of nano-luciferase from Anc80L65-NLuc was observed in vitro. A high and sustained nano-luciferase expression was also observed in vivo. Following a single dose of Anc80L65-122/5 in mice, an impressive and sustained HBV gene expression knockdown of up to 70% was observed over four months. Safety of Anc80L65 was demonstrated by absence of harmful immune-stimulatory effects and resultant liver toxicity, a fact supported by normal levels of serum alanine transaminase enzyme. There were no mice fatalities related to Anc80L65 mediated RNAi stimulation. The safety, high efficiency and the prolonged HBV inhibition by Anc80L65 makes it a useful therapeutic delivery tool and will significantly contribute to the progression of anti-HBV RNAi based therapy in clinical applications.