Approach to establishing a liver targeting gene therapeutic vector using naturally occurring defective hepatitis B viruses devoid of immunogenic T cell epitope

The development of liver-directed virus vector may play a crucial role in hepatic gene therapy. Hepatitis B virus (HBV) is the only known DNA virus that has hepatocyte specificity. In order to construct an efficient HBV-based vector for targeting the liver, we studied the potential use of naturally occurring defective HBVs obtained from hepatitis patients. The enhanced green fluorescent protein (EGFP) gene or small tag sequences (Flag) were introduced in frame into the deleted sites of the defective HBVs. One HBV defective in site for putative T cell epitope and a part of the polymerase gene tolerated EGFP insertion and was successfully packaged. This defective recombinant HBV harboring 48 bp Flag tag sequence instead of EGFP (rHBV-7-Flag) replicated well. Human primary hepatocytes could uptake rHBV-7-Flag virions, though in a low frequency, when exposed to the virions at a high density in the culture medium, and also express Flag tag sequences. This defective HBV-based vector may have a potential application in liver targeting gene therapy.