Re-Designed Recombinant Hepatitis B Virus Vectors Enable Efficient Delivery of Versatile Cargo Genes to Hepatocytes with Improved Safety

Abstract

Hepatitis B virus (HBV) takes humans as its sole natural host, and productive infection in vivo is restricted exclusively to hepatocytes in the liver. Consequently, HBV-derived viral vectors are attractive candidates for liver-targeting gene therapies. Previously, we developed a novel recombinant HBV vector, designated 5c3c, from a highly replicative clinical isolate. 5c3c was demonstrated to be capable of efficiently delivering protein or RNA expression into infected primary tupaia hepatocytes (PTH), but the design of 5c3c imposes stringent restrictions on inserted sequences, which have limited its wider adoption. In this work, we addressed issues with 5c3c by re-designing the insertion strategy. The resultant vector, designated 5dCG, was more replicative than parental 5c3c, imposed no specific restrictions on inserted sequences, and allowed insertion of a variety of cargo genes without significant loss of replication efficiency. 5dCG-based recombinant HBV effectively delivered protein and RNA expression into infected PTH. Furthermore, due to the loss of functional core ORF, 5dCG vectors depend on co-infecting wild type HBV for replication and efficient expression of cargo genes. Development of the improved 5dCG vector makes wider applications of recombinant HBV possible, while dependence on co-infecting wild type HBV results in improved safety for certain in vivo applications.

Construction of a replication-competent hepatitis B virus vector carrying secreted luciferase transgene and establishment of new hepatitis B virus replication and expression cell lines

“…The only defined data is that a little of HBV DNA was observed by Southern blot in the cell line HuH-7 but not HepG2. Bai et al[32] constructed a new kind of HBV vector by inserting transgene at the spliced HBV polymerase spacer region and proved that it could replicate in hepatocytes. But as the PreS1 region was replaced by the transgene, it should have lost the infection ability.…”

Luteolin-7-O-Glucoside Present in Lettuce Extracts Inhibits Hepatitis B Surface Antigen Production and Viral Replication by Human Hepatoma Cells in Vitro

“…Southern blot hybridization was used for the detection of intracellular HBV RI and performed as described previously ( Bai et al, 2016 ). The assay was carried out two times independently.…”

The current status and future directions of hepatitis B antiviral drug discovery

“…In addition to rationally discovering compounds that selectively target viral proteins and their interacting cellular partners essential for cccDNA function, recent technical development in HBV infection cell culture systems 19, 20 , establishment of a HepG2-derived stable cell line expressing epitope-tagged HBeAg in a cccDNA-dependent manner 85 and production of recombinant HBV reporter viruses 86 will allow for high throughput screening of compound libraries to phenotypically identify compounds that destabilize or functionally inactivate cccDNA.…”