Sustained transgene expression in vitro and in vivo using an Epstein–Barr virus replicon vector system combined with HVJ liposomes

Abstract

For long-term gene expression in tissues, we constructed an Epstein–Barr virus (EBV) replicon-based plasmid, pEB, containing the latent viral DNA replication origin (oriP) and EBV nuclear antigen-1 (EBNA-1). When pEB was transferred to human cells (HeLa-S3, HEK 293 and FS 3) and rodent cells (BHK-21) using HVJ-cationic liposomes, luciferase expression was observed in those cells for at least 10 days. Luciferase activity was two to 10 times higher in those cell lines on and after day 3 post-transfection of pEBActLuc compared with plasmids without the EBV replicon sequence. Southern blot analysis showed that the pEB vector luciferase gene was maintained extra- chromosomally in BHK-21 cells. In human cells, transformation was five to 20 times more efficient with pEBc than with pcDNA3, and 18–35% of the introduced EBV replicon plasmid was replicated autonomously. The luciferase gene or lacZ gene was introduced into mouse liver using HVJ–AVE liposomes. Luciferase gene expression was observed for at least 35 days in cells transfected with pEBActLuc, whereas it was not detected on day 14 in cells transfected with pActLuc, which lacks the EBV sequence. By the transfer of pEBActNlacF, the lacZ gene expression rate in hepatocytes was approximately 35 and 12% on days 7 and 35, respectively.