Enhancement of transgene expression by cotransfection of oriP plasmid with EBNA-1 expression vector.
We have attempted to develop a system for specific enhancement of transgene expression, which has been one of the most important issues in human gene therapy. When an Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA-1) expression vector, pCMV-trEBNA-1, was cotransfected with an origin of latent viral DNA replication (oriP)-harboring plasmid, poriP-CMV-luciferase, luciferase gene expression was up to 20 times greater than in the absence of EBNA-1. This enhancement was regulated mainly at the transcriptional level and was dependent on the oriP sequence and the amount of EBNA-1. However, cointroduction of poriP-CMV-luciferase with purified recombinant EBNA-1 inhibited luciferase gene expression whereas no inhibition was observed when pCMV-luciferase was cointroduced with recombinant EBNA-1. We also introduced poriP-CMV-luciferase into mouse liver via the use of HVJ (hemagglutinating virus of Japan)-liposomes. By 10 days after transfer, luciferase gene expression was decreased to low levels. We then introduced pCMV-trEBNA-1 to mouse liver via HVJ-liposomes on day 10. Luciferase gene expression was reactivated, whereas no reactivation was detected by the injection of EBNA-1 expression plasmid into liver injected with pCMV-luciferase lacking the oriP sequence. Thus, cotransfection of oriP-harboring expression vector with EBNA-1 expression plasmid should be promising for human gene therapy, although the safety of the system must be investigated thoroughly.
Kaneda
Y
Y
Division of Gene Therapy Science, Graduate School of Medicine, Osaka University, Suita, Japan. kaneday@gts.med.osaka-u.ac.jp
Saeki
Y
Y
Nakabayashi
M
M
Zhou
W Z
WZ
Kaneda
M W
MW
Morishita
R
R
D000818:Animals; D015152:Blotting, Northern; D015139:Blotting, Southern; D002460:Cell Line; D019309:Epstein-Barr Virus Nuclear Antigens / Q000737:chemistry / Q000235:genetics*; D018014:Gene Transfer Techniques; D017930:Genes, Reporter; D005822:Genetic Vectors; D006801:Humans; D008099:Liver / Q000378:metabolism; D008156:Luciferases / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D010957:Plasmids; D018741:Replication Origin; D018108:Respirovirus / Q000235:genetics; D019076:Transgenes
D019309:Epstein-Barr Virus Nuclear Antigens; D008156:Luciferases