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

Successful and optimized in vivo gene transfer to rabbit carotid artery mediated by electronic pulse

“…The one disadvantage of the vector is, as shown in this study, transient transgene expression. However, recent studies showed that the combination of EpsteinBarr virus (EBV) replicon vector based on the plasmid resulted in sustained gene expresion in nonlymphocytic cultured cells and the mouse liver; 27 thus we are now assessing the sustained gene expression using an EBV replicon vector in a similar animal model. In summary, we developed a method for gene transfer using electronic pulse, an approach which proved to be safe and more efficient than that using naked DNA.…”

Highly efficient gene transfer into murine liver achieved by intravenous administration of naked Epstein–Barr virus (EBV)-based plasmid vectors

“…We have recently found that these functions actually work in rodent cells, at least in some in vitro experimental systems (Kishida et al, unpublished observations). Also, Saeki et al, 32 Kaneda et al, 33 and Nishizaki et al 30 have shown that the EBVbased plasmid vector enabled significantly prolonged expression of marker genes in rodent organs that had been transfected by means of HVJ-liposomes 32,33 or a gene gun. 30 Taking into account the potential application of naked pDNA injection to gene therapy, constant and persistent expression is desired.…”

Liver-directed gene therapy of diabetic rats using an HVJ-E vector containing EBV plasmids expressing insulin and GLUT 2 transporter

“…The application of this plasmid in various cells has been developed. [10][11][12] EBV is of particular interest as a vector because of its latent infection property, which is characterized by autonomous replication and nuclear retention of the EBV genome in host cells. 13 The persistent maintenance of the EBV genome has been analyzed in depth and is mediated by the replication origin of the latent viral DNA (oriP) and EBV nuclear antigen 1 (EBNA-1).…”

“…14,15 Previously, our group showed that use of the EBV replicon vector leads to sustained transgene expression in vivo in the liver. 11 With regard to efficient vector delivery, numerous viral and nonviral vectors have been developed. 4 Adenovirus-mediated gene transfer has been successful in transiently expressing transgenes, but it also causes undesired effects such as hepatic inflammation.…”