Kaneda Y, Saeki Y, Nakabayashi M, Zhou W Z, Kaneda M W, Morishita R
Division of Gene Therapy Science, Graduate School of Medicine, Osaka University, Suita, Japan.
Hum Gene Ther. 2000 Feb 10;11(3):471-9. doi: 10.1089/10430340050015932.
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.
我们试图开发一种特异性增强转基因表达的系统，这一直是人类基因治疗中最重要的问题之一。当将爱泼斯坦 - 巴尔病毒（EBV）核抗原1（EBNA - 1）表达载体pCMV - trEBNA - 1与携带潜伏病毒DNA复制起点（oriP）的质粒poriP - CMV - 荧光素酶共转染时，荧光素酶基因表达比不存在EBNA - 1时高出20倍。这种增强主要在转录水平受到调控，并且依赖于oriP序列和EBNA - 1的量。然而，将poriP - CMV - 荧光素酶与纯化的重组EBNA - 1共同导入会抑制荧光素酶基因表达，而当pCMV - 荧光素酶与重组EBNA - 1共同导入时未观察到抑制现象。我们还通过使用日本血凝病毒（HVJ） - 脂质体将poriP - CMV - 荧光素酶导入小鼠肝脏。在转移后10天，荧光素酶基因表达降至低水平。然后在第10天通过HVJ - 脂质体将pCMV - trEBNA - 1导入小鼠肝脏。荧光素酶基因表达被重新激活，而将EBNA - 1表达质粒注射到注射了缺乏oriP序列的pCMV - 荧光素酶的肝脏中未检测到重新激活。因此，尽管必须对该系统的安全性进行彻底研究，但携带oriP的表达载体与EBNA - 1表达质粒的共转染对于人类基因治疗应该是有前景的。
