Prolonged transgene expression in glomeruli using an EBV replicon vector system combined with HVJ liposomes.
Prolonged transgene expression in glomeruli using an EBV replicon vector system combined with HVJ liposomes.
复制标题
使用 EBV 复制子载体系统与 HVJ 脂质体相结合，延长肾小球中的转基因表达。
DOI：
10.1046/j.1523-1755.2001.0590041390.x
复制
发表时间：
2001
影响因子：
19.6
通讯作者：
Masatsugu Hori
中科院分区：
文献类型：
--
作者：
Michiko Tsujie;Yoshitaka Isaka;Hiroyuki Nakamura;Y. Kaneda;E. Imai;Masatsugu Hori
背景
各种基因转移载体和递送系统已被开发出来，但仍有许多问题有待解决。我们已经实现了一种通过肾动脉血凝日本病毒(HVJ)脂质体介导的基因转移将基因导入肾小球系膜细胞的技术。这种方法的主要局限性是瞬时转基因表达。
方法
为了在肾小球中长期表达基因，采用了基于EB病毒(EBV)复制子的质粒，含有潜在的病毒DNA复制起点(ORIP)和EBV核抗原-1(EBNA-1)，这是转基因-核保持的最小EBV成分。采用EBV复制子载体pEBActLuc和对照质粒载体pActLuc，研究了EBV复制子装置对转基因在体内肾小球表达持续时间的影响。用人工病毒包膜(AVE)型脂质体将这些载体通过肾动脉导入肾组织，检测不同时间点肾小球荧光素酶活性。
结果
在第4天，pEBActLuc和pActLuc转染组的肾小球荧光素酶活性相同，并且在转染pEBActLuc的肾小球中，荧光素酶基因的表达持续了至少56天，而在转染组的第7天，荧光素酶基因的表达减弱。
结论
EBV复制子装置与HVJ脂质体的结合为体内基因的长期表达提供了一种强有力的工具，而且可能成为治疗肾脏疾病进展的一种新方法。
BACKGROUND
Various gene transfer vectors as well as delivery systems have been developed; however, many problems remain to be solved. We already achieved a technique to introduce genes into glomerular mesangial cells by hemagglutinating virus of Japan (HVJ) liposome-mediated gene transfer via renal artery. The main limitation of this method is the transient transgene expression.
METHOD
For long-term gene expression in glomeruli, Epstein-Barr virus (EBV) replicon-based plasmid was employed, containing the latent viral DNA replication origin (oriP) and EBV nuclear antigen-1 (EBNA-1), which are the minimum EBV component of transgene-nuclear retention. To examine the effect of EBV replicon apparatus on the duration of transgene expression in glomeruli in vivo, the EBV replicon vector pEBActLuc, and the control plasmid vector pActLuc were adopted. These plasmid vectors were transferred into the kidney via renal artery by using artificial viral envelope (AVE)-type HVJ liposome method, and glomerular luciferase activities were analyzed at various time points after transfection.
RESULTS
On day 4, pEBActLuc and pActLuc transfer resulted in equal glomerular luciferase activity, and the luciferase gene expression was sustained for at least 56 days in glomeruli transfected with pEBActLuc, whereas it was reduced on seven days in glomeruli transfected with pActLuc.
CONCLUSION
The combination of EBV replicon apparatus and HVJ liposomes appears to be a powerful tool for long-term gene expression in vivo, and furthermore, it may be a promising new therapeutic method for the progression of renal disease.