摘要
Semliki Forest virus (SFV) vectors have a great potential for the induction of protective immunity in a large number of clinical conditions including cancer. Such a potential accounts for the huge efforts made to improve the in vivo expression from SFV vectors. It is noteworthy that efficient in vivo expression strongly relies on the ability to deliver high-titre vectors. To achieve this, the generation of recombinant SFV particles, using independent expression systems for structural SFV genes, has been proposed. However, despite several modifications in the production process, a risk of contamination with replication-competent, or partially recombined, virus has remained. Methods
Here, we exploit the ability of the vesicular stomatitis virus glycoprotein (VSV-G), expressed in trans, to hijack full-length genomic SFV RNA into secreted virus-like particles (VLPs). To allow SFV vector mobilisation, we designed a CMV driven SFV vector in which the internal 26S promoter has been extensively mutated. With this vector, mobilisation events were monitored using the Green Fluorescent Protein (GFP). The production procedure involves a sequential transfection protocol, of plasmids expressing the VSV-G and the SFV vector respectively. Results
We show that the VLPs are effective for cellular delivery of SFV vectors in a broad range of human and non-human cellular targets. Furthermore, production of VLPs is easy and allows, through concentration, the harvest of high-titre vector. Conclusions
The present paper describes a convenient process aimed at mobilising full length SFV vectors. A major issue to consider, while developing clinically relevant gene transfer vectors, is the risk of undesirable generation of replication competent by-products. Importantly, as the VSV-G gene shares no homology with the SFV genome, our VLPs offer a strong guarantee of biosafety. Copyright © 2004 John Wiley & Sons, Ltd.
摘要译文
Semliki森林病毒（SFV）的载体具有用于诱导保护性免疫中有大量的临床病症，包括癌症的巨大潜力。这样的潜在帐户为改进的体内表达从SFV载体的巨大努力。值得注意的是，有效的体内表达强烈依赖于提供高滴度载体的能力。为了实现这一目标，重组SFV颗粒的产生，使用独立的表达系统对结构SFV基因，已提议。然而，尽管在生产过程中的几个变型，污染有复制能力的风险，或部分重组，病毒一直保持。方法在这里，我们利用的水泡性口炎病毒糖蛋白（VSV-G）的能力，表示反式，劫持全长基因组的SFV RNA导入分泌病毒样颗粒（VLPs）。为了让SFV矢量动员，我们设计了一个CMV驱动的SFV载体，其中内部26S启动子已经被广泛突变。与此矢量，动员事件使用绿色荧光蛋白（GFP）的监测。生产过程包括连续的转染方案的质粒分别表达VSV-G和SFV载体。结果我们表明，病毒样颗粒是有效的SFV载体在广泛的人类和非人类细胞靶细胞递送。此外，生产VLP的容易，并允许，通过浓缩，高滴度载体的收获。结论本文介绍了一种方便的进程，旨在动员全长SFV载体。一个主要需要考虑的问题，而发展的临床相关基因转移载体，是不希望产生有复制能力的副产物的风险。重要的是，作为VSV-G基因股的基因组SFV无同源性，我们提供的VLP生物安全的有力保障。版权
Fabien Dorange, Eric Piver, Thierry Bru, Christine Collin, Philippe Roingeard and Jean-Christophe Pagès[*]. Vesicular stomatitis virus glycoprotein: a transducing coat for SFV-based RNA vectors[J]. The Journal of Gene Medicine, 2004,6(9): 1014-1022