Matsushita T, Elliger S, Elliger C, Podsakoff G, Villarreal L, Kurtzman G J, Iwaki Y, Colosi P
Avigen, Inc., Alameda, CA, USA.
Gene Ther. 1998 Jul;5(7):938-45. doi: 10.1038/sj.gt.3300680.
The purpose of this work was to develop an efficient method for the production of adeno-associated virus (AAV) vectors in the absence of helper virus. The adenovirus regions that mediate AAV vector replication were identified and assembled into a helper plasmid. These included the VA, E2A and E4 regions. When this helper plasmid was cotransfected into 293 cells, along with plasmids encoding the AAV vector, and rep and cap genes, AAV vector was produced as efficiently as when using adenovirus infection as a source of help. CMV-driven constructs expressing the E4orf6 and the 72-M(r), E2A proteins were able to functionally replace the E4 and E2A regions, respectively. Therefore the minimum set of genes required to produce AAV helper activity equivalent to that provided by adenovirus infection consists of, or is a subset of, the following genes: the E4orf6 gene, the 72-M(r), E2A protein gene, the VA RNA genes and the E1 region. AAV vector preparations made with adenovirus and by the helper virus-free method were essentially indistinguishable with respect to particle density, particle to infectivity ratio, capsimer ratio and efficiency of muscle transduction in vivo. Only AAV vector preparations made by the helper virus-free method were not reactive with anti-adenovirus sera.
这项工作的目的是开发一种在无辅助病毒的情况下高效生产腺相关病毒（AAV）载体的方法。确定了介导AAV载体复制的腺病毒区域，并将其组装成一个辅助质粒。这些区域包括VA、E2A和E4区域。当将该辅助质粒与编码AAV载体、rep和cap基因的质粒共转染到293细胞中时，AAV载体的产生效率与使用腺病毒感染作为辅助来源时一样高。表达E4orf6和72-M（r）、E2A蛋白的CMV驱动构建体能够分别在功能上替代E4和E2A区域。因此，产生与腺病毒感染提供的AAV辅助活性相当的辅助活性所需的最小基因集由以下基因组成或为其一个子集：E4orf6基因、72-M（r）、E2A蛋白基因、VA RNA基因和E1区域。用腺病毒和无辅助病毒方法制备的AAV载体制剂在颗粒密度、颗粒与感染性比率、衣壳粒比率和体内肌肉转导效率方面基本无法区分。只有用无辅助病毒方法制备的AAV载体制剂与抗腺病毒血清无反应。
