Fan P D, Dong J Y
Department of Laboratory Medicine, University of California, San Francicso 94143-0724, USA.
Hum Gene Ther. 1997 Jan 1;8(1):87-98. doi: 10.1089/hum.1997.8.1-87.
Adenoassociated virus (AAV) has been developed as a vector for gene transfer because of its advantageous features: it is nonpathogenic, naturally replication-defective; it infects growth-arrested cells, and can transfer the therapeutic gene without co-delivery of any viral genes. However, a major obstacle in conducting systematic studies of AAV-mediated gene transfer in animal models is the difficulty of obtaining large quantities of recombinant virus. Recent development of AAV packaging cell lines has simplified the procedure of producing recombinant AAV (rAAV). However, the efficacy of producing large quantities of rAAV with these cell lines is yet to be demonstrated. In this study we have analyzed the difference between the replication of wild-type AAV and the production of rAAV. Using a combined single-plasmid system that carries both an AAV vector and the rep-cap genes, we have demonstrated that the AAV vector replicates to high number of copies whereas the rep-cap sequences remain unamplified in the virus-producing cells, When the copy number of rep-cap genes was increased by varying the vector/rep-cap ratio in the transfection mixture, the titer of rAAV increased proportionally. Thus, the titer of rAAV is limited by the low copy number of the rep-cap genes that results in an insufficient expression of the Rep and Cap proteins. We have also shown that generation of double-stranded replicating form of the vector DNA is accompanied by an amplified transgene expression. We propose that the increased gene expression from the accumulating double-stranded viral DNA is likely to be the mechanism by which wild-type AAV produces a large number of particles necessary to package the self-replicating AAV genomes. We conclude that mimicking this amplified expression of rep-cap genes may provide the key to produce high titers of rAAV.
腺相关病毒（AAV）因其具有以下优势特性而被开发用作基因转移载体：它无致病性，天然存在复制缺陷；能感染生长停滞的细胞，且可在不共递送任何病毒基因的情况下转移治疗性基因。然而，在动物模型中对AAV介导的基因转移进行系统研究的一个主要障碍是难以获得大量重组病毒。AAV包装细胞系的最新进展简化了重组AAV（rAAV）的生产过程。然而，利用这些细胞系大量生产rAAV的效率还有待证明。在本研究中，我们分析了野生型AAV的复制与rAAV生产之间的差异。通过使用携带AAV载体和rep-cap基因的组合单质粒系统，我们证明了AAV载体能复制出大量拷贝，而rep-cap序列在病毒生产细胞中未得到扩增。当通过改变转染混合物中的载体/rep-cap比例来增加rep-cap基因的拷贝数时，rAAV的滴度成比例增加。因此，rAAV的滴度受到rep-cap基因低拷贝数的限制，这导致Rep和Cap蛋白表达不足。我们还表明，载体DNA双链复制形式的产生伴随着转基因表达的增强。我们提出，积累的双链病毒DNA导致基因表达增加，这可能是野生型AAV产生大量包装自我复制AAV基因组所需颗粒的机制。我们得出结论，模拟rep-cap基因的这种增强表达可能是生产高滴度rAAV的关键。
