Ogasawara Y, Urabe M, Ozawa K
Department of Molecular Biology, Institute of Hematology, Jichi Medical School, Tochigi, Japan.
Microbiol Immunol. 1998;42(3):177-85. doi: 10.1111/j.1348-0421.1998.tb02269.x.
Although adeno-associated virus (AAV) vectors are potentially useful gene transfer vehicles for gene therapy, the vector production system is currently at the developmental stage. We constructed AAV helper plasmids (Rep and Cap expression plasmids) by replacing a native AAV promoter, p5, with various heterologous promoters to examine whether the efficiency of AAV vector production was influenced by modulating the AAV protein expression pattern. The helper plasmids containing heterologous promoters (EF, CMV, SV40, B19p6, and CAG promoters, respectively) expressed Rep78/68 more efficiently than a conventional helper plasmid (pIM45), but the expression of Rep52/40 and Cap decreased, resulting in a significant reduction in AAV vector production. Furthermore, the efficiency of vector production never fully recovered even if the Cap proteins were supplied by an additional expression plasmid. A large amount of Rep78/68 and/or a reduced level of Rep52/40 may have deleterious effects on AAV vector production. The present findings will aid in the development of a more efficient AAV vector production system.
尽管腺相关病毒（AAV）载体作为基因治疗中潜在有用的基因转移工具，但载体生产系统目前仍处于发展阶段。我们通过用各种异源启动子替换天然AAV启动子p5来构建AAV辅助质粒（Rep和Cap表达质粒），以研究AAV载体生产效率是否受AAV蛋白表达模式调控的影响。分别含有异源启动子（EF、CMV、SV40、B19p6和CAG启动子）的辅助质粒比传统辅助质粒（pIM45）更有效地表达Rep78/68，但Rep52/40和Cap的表达下降，导致AAV载体产量显著降低。此外，即使通过额外的表达质粒提供Cap蛋白，载体生产效率也从未完全恢复。大量的Rep78/68和/或Rep52/40水平降低可能对AAV载体生产产生有害影响。目前的研究结果将有助于开发更高效的AAV载体生产系统。
