Généthon, Evry, France.
Université d'Evry Val d'Essonne, Evry, France.
PLoS One. 2018 Jul 5;13(7):e0199866. doi: 10.1371/journal.pone.0199866. eCollection 2018.
The human Adeno-Associated Virus serotype 2 (wtAAV2) is a common non-pathological virus and its recombinant form (rAAV) is widely used as gene therapy vector. Although rAAVs are routinely produced in the Baculovirus/Sf9 cell system, wtAAV2 has never been studied in this context. We tried to produce wtAAV2 in the baculovirus/Sf9 cell system hypothesizing that the wtAAV2 may be considered as a normal recombinant AAV transgene. Through our attempts to produce wtAAV2 in Baculovirus/Sf9, we found that wtAAV2 p5 promoter, which controls the expression of large Rep proteins in mammalian cells, was active in this system. p5 promoter activity in the baculovirus/Sf9 cell system leads to the expression of Rep78 that finally excises wtAAV2 genome from the baculovirus genome during the earliest phases of baculovirus stock production. Via p5 promoter expression kinetics and strand specific RNA-Seq analysis of wtAAV2, rAAV and Rep2/Cap2 cassettes in the baculovirus context we could demonstrate that wtAAV2 native promoters, p5, p19 and p40 are all active in the context of the baculovirus system and lead to the expression of different proteins and peptides. In addition, this study has proven that the baculovirus brings at least some of the helper functions needed in the AAV replication/life cycle.
人腺相关病毒血清型 2（wtAAV2）是一种常见的非致病性病毒，其重组形式（rAAV）被广泛用作基因治疗载体。尽管 rAAV 通常在杆状病毒/ Sf9 细胞系统中生产，但从未在这种情况下研究过 wtAAV2。我们试图在杆状病毒/ Sf9 细胞系统中生产 wtAAV2，假设 wtAAV2 可以被认为是一种正常的重组 AAV 转基因。通过尝试在杆状病毒/ Sf9 中生产 wtAAV2，我们发现 wtAAV2 p5 启动子，它在哺乳动物细胞中控制大 Rep 蛋白的表达，在这个系统中是活跃的。杆状病毒/ Sf9 细胞系统中的 p5 启动子活性导致 Rep78 的表达，最终在杆状病毒库存生产的最早阶段，从杆状病毒基因组中切除 wtAAV2 基因组。通过 p5 启动子表达动力学和杆状病毒中的 wtAAV2、rAAV 和 Rep2/Cap2 盒的链特异性 RNA-Seq 分析，我们可以证明 wtAAV2 天然启动子 p5、p19 和 p40 在杆状病毒系统中均活跃，并导致不同蛋白质和肽的表达。此外，这项研究证明了杆状病毒至少带来了 AAV 复制/生命周期所需的一些辅助功能。
