重组腺相关病毒(rAAV)是主要的基因递送载体。我们通过整合必需的rAAV基因组构建了稳定的rAAV生产细胞系,病毒和辅助基因在诱导型启动子的控制下进入HEK293细胞的基因组。诱导后,该细胞系产生转导rAAV。为了深入了解提高rAAV生产率和载体质量,我们对我们的合成细胞系GX2和两个野生型AAV(wtAAV)生产系统进行了比较转录组和蛋白质组分析,一种是病毒共感染,另一种是多质粒转染。这三个系统在病毒成分合成中具有不同的动力学,但产生了相当的AAV基因组拷贝;然而,与这两种WTAAV系统相比,GX2的衣壳滴度要低一个数量级,表明其衣壳产量可能不足。GX2的基因组包装效率也较低,尽管它比任何一个wtAAV系统产生更高水平的Rep52蛋白。表明Rep52蛋白表达可能不限制基因组包装。在两个wtAAV系统中,VP是最丰富的AAV蛋白,它们的水平持续增加,而GX2具有高水平的浪费货物基因表达。此外,炎症上调,先天免疫反应,和MAPK信号,以及下调的线粒体功能,通常在rAAV或wtAAV系统中观察到。总的来说,这项比较多组学研究提供了对宿主细胞和病毒因子的丰富见解,这些因子是遗传和过程干预的潜在靶标,以提高合成rAAV生产细胞系的生产率.关键点:·wtAAV感染在产生全病毒颗粒方面比合成细胞GX2更有效。•衣壳蛋白合成,基因组复制,和包装可能会限制GX2中的rAAV生产。•GX2中的wtAAV感染和rAAV产生引发类似的宿主细胞应答。
Recombinant adeno-associated virus (
rAAV) is a major gene delivery vehicle. We have constructed a stable
rAAV producer cell line by integrating essential
rAAV genome, viral and helper genes into the genome of HEK293 cell under the control of inducible promoters. Upon induction, the cell line produces transducing
rAAV. To gain insight into enhancing rAAV productivity and vector quality, we performed a comparative transcriptomic and proteomic analysis of our synthetic cell line GX2 and two wild-type AAV (wtAAV) production systems, one by virus co-infection and the other by multi-plasmid transfection. The three systems had different kinetics in viral component synthesis but generated comparable copies of AAV genomes; however, the capsid titer of GX2 was an order of magnitude lower compared to those two wtAAV systems, indicating that its capsid production may be insufficient. The genome packaging efficiency was also lower in GX2 despite it produced higher levels of Rep52 proteins than either wtAAV systems, suggesting that Rep52 protein expression may not limit genome packaging. In the two wtAAV systems, VP were the most abundant AAV proteins and their levels continued to increase, while GX2 had high level of wasteful cargo gene expression. Furthermore, upregulated inflammation, innate immune responses, and MAPK signaling, as well as downregulated mitochondrial functions, were commonly observed in either
rAAV or wtAAV systems. Overall, this comparative multi-omics study provided rich insights into host cell and viral factors that are potential targets for genetic and process intervention to enhance the productivity of synthetic rAAV producer cell lines. KEY POINTS: • wtAAV infection was more efficient in producing full viral particles than the synthetic cell GX2. • Capsid protein synthesis, genome replication, and packaging may limit rAAV production in GX2. • wtAAV infection and
rAAV production in GX2 elicited similar host cell responses.