PDF(Pubmed)
Abstract：
Recombinant adeno-associated virus (rAAV) vectors could be manufactured by plasmid transfection into human embryonic kidney 293 (HEK293) cells or baculovirus infection of Spodoptera frugiperda (Sf9) insect cells. However, systematic comparisons between these systems using large-scale, high-quality AAV vectors are lacking. rAAV from Sf9 cells (Sf9-rAAV) at 2-50 L and HEK293 cells (HEK-rAAV) at 2-200 L scales were characterized. HEK-rAAV had ∼40-fold lower yields but ∼10-fold more host cell DNA measured by droplet digital PCR and next-generation sequencing, respectively. The electron microscope observed a lower full/empty capsid ratio in HEK-rAAV (70.8%) than Sf9-rAAV (93.2%), while dynamic light scattering and high-performance liquid chromatography analysis showed that HEK-rAAV had more aggregation. Liquid chromatography tandem mass spectrometry identified different post-translational modification profiles between Sf9-rAAV and HEK-rAAV. Furthermore, Sf9-rAAV had a higher tissue culture infectious dose/viral genome than HEK-rAAV, indicating better infectivity. Additionally, Sf9-rAAV achieved higher in vitro transgene expression, as measured by ELISA. Finally, after intravitreal dosing into a mouse laser choroidal neovascularization model, Sf9-rAAV and HEK-rAAV achieved similar efficacy. Overall, this study detected notable differences in the physiochemical characteristics of HEK-rAAV and Sf9-rAAV. However, the in vitro and in vivo biological functions of the rAAV from these systems were highly comparable. Sf9-rAAV may be preferred over HEK293-rAAV for advantages in yields, full/empty ratio, scalability, and cost.
摘要：
重组腺相关（rAAV）载体可以通过质粒转染到人HEK293细胞中或杆状病毒感染节食夜蛾（Sf9）昆虫细胞来制造。然而,使用大规模对这些系统进行系统比较，缺乏高质量的AAV载体。表征来自2-50L规模的Sf9细胞(Sf9-rAAV)和2-200L规模的HEK293细胞(HEK-rAAV)的rAAV。HEK-rAAV的产量降低了40倍，但通过ddPCR和NGS测量的宿主细胞DNA却增加了10倍，分别。电子显微镜观察到HEK-rAAV中的满/空衣壳比率(70.8%)低于Sf9-rAAV(93.2%),而动态光散射(DLS)和HPLC分析显示HEK-rAAV具有更多的聚集。LC-MS/MS鉴定了Sf9-rAAV和HEK-rAAV之间的不同翻译后修饰(PTM)谱。此外，Sf9-rAAV比HEK-rAAV具有更高的TCID50/vg,表明更好的传染性。此外,sf9-rAAV实现了更高的体外转基因表达，通过ELISA测量。最后,在玻璃体内给药进入小鼠激光脉络膜新生血管模型后，Sf9-rAAV和HEK-rAAV实现了相似的功效。总的来说,这项研究发现HEK-rAAV和Sf9-rAAV的理化特性存在显着差异。然而,来自这些系统的rAAV的体外和体内生物学功能是高度可比的。Sf9-rAAV可能优于HEK293-rAAV，因为在产量上有优势，满/空比率，可扩展性，和成本。
