Different approaches are used in the production of recombinant AAV. The two leading approaches are transiently transfected human HEK293 cells and live baculovirus infection of Sf9 insect cells. Unexplained differences in vector performance have been seen clinically and preclinically. Thus, we performed a controlled comparative production analysis varying only the host cell species but maintaining all other parameters. We characterized differences with multiple analytical approaches: proteomic profiling by mass spectrometry, isoelectric focusing, cryo-EM, denaturation assays, genomic and epigenomic sequencing of packaged genomes, human cytokine profiling, and functional transduction assessments in vitro and in vivo, including in humanized liver mice. Using these approaches we have made two major discoveries: 1) rAAV capsids have post-translational modifications including glycosylation, acetylation, phosphorylation, and methylation, and these differ between platforms. 2) rAAV genomes are methylated during production, and these are also differentially deposited between platforms. Our data find that host cell protein impurities differ between platforms and can have their own PTMs including potentially immunogenic N-linked glycans. Human-produced rAAVs are more potent than baculovirus-Sf9 vectors in various cell types in vitro (P < 0.05-0.0001), in various mouse tissues in vivo (P < 0.03-0.0001), and in human liver in vivo (P < 0.005). These differences may have clinical implications for rAAV receptor binding, trafficking, expression kinetics, expression durability, vector immunogenicity as well as cost considerations.

中文翻译：

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方法很重要-重组AAV的标准生产平台可生产化学和功能不同的载体

在重组AAV的生产中使用了不同的方法。两种主要方法是瞬时转染的人HEK293细胞和活杆状病毒感染Sf9昆虫细胞。在临床和临床前已发现载体性能的无法解释的差异。因此，我们进行了受控的比较生产分析，仅改变宿主细胞种类，但保持所有其他参数。我们通过多种分析方法对差异进行了表征：质谱分析，等电聚焦，低温EM，变性测定，包装基因组的基因组和表观基因组测序，人细胞因子分析以及体外和体内功能转导评估（包括在人源化肝脏中）老鼠。使用这些方法，我们取得了两个主要发现：1）rAAV衣壳具有翻译后修饰，包括糖基化，乙酰化，磷酸化和甲基化，这些在平台之间有所不同。2）rAAV基因组在生产过程中被甲基化，并且也被差异沉积在平台之间。我们的数据发现，宿主细胞蛋白质杂质在平台之间有所不同，并且可以拥有自己的PTM，包括可能具有免疫原性的N-连接聚糖。在体外（P <0.05-0.0001）的各种细胞类型，体内各种小鼠组织（P <0.03-0.0001）和人体内的肝（P <0.005）中，人产生的rAAV比杆状病毒-Sf9载体更有效）。这些差异可能对rAAV受体的结合，运输，表达动力学，表达持久性，载体免疫原性以及成本方面具有临床意义。