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首页> 外文期刊>Molecular Therapy - Methods & Clinical Development >Methods Matter: Standard Production Platforms for Recombinant AAV Produce Chemically and Functionally Distinct Vectors
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Methods Matter: Standard Production Platforms for Recombinant AAV Produce Chemically and Functionally Distinct Vectors

机译：方法问题：重组AAV的标准生产平台在化学和功能上不同的载体产生

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摘要

Different approaches are used in the production of recombinant adeno-associated virus (rAAV). The two leading approaches are transiently transfected human HEK293 cells and live baculovirus infection of Spodoptera frugiperda ( 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 (transmission electron cryomicroscopy), 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 (PTMs), including glycosylation, acetylation, phosphorylation, and methylation, and these differ between platforms; and (2) rAAV genomes are methylated during production, and these are also differentially deposited between platforms. Our data show 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.
机译：在重组腺相关病毒（RAAV）的生产中使用不同的方法。这两种主要的方法是瞬时转染的人HEK293细胞和Spodoptera Rugiperda（SF9）昆虫细胞的活杆病毒感染。临床和临界在临床上已经看到了载体性能的原因差异。因此，我们进行了受控的比较生产分析，只有宿主细胞物种，但保持所有其他参数。我们表征了多种分析方法的差异：通过质谱法，等电聚焦，Cryo-em（透射电子冷冻镜），变性测定，包装基因组，人细胞因子分析和功能性转导评估的蛋白质组学分析。 ？体内，包括人源化肝小鼠。使用这些方法，我们已经进行了两种主要发现：（1）rav帽具有翻译后修饰（PTM），包括糖基化，乙酰化，磷酸化和甲基化，这些平台之间存在差异; （2）rAAV基因组在生产过程中甲基化，并且这些也在平台之间差异沉积。我们的数据表明，宿主细胞蛋白质杂质在平台之间不同，并且可以具有它们自己的PTM，包括潜在的免疫原性N-连接的聚糖。人类产生的rAAV在β体外（p？<0.05-0.0001）中的各种细胞类型中的杆状病毒族载体更有效（p？<0.05-0.0001），在α体内（p？<0.03-0.0001），以及人肝中？体内（p？<0.005）。这些差异可能具有对RAAV受体结合，贩运，表达动力学，表达耐久性，染料免疫原性以及成本考虑的临床意义。

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