Methods Matter: Standard Production Platforms for Recombinant AAV Produce Chemically and Functionally Distinct Vectors
Methods Matter: Standard Production Platforms for Recombinant AAV Produce Chemically and Functionally Distinct Vectors
复制标题
DOI：
10.1016/j.omtm.2020.05.018
复制
发表时间：
2020-09-11
影响因子：
4.7
通讯作者：
Paulk, Nicole K.
中科院分区：
文献类型：
--
作者：
Rumachik, Neil G.;Malaker, Stacy A.;Paulk, Nicole K.
不同的方法用于生产重组腺相关病毒（rAAV）。两种主要的方法是瞬时转染的人HEK 293细胞和草地贪夜蛾（Spodoptera frugiperda，Sf 9）昆虫细胞的活杆状病毒感染。在临床和临床前观察到无法解释的载体性能差异。因此，我们进行了对照比较生产分析，仅改变宿主细胞种类，但保持所有其他参数。我们用多种分析方法表征差异：质谱法蛋白质组分析、等电聚焦、冷冻EM（透射电子冷冻显微镜）、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（包括人源化肝小鼠）功能转导评估。使用这些方法，我们已经取得了两个重大发现：（1）rAAV cap sid具有翻译后修饰（PTM），包括糖基化、乙酰化、磷酸化和甲基化，并且这些在平台之间不同;和（2）rAAV基因组在生产期间被甲基化，并且这些在平台之间也差异沉积。我们的数据表明，不同平台的宿主细胞蛋白质杂质不同，并且可能具有自己的PTM，包括潜在免疫原性的N-连接聚糖。人产生的rAAV比杆状病毒-Sf 9载体在体外的各种细胞类型中（p < 0.05-0.0001）、在体内的各种小鼠组织中（p < 0.03-0.0001）和在体内的人肝脏中（p < 0.005）更有效。这些差异可能对rAAV受体结合、运输、表达动力学、表达持久性、载体免疫原性以及成本考虑具有临床意义。
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 cap sids 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.