Strobel Benjamin, Zuckschwerdt Kai, Zimmermann Gudrun, Mayer Christine, Eytner Ruth, Rechtsteiner Philipp, Kreuz Sebastian, Lamla Thorsten
1 Research Beyond Borders, Boehringer Ingelheim Pharma GmbH & Co. KG, Biberach an der Riss, Germany.
2 Drug Discovery Sciences, Boehringer Ingelheim Pharma GmbH & Co. KG, Biberach an der Riss, Germany.
Hum Gene Ther Methods. 2019 Feb;30(1):23-33. doi: 10.1089/hgtb.2018.228.
Adeno-associated virus (AAV) vectors currently represent the most attractive platform for viral gene therapy and are also valuable research tools to study gene function or establish disease models. Consequently, many academic labs, core facilities, and biotech/pharma companies meanwhile produce AAVs for research and early clinical development. Whereas fast, universal protocols for vector purification (downstream processing) are available, AAV production using adherent HEK-293 cells still requires time-consuming passaging and extensive culture expansion before transfection. Moreover, most scalable culture platforms require special equipment or extensive method development. To tackle these limitations in upstream processing, this study evaluated frozen high-density cell stocks as a ready-to-seed source of producer cells, and further investigated the multilayered CELLdisc culture system for upscaling. The results demonstrate equal AAV productivity using frozen cell stock-derived cultures compared to conventionally cultured cells, as well as scalability using CELLdiscs. Thus, by directly seeding freshly thawed cells into CELLdiscs, AAV production can be easily upscaled and efficiently standardized to low-passage, high-viability cells in a timely flexible manner, potentially dismissing time-consuming routine cell culture work. In conjunction with a further optimized iodixanol protocol, this process enabled supply to a large-animal study with two high-yield AAV2 capsid variant batches (0.6-1.2 × 10 vector genomes) in as little as 4 weeks.
腺相关病毒（AAV）载体目前是病毒基因治疗中最具吸引力的平台，也是研究基因功能或建立疾病模型的宝贵研究工具。因此，许多学术实验室、核心设施以及生物科技/制药公司同时生产用于研究和早期临床开发的AAV。虽然有快速、通用的载体纯化（下游处理）方案，但使用贴壁HEK-293细胞生产AAV在转染前仍需要耗时的传代和大量的培养扩增。此外，大多数可扩展的培养平台需要特殊设备或大量的方法开发。为了解决上游处理中的这些限制，本研究评估了冷冻高密度细胞库作为随时可用的生产细胞接种源，并进一步研究了用于扩大规模的多层CELLdisc培养系统。结果表明，与传统培养的细胞相比，使用冷冻细胞库来源的培养物生产AAV的效率相同，并且使用CELLdiscs可以实现可扩展性。因此，通过将刚解冻的细胞直接接种到CELLdiscs中，可以轻松扩大AAV的生产规模，并及时灵活地将其高效标准化为低传代、高活力的细胞，这可能省去耗时的常规细胞培养工作。结合进一步优化的碘克沙醇方案，该流程能够在短短4周内为一项大型动物研究提供两批高产AAV2衣壳变体（0.6-1.2×10载体基因组）。
