Department of Production Platforms & Analytics, National Research Council Canada, Building Montreal, Montreal, QC H4P 2R2, Canada.
Department of Bioengineering, McGill University, Montreal, QC H3A 0E9, Canada.
Cells. 2023 Feb 14;12(4):619. doi: 10.3390/cells12040619.
To increase the safety of adenovirus vector (AdV)-based therapy without reducing its efficacy, a single-cycle adenovirus vector (SC-AdV) with a deletion in the protease gene (PS) was developed in order to be used as a substitute for the replication-competent adenovirus (RC-AdV). Since no infectious viral particles are assembled, there is no risk of viral shedding. The complementary cell lines for this developed AdV proved to be suboptimal for the production of viral particles and require the presence of fetal bovine serum (FBS) to grow. In the current study, we produced both stable pools and clones using adherent and suspension cells expressing the PS gene. The best adherent cell pool can be used in the early stages for the generation of protease-deleted adenovirus, plaque purification, and titration. Using this, we produced over 3400 infectious viral particles per cell. Additionally, the best suspension subclone that was cultured in the absence of FBS yielded over 4000 infectious viral particles per cell. Harvesting time, culture media, and concentration of the inducer for the best suspension subclone were further characterized. With these two types of stable cells (pool and subclone), we successfully improved the titer of protease-deleted adenovirus in adherent and suspension cultures and eliminated the need for FBS during the scale-up production. Eight lots of SC-AdV were produced in the best suspension subclone at a scale of 2 to 8.2 L. The viral and infectious particle titers were influenced by the virus backbone and expressed transgene.
为了提高腺病毒载体（AdV）治疗的安全性而不降低其疗效，开发了一种缺失蛋白酶基因（PS）的单周期腺病毒载体（SC-AdV），用作复制缺陷型腺病毒（RC-AdV）的替代品。由于没有组装传染性病毒颗粒，因此不存在病毒脱落的风险。用于开发的这种 AdV 的互补细胞系证明不适合生产病毒颗粒，并且需要存在胎牛血清（FBS）才能生长。在当前的研究中，我们使用表达 PS 基因的贴壁细胞和悬浮细胞生产了稳定池和克隆。最佳的贴壁细胞池可用于早期生成蛋白酶缺失的腺病毒、噬斑纯化和滴定。使用这种方法，我们每细胞产生了超过 3400 个感染性病毒颗粒。此外，在没有 FBS 的情况下培养的最佳悬浮亚克隆可产生每细胞超过 4000 个感染性病毒颗粒。进一步对最佳悬浮亚克隆的收获时间、培养介质和诱导剂浓度进行了表征。使用这两种类型的稳定细胞（池和亚克隆），我们成功地提高了贴壁和悬浮培养中蛋白酶缺失型腺病毒的滴度，并在扩大生产过程中消除了对 FBS 的需求。在最佳悬浮亚克隆中以 2 到 8.2 L 的规模生产了 8 批 SC-AdV。病毒和感染性颗粒的滴度受病毒骨架和表达的转基因的影响。
