Meade Oliver, Clark Jeffrey, McCutchen Michael, Kerwin John
Gene Therapy Process Development, Resilience, Boston, MA, United States.
Gene Therapy Process Development, Resilience, Boston, MA, United States.
Methods Enzymol. 2021;660:341-360. doi: 10.1016/bs.mie.2021.08.003. Epub 2021 Sep 22.
The safety and utility of adeno-associated virus (AAV) to modulate target gene expression has been well demonstrated, and AAV vectors are a leading gene therapy platform. However, manufacturing presents challenges in terms of productivity and scalability as compared to incumbent therapeutic modalities. In particular, a pivot from adherent cell- to suspension culture-based AAV manufacturing processes requires enhanced study of the transfection step. For the method proposed herein, a Response Surface Design of Experiments is suggested to explore the role of five transfection factors-cell density at transfection, DNA concentration, ratio of complexing reagent to DNA, and molar ratios of the transfecting plasmids-influencing viral genome titer and biological potency. Additionally, an AAV categorical factor matrix is presented for developing a workflow to interrogate the impact of AAV permutations for different capsid serotypes, harbored genes of interest, and inverted terminal repeat configurations on transfection process parameters.
腺相关病毒（AAV）调节靶基因表达的安全性和实用性已得到充分证明，AAV载体是领先的基因治疗平台。然而，与现有治疗方式相比，其生产在生产率和可扩展性方面存在挑战。特别是，从基于贴壁细胞培养到基于悬浮培养的AAV生产工艺的转变需要加强对转染步骤的研究。对于本文提出的方法，建议采用实验的响应面设计来探索五个转染因素——转染时的细胞密度、DNA浓度、络合试剂与DNA的比例以及转染质粒的摩尔比——对病毒基因组滴度和生物学效力的作用。此外，还提出了一个AAV分类因子矩阵，用于开发一个工作流程，以研究不同衣壳血清型、感兴趣的携带基因和反向末端重复序列构型的AAV排列对转染工艺参数的影响。
