Exploring the design space of AAV transient-transfection in suspension cell lines.

Abstract

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.