Factors affecting recombinant Adeno-Associated Virus titers during triple-plasmid transient transfection in HEK-293 cells

Abstract The efficiency of triple-plasmid transfection in recombinant Adeno-Associated Virus (rAAV) production was analyzed by examining two distinct strains of HEK-293 cells. These strains were categorized as high producer (HP) and low producer (LP) based on their differing levels of productivity under identical conditions. Analysis of RNA expression levels of viral genes revealed disparities in plasmid derived gene expression between the cell lines. Further assessment of transfection efficacy utilizing labeled plasmids revealed lower plasmid uptake and less efficient nuclear transport in LP cell line. Additionally, we observed inferior translation activity in LP, contributing to its shortcomings in overall productivity. In our attempt to optimize plasmid ratios to enhanced fully packaged rAAV particle yield, we discovered cell-strain-specific optimization potential. The findings highlight the transfection's complexity, urging tailored strategies for improved rAAV production based on each cell line's characteristics, enhancing understanding and guiding further efficiency optimization in rAAV production.