Recombinant adeno-associated virus (rAAV) vectors are among the most effective for gene therapy. A significant advancement in rAAV vector production is developing the triple-plasmid transfection method, which remains the most widely used technique. In this study, we used Expi293FTM (Expi293F) and Viral Production Cells 2.0 (VPC2.0 cells) to evaluate various transfection reagents, comparing transgene protein expression levels and intracellular plasmid copy numbers to optimize rAAV production. Our findings indicated that the effectiveness of transfection reagents in promoting higher rAAV production was cell-dependent and that rAAV productivity correlated more with plasmid levels in the cell nucleus than with transgene protein expression levels. Confocal laser microscopy revealed that in cells transfected with the high-yield transfection reagent, a large amount of free plasmid DNA entered the nucleus, whereas the transfection reagents themselves did not. These results provide new insights into the intracellular mechanisms underlying efficient rAAV vector production. Furthermore, identifying transfection reagents that facilitate nuclear plasmid delivery will aid in the selection of optimal reagents for high-yield AAV production.
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