Comparative Analysis of rAAV Production from Plasmid-Encoded Versus Chromosomally Integrated rAAV Transgene in HEK293 Cells

Stable cell lines have recently achieved recombinant adeno-associated virus (rAAV) titers comparable to the standard triple transfection approach, making them a promising alternative to plasmid-based production systems. However, whether integration of the rAAV transgene into the host genome influences packaging efficiency and vector quality remains unclear. In this study, we generated stable HEK293 cell lines carrying the rAAV transgene in their genome. rAAV production was enabled by supplying the rep/cap and helper genes on two plasmids, rendering vector genome generation dependent on the chromosomally integrated transgene. Although the stable cell lines produced a 4.5-fold lower titer of viral genomes (VGs) compared to the standard triple transfection method, VG-normalized potency was four times higher. Detailed particle characterization further revealed 3-fold lower plasmid backbone DNA packaging in rAAVs produced by stable cell lines relative to triple transfection. Consistent results were obtained from mass photometry and ELISA/ddPCR analyses for the double transfection condition, while discrepancies emerged under triple transfection. These findings emphasize the importance of functional and qualitative assessments for evaluating different rAAV production approaches.