Enhancement of Recombinant Adeno‐Associated Virus Production in HEK293 Cells by Application of Small Molecule Additives and Genetic Regulation Based on Perfusion Technique

Adeno-associated virus (AAV) vectors offer numerous advantages, including low immunogenicity and a high safety profile, but their development and wide application are still hindered by some technical limitations. Recent studies have shown that small molecule chemical additives can significantly increase the yield of rAAV vectors in HEK293. Our study found that the antimitotic nocodazole, a positive regulator of the rAAV genomic titer, approximately doubled the yield of rAAV vectors. In triple-transfected HEK293 suspension cells used for rAAV production, the addition of nocodazole caused the cells to arrest at G2/M phase. Compared to untreated cells, nocodazole-treated cells-initiated mitosis but were unable to undergo cytokinesis, resulting in prolonged mitotic arrest and apoptosis, this reduced the viable cell density at harvest. The final crude genomic vector titer of nocodazole-treated cultures was more than 1.7-fold higher than that of untreated controls. Optimal enhancement was observed when nocodazole was administered 2 hours post-transfection (hpt). Subsequent transcriptome analyses comparing cultures with and without nocodazole identified the key genes ZFP91 and SFRP5. Overexpression of ZFP91 and silencing of SFRP5 led to an increase in the G2/M phase arrested cells, reflecting the effect of nocodazole treatment. This delay in spindle formation increased packaging time and significantly increased rAAV vector yield by 2 to 3-fold. These findings highlight the potential for optimizing cellular conditions through small molecule additives and genetic modifications to overcome existing bottlenecks in AAV production.