Enhanced rAAV Production in HEK293 Cells by Promoting Helper Plasmid Nuclear Import Under HDAC6 Inhibition

Recombinant adeno-associated virus (rAAV) vectors have emerged as leading platforms for gene therapy owing to their safety, long-term transgene expression, and broad tissue tropism. However, rAAV production in HEK293 cells via triple-plasmid transfection remains limited by low yield, partly due to inefficient nuclear import of plasmids. In this study, we enhanced rAAV production by promoting plasmid nuclear delivery. DNA-targeting sequence (DTS) insertion alone did not improve plasmid nuclear import or rAAV yield, whereas combining DTS insertion with tubacin, an HDAC6 inhibitor, significantly increased nuclear plasmid accumulation and rAAV productivity. Among the three plasmids, inserting the 5NF sequence into pHelper produced the greatest effect. Under tubacin treatment, pHelper-5NF increased rAAV5 yield from 1.08 × 10¹¹ to 2.44 × 10¹¹ vg/mL (2.26-fold), without altering the full capsid percentage. This improvement was associated with increased Rep/Cap expression and viral DNA replication. Moreover, the pHelper-5NF enhanced rAAV production across multiple capsid serotypes, including rAAV1, rAAV2, rAAV6, rAAV8, rAAV9, and rAAVrh10. Unexpectedly, insertion of 5NF into two or three plasmids did not further increase rAAV yield via triple-plasmid transfection. These results identify pHelper-5NF combined with HDAC6 inhibition as an effective strategy to enhance triple-plasmid transfection-based rAAV manufacturing.