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However, rAAV production in HEK293 cells via triple\u2010plasmid 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\u2010targeting 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\u20105NF increased rAAV5 yield from 1.08 \u00d7 10\n                    <jats:sup>11<\/jats:sup>\n                    to 2.44 \u00d7 10\n                    <jats:sup>11<\/jats:sup>\n                    \u00a0vg\/mL (2.26\u2010fold), without altering the full capsid percentage. This improvement was associated with increased Rep\/Cap expression and viral DNA replication. Moreover, the pHelper\u20105NF 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\u2010plasmid transfection. These results identify pHelper\u20105NF combined with HDAC6 inhibition as an effective strategy to enhance triple\u2010plasmid transfection\u2010based rAAV manufacturing.\n                  <\/jats:p>","DOI":"10.1002\/biot.70313","type":"journal-article","created":{"date-parts":[[2026,9,29]],"date-time":"2026-09-29T06:28:18Z","timestamp":1790663298000},"source":"Crossref","is-referenced-by-count":0,"title":["Enhanced rAAV Production in HEK293 Cells by Promoting Helper Plasmid Nuclear Import Under HDAC6 Inhibition"],"prefix":"10.1002","volume":"21","author":[{"ORCID":"https:\/\/orcid.org\/0009-0006-9066-2615","authenticated-orcid":false,"given":"He","family":"Ren","sequence":"first","affiliation":[{"name":"School of Biotechnology and Key Laboratory of Industrial Biotechnology of Ministry of Education Jiangnan University  Wuxi China"},{"name":"National Engineering Research Center of Cereal Fermentation and Food Biomanufacturing Jiangnan University  Wuxi 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