Modulation of the cell cycle and inhibition of histone deacetylases by small molecules increase recombinant adeno‐associated virus productivity across different <scp>HEK293</scp> cell lines

Recombinant adeno-associated viruses (rAAV) are one of the most popular gene therapy vectors. To date, low-product yields are limiting a broader clinical application. To identify targets for improving productivity, two human embryonic kidney cell lines (HEK293) with varying productive profiles were transiently transfected for rAAV2 production and transcriptomes were compared at 18 h after transfection. As expected, high-producing cell lines exhibited elevated levels of plasmid-derived viral gene expression. Gene set enrichment analysis indicated that these cells demonstrated increased transcriptional activity and upregulation of mRNA-processing mechanisms. Furthermore, transcriptomic analysis suggested increased transcription of histone-coding genes and a modulated cell cycle under the influence of viral gene expression, with differences being more prominent in the high-producer cell line. Aiming to increase rAAV yield, cyclin-dependent kinases and histone deacetylases were targeted by treatment with the small molecule inhibitors Flavopiridol and M344, respectively. Without compromising biological activity, Flavopiridol increased rAAV titer by 2-fold, and M344 increased it up to 8-fold in a cell line-independent manner, while also enhancing the percentage of filled capsids. A DoE-based approach also revealed the potential for combining both molecules to enhance rAAV production, exhibiting an additive effect across three different HEK293 derivatives. Consequently, novel functions of M344 and Flavopiridol as enhancers of rAAV production were unraveled, which can be employed to enhance the accessibility of in vivo gene therapy applications.