Interferon inhibitors increase rAAV production in HEK293 cells

Recombinant adeno-associated viruses (rAAVs) comprise a promising viral vector for therapeutic gene delivery to treat disease. However, the current manufacturing capability of rAAVs must be improved to meet commercial demand. Previously published omics studies indicate that rAAV production through transient transfection triggers antiviral responses and endoplasmic reticulum stress responses in the host cell. Both responses negatively regulate viral production. We demonstrate that the modulation of the antiviral immune response (by blocking interferon signaling pathways) can effectively lower the production of interferon and enhance viral genome production. The use of interferon inhibitors before transfection can significantly increase rAAV production in HEK293 cells, with up to a 2-fold increase in productivity and up to a 6-fold increase in specific productivity. Compared to the untreated groups, the addition of these small molecules generally reduced viable cell density but increased vector productivity. The positive candidates were BX795 (a TBK inhibitor), TPCA-1 (an IKK2 inhibitor), Cyt387 (a JAK1 inhibitor), and ruxolitinib (another JAK1 inhibitor). These candidates were identified using deep well screening, and reproducible titer improvement was achieved in a 30 mL shake flask scale. Additionally, genome titer improvement is feasible and scalable in two different media, but the extent of improvement may vary.