show Abstracthide AbstractManufacturing of Adeno-associated viruses (AAV) for gene and cell therapyapplications has increased significantly and spurred development of improvedmammalian and insect cell-based production systems. We developed a baculovirusbased insect cell production system- the SGMO Helper- with a novel gene architectureand greater flexibility to modulate the expression level and content of individual Repand Cap proteins. In addition, we incorporated modifications to the AAV6 capsidsequence that improves yield, capsid integrity and potency. Production of rAAV6 usingthe SGMO Helper had improved yields compared to the Bac-RepCap helper from theKotin lab. SGMO Helper-derived rAAV6 is resistant to a previously describedproteolytic cleavage unique to baculovirus-insect cell production systems and hasimproved capsid ratios and potency, in vitro and in vivo, compared to rAAV6 producedusing Bac-RepCap. NGS sequence analysis demonstrated that the SGMO Helper isstable over six serial passages and rAAV6 capsids contain comparable amounts ofnon-transgene DNA as rAAV6 produced using Bac-RepCap. AAV production using theSGMO Helper is scalable using bioreactors and has improved yield, capsid ratio and invitro potency. Our studies demonstrate that the SGMO Helper is an improved platformfor AAV manufacturing to enable delivery of cutting-edge gene and cell therapies.