Viral gene replication enhances AAV vector quality and reduces manufacturing costs

Viral vectors for gene and cell therapy are typically produced by transfecting plasmids into mammalian cells. Because plasmid DNA is not replicated in these cells, large quantities are required to express sufficient viral proteins for vector packaging, contributing significantly to manufacturing costs and plasmid-derived contaminants. Here, we harness the DNA replication machinery of human adenovirus to efficiently amplify plasmid DNA within HEK293 cells for adeno-associated virus (AAV) vector production. This approach enables vector manufacturing with 10- to 20-fold less plasmid encoding AAV viral genes, while replication in cells achieves up to 400-fold amplification to support packaging. Coordinated amplification and expression of viral genes recapitulate wild-type AAV biology, conferring gene therapy vectors with beneficial attributes such as high packaging efficiency and infectivity. Implementing this process during AAV vector production increases yield, full capsid ratio, reduces plasmid backbone contaminants, and notably, enhances vector potency up to 3-fold both in vitro and in vivo. AAV vector production with robust plasmid DNA in cellulo replication (AAVPCR) proves effective across serotypes and transgenes, offering broad potential to improve the safety, efficacy, and affordability of AAV gene therapies. Moreover, plasmid DNA replication in mammalian cells may enable diverse applications in the biomanufacturing of gene and cell therapy vectors.