Deciphering Key Adenoviral Elements in the Production of Recombinant Adeno-Associated Virus Vectors

Over the last two decades, adeno-associated viruses (AAVs) have been widely used as viral vectors in gene therapy due to their ability to infect both dividing and nondividing cells, broad tissue specificity, and favorable safety profile. Recombinant AAV (rAAV) production requires a helper virus, typically adenovirus (AdV), which provides essential genes for AAV replication. However, the increasing demand for safer and more efficient rAAV production methods led to the need to develop helper plasmids with minimal AdV components. In this study, we evaluate the impact of AdV E2 and E4 in the productivity and genome packaging of rAAV serotypes 2, 5, 8, and 9, produced by transient transfection. We designed and tested eight novel helper plasmids with different deletions in E2 and E4 genes. Results indicated that deletions in these genes significantly affected rAAV productivity and packaging, particularly for serotypes 8 and 9. Helper plasmids containing minimal essential genes-E2-DBP, E4orf6, and VA RNA-showed near to 10-fold reduction in viral genome packaging compared to the control. However, including E2 L4-22/33K and E4orf3 regions significantly improved viral production, particularly for serotypes 8, and 9. In this study, we also demonstrated that the full E4 gene is crucial to achieving high full-empty ratios, minimizing the production of empty capsids, and enhancing viral release into the culture medium of rAAV8. Accordingly, we created a smaller plasmid, without adenoviral structural proteins that allows a similar rAAV production across all tested serotypes. Overall, these findings provide insights into the genetic requirements for efficient rAAV production and highlight the importance of the E2 and E4 regions for optimizing viral yield and quality. This approach could lead to the development of improved strategies for large-scale rAAV vector production by enabling safer and more cost-effective systems.