Gene therapy holds great promise for the treatment of severe diseases, and adeno-associated virus (AAV) vectors have emerged as valuable tools in this field. However, challenges such as immunogenicity and high production costs complicate the commercial viability of AAV-based therapies. To overcome these barriers, improvements in production yield, driven through the availability of robust and sensitive characterization techniques that allow for the monitoring of critical quality attributes to deepen product and process understanding are crucial. Among the main attributes affecting viral production and performance, the ratio between empty and full capsids along with capsid protein stoichiometry are emerging as potential parameters affecting product quality and safety. This study focused on the production of AAV vectors using the baculovirus expression vector system (BEVS) in Sf9 cells and the complete characterization of AAV5 variants using novel liquid chromatography and mass spectrometry techniques (LC-MS) that, up to this point, had only been applied to reference commercially produced virions. When comparing virions produced using ATG, CTG or ACG start codons of the cap gene, we determined that although ACG was the most productive in terms of virus yield, it was also the least effective in transducing mammalian cells. This correlated with a low VP1/VP2 ratio and a higher percentage of empty capsids. Overall, this study provides insights into the impact of translational start codon modifications during rAAV5 production using the BEVS, the associated relationship with capsid packaging, capsid protein stoichiometry and potency. The developed characterization workflow using LC-MS offers a comprehensive and transferable analysis of AAV-based gene therapies, with the potential to aid in process optimization and facilitate the large-scale commercial manufacturing of these promising treatments.

Recombinant Adeno-associated virus (rAAV) is a popular vector for treating genetic diseases caused by absent or defective genes. rAAVs can be produced that contain a therapeutic transgene, i.e., a correct copy of the affected gene, which is then delivered into target cells. A further application of rAAV is to deliver pro-apoptotic genes such as TNF-related apoptosis-inducing ligand (TRAIL) into cancer cells, leading to tumor regression. However, rAAV production is expensive and insufficient yields may hinder wide-spread adoption especially in systemic conditions. During rAAV production, the therapeutic transgene may be expressed in the producer cell line, and in the case of an oncolytic gene, this would likely lead to cell death thus reducing rAAV yields. Here we demonstrate that expression of TRAIL during rAAV production in HEK293F cells negatively impacts rAAV yield. A shRNA-based strategy was developed to suppress the expression of TRAIL in rAAV-producing cells specifically during the production process. Incorporating a TRAIL-targeting shRNA expression cassette within the backbone of the rAAV genome-encoding plasmid during triple-transfection of HEK293F cells reduced transgene expression and led to a 60% increase in the yield of rAAV-TRAIL compared to controls.