The increasing demand for adeno-associated virus (AAV) vectors, a result from the surging interest for their potential to cure human genetic diseases by gene transfer, tumbled on low-performing production systems. Innovative improvements to increase both yield and quality of the vector produced have become a priority undertaking in the field. In a previous study, we showed that adding a specific concentration of sodium chloride (NaCl) to the production medium resulted in a dramatic increase of AAV vector particle and infectious titers when using the herpes simplex virus (HSV) production system, both in adherent or suspension platforms. In this work, we studied additional salts and their impact on AAV vector production. We found that potassium chloride (KCl), or a combination of KCl and NaCl, resulted in the highest increase in AAV vector production. We determined that the salt-mediated effect was the most impactful when the salt was present between 8 and approximately 16 h post-infection, with the highest rate increase occurring within the first 24 h of the production cycle. We showed that the AAV vector yield increase did not result from an increase in cell growth, size, or viability. Furthermore, we demonstrated that the impact on AAV vector production was specifically mediated by NaCl and KCl independently of their impact on the osmolality of the production media. Our findings convincingly showed that NaCl and KCl were uniquely efficacious to promote up to a 10-fold increase in the production of highly infectious AAV vectors when produced in the presence of HSV. We think that this study will provide unique and important new insights in AAV biology toward the establishment of more successful production protocols.

Recent clinical successes have propelled recombinant adenoassociated virus vectors (rAAV) to the center stage for human gene therapy applications. However, the exploding demand for high titers of highly pure rAAV vectors for clinical applications and market needs remains hindered by challenges met at the manufacturing stage. The production of rAAV by transfection in suspension cells remains one of the most commonly used production platforms. In this study, we describe our optimized protocol to produce rAAV by polyethyleneimine (PEI)mediated transfection in suspension HEK293 cells, along with a side-by-side comparison to our high-performing system using the herpes simplex virus (HSV). Further, we detail a new, robust, and highly efficient downstream purification protocol compatible with both transfection and infection-based harvests that generated rAAV9 stocks of high purity. Our in-depth comparison revealed quantitative, qualitative, and biological differences between PEI-mediated transfection and HSV infection. The HSV production system yielded to higher rAAV vector titers, higher specific yields, and a higher percentage of full capsids than transfection. Furthermore, HSV-produced stocks had a significantly lower concentration of residual host cell proteins and helper DNA impurities, but contained detectable levels of HSV DNA. Importantly, the potency of PEI-produced and HSV-produced rAAV stocks were identical. Analyses of AAV Rep and Cap expression levels and replication showed that HSV-mediated production led to a lower expression of Rep and Cap, but increased levels of AAV genome replication. Our methodology enables high-yield, high purity rAAV production and a biological framework to improve transfection quality and yields by mimicking HSV-induced biological outcomes.