Probing aspects of extracellular vesicle (EV)-associated AAV biology and composition allows increased vector yield and insight into its transduction and immune-evasive properties

Extracellular vesicle-associated adeno-associated virus vectors (EV-AAV) are generated during production in 293 cells. EV-AAV provides desirable gene delivery traits such as greater resistance to antibody neutralization and increased transduction of organs in vivo compared to conventional AAV. Despite these promising data, better characterization of EV-AAV is needed. We used density gradient ultracentrifugation to separate EV-AAV from free AAV to determine yields and functional activity of EV-AAV. We found that the fraction of EV-AAV to conventional AAV in culture media from six AAV serotypes ranged from 0.5 to 12%. Next, we assessed whether intraluminal EV-AAV9 could mediate functional transduction of cells and observed that a portion of EV-AAV9 are intraluminal and mediated transduction of cultured cells in vitro and in vivo and evade antibodies compared to conventional AAV9. We tested whether trans expression of membrane-associated accessory protein (MAAP) from AAV8 (MAAP8) or AAV9 (MAAP9) with AAV9 Cap/AAV9 MAAP null would alter yields of EV-AAV9. Trans expression of MAAP8 or MAAP9 increased yields of EV-AAV9 compared to the cis-expression of AAV9 Cap/AAV9 MAAP. Finally, we found that the capsid was required for efficient transduction of cultured cells by EV-AAV. In sum, these data provide a foundation for the development of EV-AAV vectors. [Display omitted] Maguire and colleagues explore properties of highly purified extracellular vesicle-associated AAV vectors. They find that intraluminal AAV inside EVs is functional at transgene expression and antibody evasion, MAAP transcomplementation can increase EV-AAV yields, and that the capsid is required for the highest transduction efficiency.