Probing aspects of extracellular vesicle associated AAV allows increased vector yield and insight into its transduction and immune-evasive properties.

Extracellular vesicle-associated adeno-associated virus vectors (EV-AAVs) 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 compared with 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 the 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 and and evade antibodies compared with conventional AAV9. We tested whether -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. -expression of MAAP8 or MAAP9 increased yields of EV-AAV9 compared with the -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.