Distinguishing Protein and Gene Delivery Enables Characterization and Bioengineering of Extracellular Vesicle-Adeno-Associated Virus Vectors

ABSTRACT Adeno-associated virus (AAV) gene therapies have achieved clinical success, with multiple products reaching regulatory approval. Encapsulation of AAV vectors within engineered extracellular vesicles (EVs) is an emerging strategy which could help overcome challenges including pre-existing anti- capsid immunity and the need for controlling targeting and tropism. To guide the development of EV-AAV technologies, we developed an assay for quantifying and controlling for the contribution of pseudotransduction to evaluations of EV-AAV-mediated gene delivery. We developed an AAV vector that switches its transgene output from one reporter to another when acted upon by Cre recombinase expressed in a recipient cell. Using this platform, we investigated EV-AAV transduction as a function of various engineered EV surface modifications. For actively endocytic cells (HEK293FTs), modifications that enhance EV uptake and membrane fusion influence protein delivery but not gene delivery. Conversely, in less endocytic Jurkat T cells, modifications enhancing EV uptake improved both protein and gene delivery. These conclusions held across multiple AAV serotypes. Our results resolve apparent conflicts in prior reports and suggest that effects of enhancing uptake and membrane fusion of EV-AAV vectors are recipient cell type specific. The methods developed here unambiguously dissect EV-AAV transduction mechanisms and can guide future bioengineering of EV-AAV vectors.