Abstract 12178: Extracellular Vesicle-Encapsulated AAVs for Therapeutic Gene Delivery to the Heart

Background: Adeno-associated virus (AAV) has emerged as one of the best tools for cardiac gene delivery due to its cardiotropism, long-term expression, and safety. However, a significant challenge to its successful clinical use are pre-existing neutralizing antibodies (NAbs), which bind to free AAVs, prevent efficient gene transduction and reduce or negate therapeutic effects. Here we describe extracellular vesicle-encapsulated AAVs (EV-AAVs), secreted naturally by AAV-producing cells, as a superior cardiac gene delivery vector that delivers more genes and offers higher NAb resistance. Method: We developed a two-step density-gradient ultracentrifugation method to isolate highly purified EV-AAVs. Results: Using cardiotropic AAV serotypes 6 and 9 and several reporter constructs, we demonstrated that EV-AAVs deliver significantly higher quantities of genes than AAVs in the presence of NAb, both to human left ventricular and hiPSC cardiomyocytes in vitro, and to mouse hearts in vivo. Intramyocardial delivery of EV-AAV9-SERCA2a to infarcted hearts in pre-immunized mice significantly improved ejection fraction and fractional shortening compared to AAV9-SERCA2a delivery. These data validated NAb evasion by and therapeutic efficacy of EV-AAV9 vectors. Interestingly, in vivo trafficking studies in mouse hearts showed significantly higher expression of EV-AAV9-delivered genes in cardiomyocytes compared to non-cardiomyocytes, even with comparable cellular uptake. Using cellular sub-fraction analyses and pH-sensitive dyes, we discovered that EV-AAVs were internalized into acidic endosomal compartments of cardiomyocytes for releasing and acidifying AAVs for their nuclear uptake. Conclusion: Together, using six different in vitro and in vivo model systems, we demonstrate significantly higher potency and therapeutic efficacy of EV-AAV vectors compared to free AAVs. These results establish the potential of EV-AAV vectors as a gene delivery tool to treat heart failure. 1