Abstract Body (Do not enter title and authors here): Background: Adeno-associated viruses (AAV) have been widely used to deliver therapeutic genes to the heart due to their safety and efficacy. However, a major obstacle to their successful gene delivery is the presence of neutralizing antibody (NAb) which form during natural exposure to AAV or following AAV administration. Our recent publication has shown that EV-encapsulated AAVs (EV-AAVs) shields AAVs from NAb neutralization, and overcoming this key roadblock associated with AAV-mediated gene therapy.

Hypothesis: In this study, we addressed whether EV-AAVs can target cardiomyocytes in vivo.

Aims: To test whether cardiomyocytes effectively uptake EV-AAVs in vivo, in a pre-clinical swine model.

Methods and Results: Adult pigs prescreened for a lower NAb titer were randomly assigned to receive intramyocardial administration of either vehicle, AAV6-Luciferase or EV-AAV6-Luciferase. A total of six injections across three injection sites within the left ventricle (LV) were administered per animal. All animals were sacrificed at four weeks and liver and the LV tissues were collected. Luciferase activity assay, flow cytometry analysis of isolated cardiomyocytes (CM) and non-myocytes (NM), and immunofluorescence microscopy were performed to evaluate transgene expression and cellular tropism. Echocardiography was performed at baseline and at 4W to monitor cardiac function. At 4W, EV-AAV6s resulted in a higher luciferase expression in LV compared to nude AAV6 particles. Expression in the LV was higher compared to the liver. Flow cytometry analysis revealed a higher luciferase expression in CM compared to NM, which was corroborated by Immunofluorescence. No differences were detected in the global heart function across groups. Biodistribution of EV-AAV uptake and transgene expression are being studied with intramyocardial and intracoronary delivery mechanisms.

Conclusion: EV-AAVs deliver higher amounts of genes than nude AAV6 via intramyocardial administration in a pre-clinical swine model. CMs preferentially uptake EV-AAVs compared to NMs. Our studies suggest that EV-AAVs are a superior cardiac gene delivery vector for future clinical studies.