Abstract Body: Introduction: We have recently shown that extracellular vesicle-encapsulated AAVs (EV-AAVs) are resistant to neutralizing antibodies (NAbs), and compared to nude AAVs, deliver higher amounts of genes in the presence of NAbs. Interestingly, EV-AAVs exhibited a preferential delivery to cardiomyocytes (CMs), even in the absence of NAbs, suggesting potential benefits in cardiac gene therapy.
Aims: To study the cellular and molecular mechanisms of EV-AAVs cardiac uptake, cellular trafficking, and biodistribution in small and large animal models.
Methods: Cardiotropic delivery and post-lysosomal nuclear entry were compared between CMs and NM using fluorescence-tagged EV-AAVs and nude AAVs in H9C2 cells and human iPS-CMs, in vitro, and mice, in vivo. We used either AAV6-Luciferase or EV-AAV6-Luciferase to compare cardiac and liver gene delivery and cellular targeting using luciferase activity assay and immunofluorescence. Using GFP-tagged EVs, we also evaluated the cellular uptake and biodistribution of EVs via intracoronary or intramyocardial administration in pigs.
Results: Flow cytometry analysis of single cell suspensions showed a preferential uptake of EV-GFP by CMs as compared to and NMs, both in vitro and in vivo. Notably, for hiPSC-CMs, the uptaken EV-AAVs were detected in the perinuclear region, whereas in hiPSC-NMs, they appeared diffused in the cytoplasm, suggesting a distinct intracellular trafficking for CMs and NMs. EV-AAV6-Luc injections led to notably higher luciferase expression in myocardial tissue compared to nude AAV6, four weeks post-administration in pigs. Interestingly, the liver exhibited higer vector genome copy number but lower transgene expression, compared to the heart. Intracoronary administration of EV-GFP resulted in a broad labeling of the myocardium, seen by immunofluorescence.
Conclusions: CMs exhibited a higher uptake of EV-AAV6 over NMs. EV-AAV6 demonstrated an enhanced transfection efficiency over AAV6 via direct delivery to the heart of rodents and pigs. This data suggest that EV-AAVs can serve as a superior cardiac gene delivery vector, opening new avenues for understanding the mechanisms of EV-AAV-mediated cardiac gene delivery and thus supporting its clinical translation.