Robust Cardiac Gene Delivery and Evasion of Neutralizing Antibodies by Extracellular Vesicle-Associated AAV Vectors

Abstract

Introduction: Due to their excellent safety profile in clinical trials, adeno-associated virus (AAV) has become the vector of choice for gene delivery to the myocardium. However, pre-existing immunity to AAV from naturally present neutralizing antibodies (NAbs, present in >60% of the human population) significantly limits the potential candidates for the AAV-based gene therapy. NAbs bind to AAV and block its infection, greatly reducing transduction and clinical efficacy. Thus, development of novel AAV-based vectors that can circumvent the effect of NAbs is essential for clinical administration of AAVs. Objectives: We investigated the effectiveness of extracellular vesicle (EV)-associated AAV (EV-AAV) in resisting NAbs and enhancing vector transduction in the myocardium. Methods and Results: We isolated EV-AAV from the conditioned medium of infected 293 cells using a multi-step iodixanol density gradient ultracentrifugation. Electron microscopy, flow cytometry, bioluminescence imaging and echocardiography were used to quantify AAV-mediated gene delivery and transduction efficiency. EV-AAV6-mCherry and EV-AAV9-FLuc were more resistant to antibody-mediated neutralization than conventional AAV and induced significantly higher mCherry or firefly luciferase (FLuc) expression both in vitro and in vivo. To test the therapeutic efficacy, EV-AAV9-SERCA2a or AAV9-SERCA2a were injected intramyocardially in post-MI mice preinjected with human NAbs (IVIg). Remarkably, EV-AAV9-SERCA2a outperformed standard AAV, significantly improving cardiac function both in the presence and absence of NAbs (%EF 57.4 ± 4.7 vs. 29.2± 2.3, respectively; 6 weeks after surgery). Conclusion: EV-AAV highly outperforms conventional AAV in delivering beneficial genes, such as SERCA2a. The use of naturally enveloped AAV vectors represents a promising approach to evade NAbs, and to facilitate the clinical translation of AAV-based gene therapies to a larger human population.