AAV-Containing Exosomes as a Novel Vector to Improve AAV-Mediated Myocardial Gene Delivery in Resistance to Neutralizing Antibody

Abstract

Introduction: Heart failure is a major cause of morbidity and mortality. Adeno-associated virus (AAV)-based gene therapy delivering the sarcoplasmic reticulum calcium ATPase (SERCA2a) to cardiomyocytes has shown promising results improving heart functions in phase 1 and phase 2a clinical trials. However, potential blocking of AAVs by neutralizing antibody (Nab) is a major challenge limiting the efficacy of AAV-mediated gene delivery. More than 90% of the human population is naturally infected with AAV and about half have Nab against the virus. Thus, to advance gene therapies for cardiovascular treatment for a wider population, it is essential to develop new vehicles that minimize Nab neutralization, while retaining the benefits of AAV-mediated gene delivery. Exosomes are nano-sized cell-secreted vesicles that mediate intercellular communication and transfer selective proteins and RNAs to recipient cells. Recent studies have shown that exosomes can carry several types of viruses including hepatitis and AAV.Hypothesis: Exosomes can naturally envelope AAV-vectors to shield from neutralizing antibody and AAV-containing exosomes (AAVExo) can be superior agents for delivering genes to the cardiomyocytes and to the heart.Methods and Results: First we developed a robust method combining sucrose cushion and density gradient ultracentrifugation to efficiently purify AAVExo with minimal contamination of free AAVs. We evaluated gene transfer efficiency of AAVExo both in cultured and iPS-derived cardiomyocytes from human in vitro and in a rodent model in vivo. We found that AAVExo had significant enhancement in gene transduction compared to free AAVs. Remarkably, AAVExo was much more resistant to Nab than AAV both in vitro and in vivo. Finally, we generated AAV9Exo-SERCA2a and tested the therapeutic effects in a mouse model with myocardial infarction. We found AAVExo outperformed conventional AAV vectors in preserving cardiac functions both in presence and absence of Nab.Conclusion: AAVExo represents a novel vector for high-efficient myocardial gene delivery and a promising strategy to potentially overcome Nab neutralizing limitation in conventional AAV gene therapy.