AAVs Meet Exosomes: Exosomal AAVs for Shielding Neutralizing Antibodies and Improved Cardiac Gene Delivery in vivo

Abstract

Introduction:Due to their safety profile, tissue tropism and long-term transgene expression, adeno-associated viruses (AAVs) have become the vector of choice for human gene therapy. However, pre-existing neutralizing antibodies (NAbs) to many AAV serotypes pose a critical challenge for the translation of gene therapies to clinic. Here, we describe the use of exosomal AAVs (eAAV) as a robust cardiac gene delivery system that enhance transduction efficiency while shielding from pre-existing humoral immunity to the viral capsid.Methods & Results:We developed a multi-step ultracentrifugation strategy to isolate eAAVs from AAV-producing HEK-293T cells with minimal contamination of standard AAVs. Importantly, we demonstrated through electron microscopy-based visualization, immunoblotting and proteinase protection assay that iodixanol gradient-purified eAAVs contain AAV capsids inside. Efficiency of heart targeting was then evaluated for eAAV9 or eAAV6 and standard AAV9 or AAV6 vectors in human cardiomyocytes (hCM) in vitro and in passive immunity nude mouse model in vivo. Regardless of the presence or absence of NAbs, we show that eAAVs were more efficient in cell transduction in the same titer ranges as standard AAVs. To test the therapeutic efficacy, we injected eAAV9-SERCA2a or AAV9-SERCA2a intramyocardially in NAb+ post-myocardial infarction (MI) mice. Remarkably, eAAV9-SERCA2a outperformed standard AAVs significantly improving cardiac function in presence of NAbs (%EF 55.14 ? 3.50 compared to 27.31 ? 1.63 at 6 weeks, respectively). We further demonstrated in vivo that eAAV9-mediated gene delivery is more specific to CMs than to other cardiac cells, which suggests that eAAVs preserve cardiotropic properties of AAV9 serotype. By examining colocalization of eAAVs and markers specific for endosomes in hCMs in vitro, our preliminary data indicated that eAAV6 infectious entry potentially involves trafficking via endocytic compartments.Conclusions:Delivery of standard AAVs protected by carrier exosomes (i.e. eAAVs) is therefore a promising approach to evade pre-existing NAbs while still efficiently transducing myocardium, which can be applied in the broader population of MI patients and may result in higher gene delivery efficacy.