Extracellular Vesicle–Encapsulated Adeno-Associated Viruses for Therapeutic Gene Delivery to the Heart

作者：Xisheng Li, Sabrina La Salvia, Yaxuan Liang, Marta Adamiak, Erik Kohlbrenner, Dongtak Jeong, Elena Chepurko, Delaine K. Ceholski, Estrella López-Gordo, Seonghun Yoon, Prabhu Mathiyalagan, Neha Agarwal, Divya Jha, Shweta Lodha, George Daaboul, Anh Tuân Phan, Nikhil Raisinghani, Shihong Zhang, Lior Zangi, Edgar Gonzalez‐Kozlova, Nicole Dubois, Navneet Dogra, Roger J. Hajjar, Susmita Sahoo · 发表于：Circulation · 年份：2023 · DOI：10.1161/circulationaha.122.063759 · 被引用次数：88 · 研究领域：Virus-based gene therapy research、Extracellular vesicles in disease、Viral Infections and Immunology Research

BACKGROUND: Adeno-associated virus (AAV) has emerged as one of the best tools for cardiac gene delivery due to its cardiotropism, long-term expression, and safety. However, a significant challenge to its successful clinical use is preexisting neutralizing antibodies (NAbs), which bind to free AAVs, prevent efficient gene transduction, and reduce or negate therapeutic effects. Here we describe extracellular vesicle-encapsulated AAVs (EV-AAVs), secreted naturally by AAV-producing cells, as a superior cardiac gene delivery vector that delivers more genes and offers higher NAb resistance. METHODS: We developed a 2-step density-gradient ultracentrifugation method to isolate highly purified EV-AAVs. We compared the gene delivery and therapeutic efficacy of EV-AAVs with an equal titer of free AAVs in the presence of NAbs, both in vitro and in vivo. In addition, we investigated the mechanism of EV-AAV uptake in human left ventricular and human induced pluripotent stem cell-derived cardiomyocytes in vitro and mouse models in vivo using a combination of biochemical techniques, flow cytometry, and immunofluorescence imaging. RESULTS: Using cardiotropic AAV serotypes 6 and 9 and several reporter constructs, we demonstrated that EV-AAVs deliver significantly higher quantities of genes than AAVs in the presence of NAbs, both to human left ventricular and human induced pluripotent stem cell-derived cardiomyocytes in vitro and to mouse hearts in vivo. Intramyocardial delivery of EV-AAV9-sarc...