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

Author:

Li Xisheng1,La Salvia Sabrina1,Liang Yaxuan2,Adamiak Marta1,Kohlbrenner Erik13,Jeong Dongtak4,Chepurko Elena1,Ceholski Delaine1,Lopez-Gordo Estrella1,Yoon Seonghun1,Mathiyalagan Prabhu5ORCID,Agarwal Neha1ORCID,Jha Divya1,Lodha Shweta1,Daaboul George6,Phan Anh1,Raisinghani Nikhil1,Zhang Shihong1ORCID,Zangi Lior1ORCID,Gonzalez-Kozlova Edgar7ORCID,Dubois Nicole89,Dogra Navneet1011,Hajjar Roger J.12ORCID,Sahoo Susmita1ORCID

Affiliation:

1. Cardiovascular Research Institute (X.L., S.L.S., M.A., E.C., D.C., E.L.-G., S.Y., N.A., D.J., S.L., A.P., N.R., S.Z., L.Z., S.S.), Icahn School of Medicine at Mount Sinai, New York, NY.

2. Center for Biological Science and Technology, Advanced Institute of Natural Sciences, Beijing Normal University, Zhuhai, China (Y.L.).

3. Spark Therapeutics, Philadelphia, PA (E.K.).

4. Department of Molecular and Life Science, College of Science and Convergence Technology, Hanyang University-ERICA, Ansan, South Korea (D.J.).

5. Benthos Prime Central, Houston, TX (P.M.).

6. NanoView Biosciences, Boston, MA (G.D.).

7. Department of Oncological Sciences (E.G.-K.), Icahn School of Medicine at Mount Sinai, New York, NY.

8. Department of Cell, Developmental and Regenerative Biology (N. Dubois), Icahn School of Medicine at Mount Sinai, New York, NY.

9. Mindich Child Health and Development Institute (N. Dubois), Icahn School of Medicine at Mount Sinai, New York, NY.

10. Department of Pathology and Laboratory Medicine (N. Dogra), Icahn School of Medicine at Mount Sinai, New York, NY.

11. Icahn Genomics Institute (N.Dogra), Icahn School of Medicine at Mount Sinai, New York, NY.

12. Gene and Cell Therapy Institute, Massachusetts General Brigham, Boston (R.J.H.).

Abstract

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–sarcoplasmic reticulum calcium ATPase 2a to infarcted hearts in preimmunized mice significantly improved ejection fraction and fractional shortening compared with AAV9–sarcoplasmic reticulum calcium ATPase 2a delivery. These data validated NAb evasion by and therapeutic efficacy of EV-AAV9 vectors. Trafficking studies using human induced pluripotent stem cell–derived cells in vitro and mouse hearts in vivo showed significantly higher expression of EV-AAV6/9–delivered genes in cardiomyocytes compared with noncardiomyocytes, even with comparable cellular uptake. Using cellular subfraction analyses and pH-sensitive dyes, we discovered that EV-AAVs were internalized into acidic endosomal compartments of cardiomyocytes for releasing and acidifying AAVs for their nuclear uptake. CONCLUSIONS: Together, using 5 different in vitro and in vivo model systems, we demonstrate significantly higher potency and therapeutic efficacy of EV-AAV vectors compared with free AAVs in the presence of NAbs. These results establish the potential of EV-AAV vectors as a gene delivery tool to treat heart failure.

Publisher

Ovid Technologies (Wolters Kluwer Health)

Subject

Physiology (medical),Cardiology and Cardiovascular Medicine

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