Extracellular vesicle-encapsulated AAVs for therapeutic gene delivery to the heart

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. Researchers at the Icahn School of Medicine at Mount Sinai 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.

The researchers have developed a 2-step density-gradient ultracentrifugation method to isolate highly purified EV-AAVs. The researchers 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, they investigated the mechanism of EV-AAV uptake in human left ventricular and human induced pluripotent stem cell cardiomyocytes in vitro and mouse models in vivo using a combination of biochemical techniques, flow cytometry, and immunofluorescence imaging.

Density-gradient ultracentrifugation-based purification process successfully enriched EV-AAVs


A, Schematic of extracellular vesicle–encapsulated adeno-associated vector (EV-AAV) isolation, characterization, function, tropism, and uptake mechanism. B, Density of 12 fractions obtained after iodixanol density-gradient ultracentrifugation (n=3). C, Western blot analysis of the 12 fractions. Equal volume of fractions was loaded on SDS-PAGE gels, and membranes were blotted with Alix, Flot1, Tsg101, Gm130, and Cyc1. D, Western blot quantification for the percent of Alix-relative expression in 12 fractions (n=3). E, Determination of AAV vector genomes with quantitative polymerase chain reaction in the 12 fractions (n=3). F and G, Nano-flow cytometry analysis and quantification of surface tetraspanin proteins, including CD63, CD81, and CD9, in the 12 fractions (n=3). Mean equivalent soluble fluorochromes (MESFs) were used to calibrate the fluorescence scale in MESF units for each fraction. No MESF values were detected in fractions other than Fr3 and Fr4, in which EV-AAVs were properly separated from other contaminants. H, Tunable resistive pulse sensing (TRPS)–qNano analysis of Fr3 and Fr4 measured EV-AAV size. Values in B and D through F are presented as mean±SEM.

Using cardiotropic AAV serotypes 6 and 9 and several reporter constructs, they 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 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, the researchers discovered that EV-AAVs were internalized into acidic endosomal compartments of cardiomyocytes for releasing and acidifying AAVs for their nuclear uptake.

Together, using 5 different in vitro and in vivo model systems, these researchers 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.

Li X, La Salvia S, Liang Y, Adamiak M, Kohlbrenner E, Jeong D, Chepurko E, Ceholski D, Lopez-Gordo E, Yoon S, Mathiyalagan P, Agarwal N, Jha D, Lodha S, Daaboul G, Phan A, Raisinghani N, Zhang S, Zangi L, Gonzalez Kozlova E, Dubois N, Dogra N, Hajjar RJ, Sahoo S. (2023) Extracellular Vesicle-Encapsulated AAVs for Therapeutic Gene Delivery to the Heart. Circulation [Epub ahead of print]. [abstract]

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