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

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.

Multi-Center, Randomized, Double-Blind, Placebo-Controlled, Exploratory Study to Evaluate the Efficacy and Safety of HAD-B1 for Dose-Finding in EGFR Mutation Positive and Locally Advanced or Metastatic NSCLC Subjects Who Need Afatinib Therapy.

Afatinib is a target anticancer drug of the second-generation EGFR TKI type, showing an advantage in treatment effect compared to conventional chemotherapy. However, patients on EGFR-TKI drugs also usually progress after 9 to 13 months according to secondary resistance. HAD-B1 is composed of drugs that are effective against lung cancer. This study is an exploratory study to evaluate the efficacy and safety between dosage groups by conducting a clinical trial in subjects requiring afatinib drug treatment in non-small cell lung cancer with EGFR mutation positive to determine the optimal dosage for HAD-B1 administration. At the final visit compared to before administration, each change in the disease control rate was measured according to the HAD-B1 doses of the test group 1 (972 mg), the test group 2 (1944 mg), and the control group. The efficacy and safety of HAD-B1 were compared and evaluated through sub-evaluation variables. As a result of the study, there was no statistically significant difference in the disease control rate at 12 weeks after dosing, but complete and partial remission were evaluated as 1 patient each in the test group 1, and none in the other groups. There was no statistically significant difference between groups in the sub-evaluation variable. In addition, there was no problem of safety from taking the test drug. However, the initially planned number of subjects was 66, but the number of enrolled subjects was only 14, which may limit the results of this study.

A novel adenylyl cyclase type 5 inhibitor that reduces myocardial infarct size even when administered after coronary artery reperfusion.

We developed a novel adenylyl cyclase type 5 (AC5) inhibitor, C90, that reduces myocardial infarct size even when administered after coronary reperfusion. This is key, since it is not practical to administer a drug to a patient with myocardial infarction before revascularization, and is one reason why so many prior drugs, which reduced infarct in experimental animals, failed in clinical trials. C90 is the most potent AC5 inhibitor, as exhibited by its IC50 value for AC5 inhibition, which was 5 times lower than the next most potent AC5 inhibitor. C90 reduced cAMP in response to forskolin in wild type mice by 42%, but no longer reduced cAMP in response to forskolin in mice with disruption of AC5, indicating that the mechanism of C90 was specific for AC5 inhibition. Compared with vehicle treatment, C90 reduced infarct size by 64% at a dose of 0.6 mg/kg. Thus, C90 is a novel, selective and potent AC5 inhibitor that reduces infarct size, when delivered after coronary artery reperfusion, rendering it potentially clinically useful. It also reduces beta-adrenergic receptor signaling, which will provide additional benefit to patients with coronary artery disease or heart failure.