Successful Gene Delivery to Cardiac Allograft with Adeno-Associated Viral Vector Using Ex Vivo Storage Perfusion Platform

Abstract

Cardiac gene therapy remains limited by inefficient transgene delivery with currently available methods (intravenous infusion, transcatheter coronary artery infusion, direct myocardial injection). We previously demonstrated in a porcine heterotopic heart transplant model that cardiac allograft gene transfer can be achieved using ex vivo perfusion with adenovirus (Ad). Adeno-associated viral (AAV) vectors generally achieve lower transgene expression compared to Ad. However, AAV vectors also achieve a significantly longer duration of expression, and thus are more suitable for therapies with genes that are effective even at lower levels but require prolonged expression. The purpose of this study is to evaluate whether ex vivo perfusion can also be used as a platform for cardiac allograft gene therapy using AAV. Heart transplantation was performed using blood type-matched and swine leukocyte antigen-matched Yucatan mini pigs (n=2 transplants). Donor hearts received ex vivo perfusion for two hours with a perfusate including AAV vector bearing the firefly luciferase gene (dosed at 2 × 10^13 and 1 × 10^14 genome-containing vectors for transplants 1 and 2, respectively). The allograft was implanted heterotopically into the recipient abdomen. Recipients were survived for 30 days on oral immunosuppression and then euthanized. Transgene expression in allograft and native recipient tissues was assessed using a luciferase enzymatic assay, and transgene detection was performed using qPCR on DNA extracted using a modified Hirt protocol. Luciferase activity was detected in the allograft in both experiments. While luciferase activity was undetectable in the right and left atria and coronary arteries from the allograft exposed to the lower dose, the higher dose of AAV resulted in detectable luciferase activity throughout the entire heart and at high levels. Luciferase activity was undetectable in all recipient tissues (heart, lung, liver, spleen, and psoas muscle). Additionally, luciferase DNA was detectable on qPCR in allograft specimens (1.5-1.9 × 10^3 transgene copies per 100 ng of DNA). Ex vivo perfusion can be used as a platform for cardiac allograft gene therapy using AAV vectors, with robust transgene expression detectable 30 days following transplant.