PROJECT ABSTRACT/SUMMARY In the past few years, several gene therapy products using adeno-associated virus (AAV) have been approved for clinical use for treatment of non-cardiac diseases. However, existing data indicate extremely low cardiac uptake of AAV in human hearts, hampering clinical translation of cardiac gene therapy. My lab has recently developed a clinically applicable approach to overcome this low AAV uptake. Our preliminary data indicate that intracoronary delivery of AAV during simultaneous occlusions of coronary artery and sinus (stop- flow delivery) results in globally increased gene expression with up to 500-fold higher expression compared to the standard antegrade delivery, even in pigs with pre-existing neutralizing antibodies against AAV. Unique to our approach, we ensure safety by supporting the systemic hemodynamics and alleviating cardiac ischemia using a catheter-based cardiac assist device. Our method will thus offer minimally invasive, safe, but efficacious gene delivery to human hearts. Although our preliminary data is strong, we still lack understanding in how and which delivery-related factor(s) contributed to improved gene expression. Defining key factor(s) that led to significant improvement will allow us to rationally design AAV gene delivery for further refinement. In this application, we propose inter-connected, but independent Aims in large animal heart failure models to: 1.Identify the key mechanical factor(s), 2. Understand uptake mechanisms and 3.Establish clinically applicable delivery protocol. Based on our preliminary data, we will focus on capillary pressure and dwell time in Aim 1 as key factors, endothelial permeability and vesicular AAV uptake in Aim 2 as potential mechanisms of improvement, and clarify remaining uncertain issues for clinical realization in Aim 3, which include optimal serotype for targeting human heart, antibody inhibition, and defining optimal patient population for stop-flow gene therapy. Our proposal is conceptually novel in focusing on various factors in delivery (mechanical, biological and pathological), in contrast to the majority of delivery-focused studies only testing methods of their interest. Additional innovative points include exploring repeat AAV dosing, mechanistic studies of cardiac AAV uptake, and treating hibernating myocardium using gene therapy. By further improving our promising AAV gene delivery method that allows efficient and safe cardiac gene expression, an obstacle currently plaguing the clinical translation of cardiac AAV gene therapy will be overcome.