PROJECT SUMMARY/ABSTRACT Gene therapy is a promising therapeutic strategy to treat human diseases. While vectors can be engineered to target specific cells, the blood flow greatly favors liver and lung exposure. Delivery of vectors to confined anatomic compartments such as the eye and central nervous system, or ex vivo to cells that are modified than re-administered, are strategies successful in approved gene therapies. Many other organs cannot be effectively targeted using current vector systems. Gene therapy for heart disease has failed in clinical trials due in large part to low transduction of cardiomyocytes reported as 1% of cells. Coronary blood flow is very robust and intracoronary vector has little time to access cardiac cells. In the blood stream, vector is vulnerable to detection by pre-existing anti-AAV neutralizing antibodies that are present in up to 90% of the population. These obstacles, along with the high cost of producing vector limits the efficacy and translatability of cardiac gene therapy. The fundamental problem of therapeutic gene delivery is the focus of this proposal. Our interdisciplinary multi-PI team will approach gene therapy from a novel bioengineering approach, developing hydrogels to traverse biological barriers to vector delivery. We will engineer a hydrolysis-cleavable poly(-ethylene glycol, PEG) hydrogel to deliver gene therapy vectors to the epicardium. Our preliminary data support the idea that this mode of delivery will optimize cardiac transduction. We have previously demonstrated the efficacy of PEG hydrogels and validated a hydrogel delivery device that is able to deliver hydrogels in a minimally invasive and fast procedure using standard fluoroscopy. Our multi-PI team includes Rebecca Levit, a physician scientist with extensive expertise cardiac therapeutics and small animal models, Andrés García, an engineer and expert on biocompatible hydrogels, and Christopher Doering, a gene therapist with a track record of development, translation, and manufacturing of gene therapies. This project is organized to efficiently and rapidly assess this novel mode of delivery of gene therapy. In aim 1 we will test 6 hydrogel prototypes in vitro and in vivo to determine the most efficient formulation for vector delivery. In aim 2 we will quantify the immunologic advantages of epicardial hydrogel vector delivery to predict the performance of this strategy in patients. In aim 3 we will deliver a unique candidate gene for cardiac regeneration as proof of principle of our epicardial hydrogel delivery strategy. We hypothesize that epicardial hydrogel gene therapy can overcome fundamental circulatory and immunologic barriers to therapeutic gene delivery. These studies will result in a clear translational path for gene therapy.