This proposal is based on the novel approach of designing an engineering novel zinc finger protein transcription factors (ZFPs) to regulate the expression of endogenous genes for the treatment of cardiovascular disease. We have previously validated this approach in vivo and in vitro with engineered ZFPs that activate expression of the VEGF-A gene. We now propose further studies to more vigorously investigate the mechanism of action of our ZFP constructs and to delineate the differences in the biological effects of endogenous gene activation vs. cDNA-based transgene expression. In the context of these experiments we study the determinants of ZFP half-life and the effects of differential ZFP positional DNA- binding on the stoichiometry of VEGF splice variant expression. We will also investigate ZFP-induced gene repression using a ZFP construct engineered to target and repress the phospholamban (PLB) gene. PLB is a critical determinant of cardiac contractility, and the ability to downregulate the expression of PLB has significant clinical implications. In the context of these experiments we will also study the relationship between the physical half-life of the PLB repressor ZFP and the functional half-life of PLB repression. These experiments represent the first time an engineered transcription repressor will have been used in a clinically relevant in vivo context. Finally, we will further build on work we have recently completed demonstrating the feasibility of building cell- permeable ZFPs that are capable of crossing cell membranes, entering the nucleus and regulating the expression of specifically targeted genes. This represents a novel new therapeutic approach that also has significant clinical implications.