iNOS Gene Therapy to Prevent Allograft Vasculopathy

Based on current data from the United Network for Organ Sharing (UNOS), cardiac allograft vasculopathy (CAV) is the leading cause of death after the first year of transplantation and accounts for 25% of all deaths annually after the third year. Initial studies from our laboratory have demonstrated that the expression of inducible nitric oxide synthase (iNOS; NOS2) can suppress the development of neointimal hyperplasia. In addition, we have shown in the previous PEGT funding period that an iNOS transgene driven by the cytomegaloviral promoter can successfully prevent the development of CAV, while ubiquitous expression of iNOS in the myocardium declined myocardial contractility. The proposal described herein will test the hypothesis that viral vectors capable of transferring the human iNOS gene specifically to vascular tissue in the heart can suppress the development of CAV without causing undue toxicity to cardiac myocytes. Furthermore, this proposal will serve as the pre-clinical basis for near-term clinical trials for the future prevention of CAV in humans. In order to serve these ends, we will 1) ascertain the vascular-specific expression of adenoviral vectors containing the human iNOS gene and their respective control vectors in vascular smooth muscle cells or endothelial cells in vitro; 2) determine the efficacy and toxicity of vascular-specific expression of iNOS for suppression of CAV in a rat model; 3) determine the efficacy and toxicity of vascular-specific iNOS vector in a porcine chronic rejection model; and 4) carry out an initial toxicity study in humans. The gene transfer strategy carried out in this proposal will be performed with the third generation adenoviral vector, gutless adenovirus, which minimizes immunogenicity that limits the expression of transgene. In this proposal, we plan to address several key issues including 1) that effect of specific over-expression of iNOS in endothelial cells or smooth muscle cells on myocardial contractility; 2) the induction, if any, of apoptosis in cardiac myocytes by iNOS over-expression; 3) the effect of iNOS over-expression on acute cellular rejection; 4) the suppression of CAV in by iNOS over-expression in a rodent model; 5) the safe and efficacious delivery of iNOS to prevent CAV in a large animal model of chronic rejection. We plan to address these issues using both in vitro and in vivo models. Our goal will be to utilize the data obtained from these experiments to serve as a guideline by which to proceed with a safe and efficacious iNOS-based gene therapy trial for the therapeutic prevention of CAV.