Project Summary/Abstract: Critical limb ischemia (CLI) is the most advanced stage of peripheral arterial disease (PAD). It manifests with leg pain, decreased walking distance, non-healing wounds and gangrene. About 1/3 of CLI patients don't respond to standard therapy and ultimately undergo major limb amputation. This critical unmet need demands a solution. CLI causes >105 major limb amputations in the USA per year. Our overarching goal is to develop a novel limb-salvage therapy. Gene therapy is a promising option to induce therapeutic angiogenesis, increasing blood flow in ischemic limbs for repair. This approach has raised a great deal of hope for `no-option' CLI patients. However, previous gene therapy approaches using naked plasmids encoding angiogenic factors, such as VEGF and FGF, failed to show substantial clinical benefits. Moreover, early adenovirus-mediated gene delivery systems raised concerns over safety. Hence, more efforts for the identification of alternative effective transgenes and safe/efficient delivery vectors are crucial. Success of gene therapy for CLI requires activation and participation of endogenous healing mechanisms including recruitment/homing of stem/progenitor cells to ischemic tissue. Enhanced recruitment/homing depends upon a `hospitable' tissue microenvironment that is receptive of endogenous regenerative cells. Our previous research discovered that in ischemia, homing for these critical repair cells relies on specific cell-cell interactions via a panel of adhesion molecules, including E- selectin/ligand pairs. The levels of vascular E-selectin in the ischemic limb tissues is down-regulated. We hypothesize that E-selectin is pivotal in the recruitment of stem/progenitor cells necessary for neovascularization in ischemic tissues. E-selectin can serve as a docking site for endogenous stem/progenitor cells to anchor. The resulting downstream cell-cell signaling cascades can increase angiogenesis and tissue repair/regeneration. We have recently demonstrated feasibility, safety and superior efficacy of E-selectin/AAV- based gene therapy for improving limb perfusion, increasing neovascularization, decreasing ischemia, reducing gangrene, minimizing tissue damage, augmenting regeneration of the ischemic skeletal muscle, and extending running capacity, in murine models. In the clinical setting, we believe that such biologic effects can increase functional walking capacity and prevent limb loss. We thus propose to induce a supportive tissue microenvironment by priming endothelial cells in capillaries and other ischemic limb-tissue cells with the adhesion molecule, E-selectin, using a safe and efficient AAV2 vector. We will utilize a clinically relevant mouse model of CLI – the footpad gangrene, to test this novel gene therapy. The objectives are to validate and optimize the translational protocol for a future large-animal pre-clinical study and conduct the requisite IND-enabling GLP/GMP animal research for a subsequent pil...