Tissue reprogramming in diabetic wound healing

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ABSTRACT An estimated 2% of US population is affected by chronic wounds. About 15–20% of all diabetic patients develop skin wounds across their lifespan, the majority evolving toward chronicity. It is estimated that only 56% of diabetics with ulcerative wounds survive more than 5 years after their initial manifestation. This proposal seeks to achieve successful vascularization of diabetic wounds utilizing the novel regenerative principles of non-viral in vivo tissue reprogramming. Tissue nanotransfection technology (TNT), recently reported by the PI, achieves tissue reprogramming, under immune surveillance in vivo, using a topical electrophoretic delivery system enabled by a novel nanofabricated silicon chip hardware. The proposed studies emanate from prior work by the applicant on the role of TNT delivery of specific transcription factors Etv2, Foxc2, and Fli1 (EFF) to achieve vasculogenic reprograming in vivo. EFF was thus discovered as a vasculogenic plasmid cocktail capable of vascularizing ischemic tissue. The proposed work is based on first evidence that instead of the use of plasmid cocktails that have been used by many groups to achieve cell reprogramming, a single anti-sense oligonucleotide (ASO) is highly effective in producing induced vasculogenic cells (iV) from skin fibroblasts. Preliminary data show that under in vivo conditions, ASO can improve perfusion of diabetic wound tissue. Because diabetes is known to cause endothelial dysfunction and vasculopathy, TNT delivery of ASO will be tested to improve wound outcomes. Vasculogenic effects of ASO is achieved by turning on a major vascular switch Fli1. The proposed work will delineate the molecular mechanisms of ASO-induced perfusion of diabetic cutaneous wound perfusion (Aim 1). To enable invasive mechanistic studies, a humanized mouse model has been thus proposed in Aim 2. The following two specific aims are proposed: Aim 1: Elucidate the significance and molecular mechanisms by which ASO induces accelerated wound closure in diabetic mice. 1.1 ASO delivery by TNT rescues healing of cutaneous wounds in murine models of diabetes; 1.2 ASO delivery improves diabetic wound vascularization by rescuing Fli1 from miR-200b dependent gene silencing; 1.3 A subset of wound-site fibroblasts contribute to the pre-existing vasculature by acquiring vasculogenic endothelial-like characteristics post-ASO treatment. Aim 2: Test significance of ASO reprogramming in a humanized diabetic mouse model. 2.1 ASO delivery by TNT accelerates wound healing and improves wound vascularization in a humanized NSG diabetic mouse model. The proposed work lays the foundation stone to future trials attempting to reprogram human skin stroma towards improved perfusion of the wound-site.