171-OR: Tissue Nanotransfection–Based Endothelial-Targeted Epigenetic Gene Editing In Vivo to Rescue Diabetic Ischemic Wounds

Our previous work (PMID: 35192691) has identified that genetically silenced phospholipase Cγ2 (PLCγ2) hinders VEGF therapy of the diabetic ischemic limb. Hyperglycemia caused hypermethylation of endothelial specific gene promoter. Given that epigenetic changes are reversible, this work tests the significance of gene-targeted therapeutic DNA demethylation in improving blood flow to diabetic ischemic wounds. Bipedicle ischemic wounds were placed in streptozotocin (STZ) induced acute diabetic C57BL6 mice. In wound tissue, PLCγ2 promoter CpG methylation levels were analyzed using bisulfite sequencing. Next, to specifically demethylate PLCγ2 promoter in endothelial cells, a demethylation cocktail was designed: (i) (scFv)-TET1 catalytic domain (TET1CD) system capable of inducing targeted DNA methylation, and (ii) endothelial specific guide RNAs. This demethylation cocktail was delivered at the diabetic ischemic wound-edge employing topical tissue nano-transfection (TNT) technology. PLCγ2 protein expression outcomes were assessed using flow cytometry. Functional outcome of such demethylation was assessed using Laser Speckle Perfusion imaging and ultrasonography (Vevo 2100) at days 3, 7&10 post-surgery. Overall DNA hyper methylation was prominent in murine ischemic flaps as demonstrated by the increased ratio of 5-methylcytosine (5-mc, methylation mark) to 5-hydroxymethylcytosine (5-hmC, demethylation mark) (n = 5). Specifically, the PLCγ2 promoter was hypermethylated (n=5). TNT mediated endothelial-targeted demethylation of the PLCγ2 promoter increased the expression of this gene in endothelial cells (n=4). Such demethylation-based upregulation of PLCγ2 improved wound tissue blood flow with increased abundance of VWF⁺/PLCG2⁺ vascular elements (n=4). Taken together, topical TNT-based endothelial demethylation of the PLCγ2 gene promoter improved perfusion of cutaneous diabetic wounds resulting in improved closure.