Tissue nanotransfection-based endothelial PLCγ2-targeted epigenetic gene editing in vivo rescues diabetic ischemic wound healing

Diabetic wounds often suffer from poor perfusion. Reliance on VEGF therapy to improve perfusion makes logical sense, yet clinical study outcomes fall far short of expectations. Our previous work has identified that genetically silenced phospholipase Cγ2 (PLCγ2) hinders VEGF therapy of the diabetic ischemic limb. However, the underlying mechanisms remained unknown. Emerging evidence supports the notion that hyperglycemia leads to DNA methylation-based gene silencing. Given that epigenetic changes are reversible, this work guided by scRNA sequencing of human wound-edge, tests the efficacy of gene-targeted therapeutic demethylation with respect to enabling VEGF therapy. Bipedicle ischemic wounds were placed in diabetic mice. PLCγ2 promoter CpG methylation were analyzed using bisulfite sequencing. To specifically demethylate endothelial PLCγ2 promoter during VEGF therapy, a CRISPR/dCas9-based demethylation cocktail was delivered to the wound-edge using tissue nanotransfection (TNT) technology. The functional outcome of such demethylation was assessed using perfusion imaging. PLCγ2 promoter was hypermethylated at murine diabetic ischemic wound-edge. Demethylation-based upregulation of PLCγ2 during VEGF therapy improved wound tissue blood flow with increased abundance of vWF+/PLCγ2+ vascular tissue elements by activating p42/p44-MAPK→HIF1α pathway. Taken together, TNT-based endothelial demethylation of the PLCγ2 gene promoter improved VEGF therapy on the perfusion of cutaneous diabetic wounds resulting in improved closure.