Tissue nanotransfection-based endothelial PLC?2-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 C?2 (PLC?2) 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. PLC?2 promoter CpG methylation were analyzed using bisulfite sequencing. To specifically demethylate endothelial PLC?2 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. PLC?2 promoter was hypermethylated at murine diabetic ischemic wound-edge. Demethylation-based upregulation of PLC?2 during VEGF therapy improved wound tissue blood flow with increased abundance of vWF+/PLC?2+ vascular tissue elements by activating p42/p44-MAPK?HIF1a pathway. Taken together, TNT-based endothelial demethylation of the PLC?2 gene promoter improved VEGF therapy on the perfusion of cutaneous diabetic wounds resulting in improved closure. Overall design: single cell RNA sequeing from chronic wound-edge samples from diabetic and non diabetic subjects