Targeted Epigenetic Gene Editing In Vivo to Rescue Diabetic Ischemic Wounds
Abstract
This proposal addresses the Peer Reviewed Medical Research Program Topic Area: Diabetes – research on interventions to prevent or treat diabetes wound healing impairment. Rationale and Critical Problem to Be Addressed: Arm and leg extremity injuries sustained during military combat often include multiple tissue types, e.g., bone, blood vessels, nerves, and muscles. In the cohort of patients with such injuries, the principal aim of wound care is to reduce the amount of time necessary for wound closure, decrease the rate of infection, and reduce scarring that may be functionally limiting. A critical challenge in wound healing is to restore blood supply to the ischemic wound site. Because of high rates of trauma exposures, such as combat, Veterans have increased risk of diabetes, due to accumulation of global DNA methylation. In diabetes, gene-environment interaction happens through epigenetic modifications like DNA methylation, which can silence important vasculogenic genes, causing endothelial dysfunction. In addition, diabetic foot ulcer patients display presence of DNA hypermethylation-controlled metabolic memory, in which prior episodes of poor glycemic control can confer continued risk of complications despite subsequent glucose normalization. This DNA methylation-dependent metabolic memory results in disturbed blood flow. Given that epigenetic changes are reversible, gene-targeted DNA demethylations have a potential to prevent diabetic wound-related amputations in Veterans and other at-risk populations. Based on this premise, this proposal seeks to achieve successful vascularization of wounds utilizing novel epigenetic gene editing principles. Innovation: The proposed work seeks to lay the foundation stone to a new line of investigation on CRISPR/dCas9-based targeted epigenetic editing of silenced vasculogenic genes towards improved perfusion. This technology uses deactivated Cas9 (dCas9) conjugated with a DNA demethylating enzyme (TET1) with endothelial cell-specific guide RNAs as an epigenetic editing cocktail. For example, one can specifically demethylate a gene of interest (PLCG2 in this proposal) in endothelial cells only (guide RNAs are cadherin 5 promoter driven) in vivo. For the efficient delivery of the DNA demethylation cocktail, tissue nanotransfection (TNT) process will be used, through which biological cargo is inserted electrically into the tissue to trigger conversion of cells within the live body. TNT is simple and rapid and may be implemented at the point of care. Ultimate Applicability and Impact of the Research: Our strategy will focus on improving outcomes from extremity injury and blood flow defects, which strongly impact quality of life of affected civilians and combat personnel. Direct in vivo epigenetic editing has the potential to revolutionize treatments for extremity injuries with loss of blood flow by erasing the DNA methylation-dependent metabolic memory results in disturbed blood flow. The proposed work seeks to lay the foundation stone to a new line of investigation on CRISPR/dCas9-based targeted epigenetic editing of silenced vasculogenic genes towards improved perfusion. Importantly, such intervention can be delivered at the point of care and does not require Good Manufacturing Practice (GMP) facilities. This approach is safe compared to traditional genetic engineering, where either knock-in or knock-out of specific genes can induce unknown and undesired regulatory effects due to the altered genome or position specific effects. Advantages includes rapid action, great stability, limited antigenicity, and multiple loci editing simultaneously. This switch should work across all organ systems, enhancing the functional significance. This technology could potentially be used for many different types of applications from healing injuries to combating aging.
Document Details
- Document Type
- DoD Grant Award
- Publication Date
- Dec 28, 2022
- Source ID
- W81XWH2210146
Entities
People
- Kanhaiya Singh
Organizations
- Indiana University
- United States Army