Combined epigenetic treatment of human organ-cultured diabetic corneas with targeted nanoconjugate and a DNA demethylating agent ameliorates epithelial wound healing and stem cell marker expression

Abstract

Purpose : Diabetes mellitus (DM) is a major cause of vision loss in working-age adults. Diabetic cornea has frequent epithelial alterations including impaired wound healing, possibly due to the dysfunction of limbal epithelial stem cells (LESC). Previously, we used gene therapy and DNA demethylation to modulate the expression of DM-altered proteins such as cathepsin F (increased in DM), c-Met (decreased in DM), and Wnt-5a (decreased in DM) to stimulate diabetic epithelial wound healing and LESC marker expression. Here, we studied the effect of combined therapy using a nanobioconjugate targeting cathepsin F and c-Met together with a DNA methylation inhibitor.

Methods : Pairs of diabetic organ-cultured corneas (n=6) from postmortem human donor eyes were pre-treated with 20 µM of biodegradable nanobioconjugates (NBC) based on polymalic acid scaffold containing antisense oligonucleotides (AON) to cathepsin F and miR-409 (that inhibits c-Met) or control scrambled AON twice for 2 days, together with 20 µM of DNA methylation inhibitor zebularine or DMSO control for 2 days. Epithelial 5-mm wounding with 1-heptanol in organ-cultured corneas was performed, and wound healing was monitored over time. LESC and diabetic marker expression was determined using immunostaining or western blot.

Results : Diabetic organ-cultured corneas treated with a combination of zebularine and NBC suppressed DNA methyltransferase 1 and cathepsin F, and increased Wnt-5a and c-Met expression. This resulted in significant decrease (by 38.4%, p<0.02) of corneal epithelial wound healing time vs. DMSO and NBC with scrambled AON. Treatment with zebularine alone stimulated healing by 29.4%, and with NBC alone, by 20.4%. Combination treatment increased the expression of putative LESC markers, keratins K15 and K17. Diabetic markers integrin α3β1 and nidogen-1, and the wound healing mediators p-Akt and p-p38 also had increased staining. There was no significant change in activated caspase-3 staining after treatment, indicating lack of toxicity for corneal cells.

Conclusions : Our results show that combined therapy with DNA methylation inhibitor and NBC targeting diabetes-altered protein markers may have a potential for the treatment of impaired diabetic corneal epithelial wound healing.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.