Poster #087, Cedars Sinai Medical Center
AAV-based gene therapy targeting Wnt-5a improves diabetic corneal epithelial wound healing and stem cell marker expression.
Mentor: Ruchi Shah, PhD
Diabetes, an epigenetic disease, can cause delayed corneal epithelial wound healing due to dysfunctional limbal epithelial stem cells (LESC). Previously, we discovered that non-canonical Wnt-5a is suppressed in diabetic corneas due to hypermethylation of WNT5A gene promoter and upregulation of Wnt-5a inhibiting miR-203a. Addition of recombinant Wnt-5a stimulated diabetic corneal epithelial wound healing and normalized LESC marker expression. In this study, we investigated the use of recombinant adeno-associated virus (AAV) as a vector to deliver WNT5A to cultured human limbal epithelial cells (LEC) enriched in LESC. We used research-consented postmortem donor eyes obtained with IRB approval for culturing human diabetic and non-diabetic primary LEC (from 5 donors) that we virally transduced with AAV with CMV promoter to deliver eGFP or WNT5A. We first investigated the transduction of various AAV serotypes, including AAV2, AAV5, AAV6, AAV8, AAV9, and AAV/DJ in diabetic and non-diabetic LEC. We found that AAV6 showed the highest transduction efficiency as compared to all other serotypes with minimal cell death by annexin V staining (for apoptosis) and propidium iodide staining (for necrosis). Scratch wound healing in diabetic LEC showed that therapeutic AAV6 (AAV6-WNT5A) significantly accelerated wound healing by 45% (p=0.04) at 24 hours vs. control AAV6. Therapeutic AAV6 also increased the expression of putative LESC markers, K15 and K17 by 1.8-fold compared to control AAV6 by western blot. This increase in K15 and K17 expression was confirmed by immunostaining. In conclusion, AAV-based gene therapy to target Wnt-5a may be a new, safe, and efficient approach for the treatment of diabetic corneal disease.