Use of Modified mRNA for the Treatment of Diabetic Foot Ulceration - ABSTRACT Impaired wound healing is an alarming problem in diabetes, attributed to the development of over 750,000 diabetic foot ulcerations (DFU) and 70,000 lower extremity amputations per year in the USA. Single-cell RNA- sequencing (scRNASeq) analysis provides valuable insights into transcriptional features and disease pathophysiology by allowing the profiling of individual cells in heterogeneous tissues. A recently conducted study in our unit primarily focused on divergent characteristics between DFU patients who healed their ulcers (Healers) and those who failed to heal them (non-Healers) and investigated molecular changes via scRNASeq analysis of surgically removed DFUs. Our analysis revealed the enrichment of a unique population of fibroblasts, overexpressing MMP1, MMP3, MMP11, HIF1A, CHI3L1, and TNFAIP6 genes in the Healers. Ongoing work in our lab has shown that specifically designed alginate bandages that can provide controlled lipid nanoparticle (LNPs) delivery of modified CHI3L1mRNA, IL-17AmRNA, FGF-2 and IL-2mRNA considerably improve wound healing in diabetic db/db mice. Based on these findings, we hypothesize that topical wound treatment with new biomaterials that can release the above mRNAs can lead to the development of new therapeutic approaches. Furthermore, we hypothesize that combination of various mRNAs with specific temporal delivery can physiologically imitate the activation of pathways that are associated with DFU healing. To fully explore our hypotheses, we propose the following Specific Aims: 1.) Develop a modified-mRNA-LNPs therapeutics delivery system to target different stages of diabetic wound healing and provide proof of concept regarding their efficacy in wounds of diabetic mice. Alginate hydrogels fabricated into bandages capable of sustained release will be employed to deliver mRNA-LNPs in 3D human skin equivalents and wounds of db/db mice. We will identify the most efficient mRNA or combination and the most appropriate dosing and timing of delivery in improving diabetic wound repair. 2.) Investigate efficacy in a larger animal model, the diabetic Yucatan minipig and confirm mechanisms of action in both animal models. We will also examine mechanisms of action of pro-healing mRNA(s) and will compare them to the results from the rodent studies. 3.) Conduct translational studies in human diabetic wound healing to verify the therapeutic potential. We will conduct ex vivo wound healing studies with discarded skin from patients with and without diabetes and in vivo studies with wounding of human skin engrafted onto diabetic nude mice. We will also employ targeted multi-omics analyses to establish molecular mechanisms of action and compare them with the ones that have been observed in our previous extensive human studies. We believe that our proposal involves state-of-the-art “bench to bedside” research that can culminate in the development of innovative new therapeutic approaches. Successful completion of the project will facilitate the conduction of Phase I/II clinical trials in DFU patients.
