Targeted mRNA Delivery to Improve Diabetic Wound Healing

ABSTRACT The career development activities and studies delineated in this K01 application are designed to equip Dr. Theocharidis, the Principal Investigator (PI), with expertise in biomaterials and multi-omics in order to become an independent investigator. Non-healing diabetic foot ulcers (DFUs) affect millions of Americans and lead to devastating consequences. Existing treatments are inefficient in promoting wound closure. The ongoing steep surge in diabetic population necessitates new strategies to accelerate healing of diabetic wounds. Unlike acute uncomplicated wounds, the linear progression from one phase of wound healing to the next is impaired in DFU, which is characterized by a chronic low-grade inflammation. This could be the primary reason that growth factor treatments that act during the proliferative phase have been unsuccessful. Single-cell RNA-sequencing (sc- RNAseq) offers extensive insights into cell function and disease pathophysiology by allowing the mapping of the transcriptomic landscape of individual cells in heterogeneous tissues like DFUs. A recently completed study led by the PI focused on differences between DFU patients who healed their ulcers and those who failed to heal them and investigated molecular changes via sc-RNAseq of surgically removed DFUs. Comparative analysis unveiled genes and pathways significantly associated with successful wound repair. Delivery of messenger RNA (mRNA) into recipient cells has the potential to enable functional protein expression with tremendous therapeutic implications. Ongoing work by the PI has revealed that alginate hydrogel dressings that facilitate lipid nanoparticle (LNPs)-mediated delivery of healing associated modified CHI3L1 mRNA and IL-2 mRNA to the injury site, markedly improve wound healing in diabetic mice. Based on these findings, I hypothesize that (i) topical wound treatment with advanced biomaterials capable of tailorable delivery of mRNAs, corresponding to genes shown to promote DFU healing can lead to novel treatments. Further, I hypothesize that (ii) altering the LNPs’ surface by conjugating antibodies targeting specific cell type receptors can streamline their application and maximize their pro-reparative impact. These hypotheses will be explored across three aims. The first aim will focus on fabricating alginate hydrogels for effective topical delivery of mRNA loaded LNPs. During the second aim, I will employ multi-omics approaches to establish LNP cell internalization and intracellular mRNA translation processes, together with particle biodistribution, as well as to identify mechanisms of action. In the third aim, LNPs will be endowed with cell specificity by conjugation of antibodies on their surface to maximize the platform’s efficiency and also evaluated in a mouse model of increased clinical relevance. Successful completion of this multidisciplinary proposal will result in highly novel data that will substantially expand our knowledge on DFU pathophysiology and advance the fields of biomaterials, RNA therapeutics and cutaneous wound repair. This can lead to the development of innovative much-needed interventions for the management of DFU.