Cell-free regenerative approach in wound healing

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Wounds, which are breaks in structure and function of skin, are healed through a regulated process initiated with tissue homeostasis followed by repair response involving inflammation, proliferation and remodeling. Wounds may be due to cuts and lacerations or incisional wounds post surgeries. At times, this healing is delayed or slowed resulting in chronic recalcitrant wounds. Risk factors for impaired wound healing include surgical site infection and comorbidities such as aging, smoking, diabetes. Strikingly, ongoing research with COVID19 patients showcased increased severity in wounds and lesions compared to normal patients(1-4). Recalcitrant wounds per se are not life-threatening but often lead to chronic wound related sequelae. The long-term outcome of impaired wound healing may lead to scarring or amputations, and often has a substantial psychological impact. The treatment of recalcitrant wounds and stimulation of healing is a critical need and consistent with priorities outlined by Department of Veterans Affairs (2018-2024 Strategic plan). While multiple advances in wound dressing materials promote healing, a noteworthy gap remains in the interventions available which considerably improve the outcome of injury. Towards this goal, a therapeutic approach was undertaken to harvest the potential of exosomes derived from human adipose stem cells (hASCexo) mediating tissue repair and regeneration. Thus, the overarching hypothesis is that hASCexo enable wound healing and recovery of function by modulating genomic pathways following injury. This project will elucidate the safety and efficacy of hASC exosomes applied topically to repair dermal wounds to move the therapy closer to the clinic. Towards this goal, the proposal will undertake an in vitro evaluation of genomic targets complemented with an in vivo approach to determine response to treatment. These hypotheses will be validated using a multi-disciplinary approach including pre- clinical, physiological, cellular and biochemical experiments. Specific Aim 1: Determine the molecular targets of the lncRNA cargo of hASC exosomes that promote wound healing: Using RNAseq to examine the contents of hASC exosomes, two long noncoding RNAs (lncRNAs) were identified which are highly enriched in the hASCexo and are pivotal in recovery post injury. Human dermal fibroblasts (HDF) and human epidermal keratinocytes (HEK) will be used with siRNA mediated knockdown or over-expression of these lncRNA in hASCexo. Cellular and biochemical outcomes will be measured in in vitro wound models along with elucidation of genomic changes in response to hASCexo treatment. These will be validated ex vivo in human skin explants. Specific Aim 2: Evaluation of response to hASC exosomes’ treatment in an in vivo wound model: The hypothesis is that repair and regeneration of wounds by topical application of hASCexo depends on its pharmacodynamics and efficacy. To elucidate this along with its genomic impact, an in vivo rodent ischemic wound model will be used to determine physiological response of hASCexo treatment to improve healing outcomes. Its efficacy to promote healing in conditions of underlying infection will be evaluated The hASCexo provide a novel, cell-free regenerative approach to accelerate wound closure and to alleviate chronic effects of impaired wound healing. Robust preliminary data supports feasibility and successful implementation of hASC exosomes’ topical treatment in wound healing. The research design is expected to greatly enhance translatability by the “learn and confirm” approach by integrating the in vitro results in the in vivo model. The therapeutic potential of hASCexo will be thoroughly evaluated for safety and scalability such that it may be translated successfully to the treatment of Veterans’ wounds and substantially improve outcome of chronic wound related sequelae.