Project Summary Exosomes have emerged as potential therapeutic vectors for repair of non-healing wounds, which continue to be a significant source of morbidity and represent a substantial financial burden to patients and health care systems in general. However, the therapeutic potency – and thus the translational potential – of exosomes for wound repair is limited, as exosomes contain low amounts of nucleic acid cargo, such as microRNAs (miRNAs), which are critical to defining exosome bioactivity. Towards overcoming this limitation, we have developed a novel ethanol conditioning approach for endothelial cell-derived exosomes. This method reduces anti- angiogenic miRNA cargo and increases pro-angiogenic long non-coding RNA cargo in these exosomes, leading to an overall increase in angiogenic bioactivity. Angiogenic dysfunction is a hallmark of non-healing wounds, thus these enhanced exosome formulations have therapeutic potential in wound repair. Our team of bioengineers and clinicians will test approaches to further exert control over endothelial exosome non-coding RNA cargo and bioactivity via environmental conditioning approaches (Aim 1) and exogenous loading techniques (Aim 2). We will further test the therapeutic efficacy of enhanced exosomes in excisional, diabetic, and burn injury wound repair models (Aim 3) to identify the most promising avenue for further translational investigations. Overall, these studies will promote the development of a new class of therapeutics for non-healing wounds that could benefit millions of patients.