Acute myocardial infarction (MI) continues to be a leading cause of morbidity and mortality worldwide. The adult human heart has minimal capacity for regeneration. Once damaged, cardiomyocytes (CMs) cannot be replaced efficiently, leading to a decline in heart function and adverse remodeling. Our long-term goal is to develop novel, clinically translatable therapeutic strategies for systemic administration that protect CMs following ischemia- reperfusion (I/R) injury to reduce infarct size and improve clinical outcomes. Exosomes have emerged as potent vectors for delivery of bioactive cargo for the treatment of various diseases including CVD. In large animal models of I/R injury, exosomes derived from cardiosphere-derived cells (CDCs) promote cardiac repair but require intramyocardial delivery, limiting their clinical potential. Likewise, while exosomes have demonstrated pre-clinical efficacy, it is unclear what cargo is necessary to mediate these benefits and how it can be enhanced. To overcome these limitations, we seek to identify innovative strategies to optimize therapeutic exosome cargo and improve tissue and cell-specific delivery. In preliminary studies, we determined that microRNA (miR) cargo is required for the regenerative potential of CDC-exosomes following acute MI. Furthermore, using surface engineering, we developed CM-targeted CDC-exosomes (CMP-exos) which showed increased cardiac honing, retention and repair compared with unmodified CDC-exos post-MI. Together, these data support the central hypothesis that modifying the targeting capabilities and bioactive cargo of therapeutic exosomes will allow for development of a clinically relevant systemic based therapy with enhanced efficacy in CVD. The objectives of this proposal are to determine the specific rate-limiting miR cargo that reduces CM apoptosis in I/R, optimize therapeutic systemic delivery using CMP-exos, and determine if CMP-exos improve efficacy in a large animal model of I/R. The central hypothesis will be tested by pursuing three specific aims: 1) Define the role of specific exo miRNA cargo in modulating CM cell death and cardiac function following I/R; 2) Determine the optimal dose and route of delivery of CMP-exosomes; 3) Determine the effect of targeting CMP-exosomes to CMs on cardiac function in a pre-clinical model of I/R. We will pursue these aims using an innovative combination of mechanistic studies to investigate the necessity and sufficiency of specific miRs on CM cell death, novel exosome engineering strategies with the potential for development of a systemic based strategy, comprehensive pharmacokinetic studies, and rigorous pre-clinical evaluation in a relevant large animal model of I/R. The proposed research is significant because it will elucidate the mechanism/s of action of a potential translational therapy and identify therapeutic targets to reduce CM cell death in pathways operant in I/R. The proximate expected outcome of this work is an understanding ...