Project Summary Current paradigms of drug development have made remarkable progress in treating and preventing disease by effecting the function of a single target enzyme, or receptor with specificity. However, many chronic diseases like type 2 diabetes, Alzheimer’s disease, and cardiovascular disease are multifaceted and may require alterations in multiple pathways, or delivery to intracellular targets, to effectively treat the condition. For this reason, extracellular vesicles (EVs) have gained attention as potential therapeutic carriers and endogenous modulators of disease. EVs are nano-sized vesicles produced in all cells and carry macromolecules that are capable of modulating multiple pathways simultaneously in the receiving cell. Their cargo includes miRNAs, mRNA, DNA, signaling proteins, enzymes metabolites and receptors. Mesenchymal stem cells (MSCs) have been extensively used as the parent cells for therapeutic EVs as the cargo of these cells is broadly protective. Many groups have engineered MSC EVs to carry therapeutic compounds through permeabilization techniques which rely on passive diffusion of compounds into isolated EVs. However, the current engineering techniques are exceptionally inefficient, which is a major factor in preventing the translation of engineered EVs into the clinic. In this proposal we utilize vascular endothelial cells (ECs) to actively package desired cargo into EVs. This is possible through a novel transcytosis-like process we recently published. We found that ECs take up exogenous albumin-bound material from the cell culture media, load the endocytosed material into newly formed EVs, and export those EVs at the basolateral side of the cell. Interestingly, the released EVs seem to target specific cells in the tissue. Therefore, we have designed experiments to test and optimize the capacity of ECs to load EVs with exogenous albumin-bound miRNA, peptides, and drugs. We will test the therapeutic efficacy of customized EVs in an in vitro and in vivo myocardial infarction mouse model system. We expect that utilizing ECs will allow for extremely efficient, active loading of EVs, unmatched targeting to the desired cell type and improved cargo offloading efficiency in receiving cells. This work has the potential to transform the EV engineering field and bring us closer to clinically applicable EV- based therapeutics.