Extracellular vesicle therapeutics: from mechanism to application in cardiac repair

Abstract

Extracellular vesicles (EVs) are membrane-enclosed particles containing a vast array of biomolecules from the parental cells, such as proteins, lipids, various nucleic acids, and soluble small molecules. There is increasing evidence that EVs play active roles in intercellular communication in various biological and pathological processes and offer promise as novel biomarkers and therapeutic agents. EVs were identified as important carriers of bioactive paracrine factors in the regenerative stem and progenitor cell secretome. Especially for patients suffering from end-stage heart failure caused by myocardial infarction and ischemia-reperfusion injury, for which treatment options are limited to heart transplantation, these newly discovered therapeutic entities have opened novel treatment possibilities. EVs isolated from cardiac progenitor cells (CPC-EVs) were demonstrated to reduce infarct size and improve cardiac function in several preclinical models. Their functionality was attributed to stimulating different cardiac repair processes in the ischemic myocardium, such as inducing angiogenesis. In this thesis, various aspects to improve the application of CPC-EVs as biological therapeutics and as natural endogenous drug delivery vehicles are addressed. We identified the functional protein components of CPC-EVs involved in endothelial cell activation and migration by employing in-depth EV characterization techniques. This provides insights into the localization of functional content in EV preparations and mechanistic insights into CPC-EV-mediated recipient cell activation. Moreover, we demonstrated that interfering with endolysosomal trafficking in donor cells leads to an increase in EV yield with maintained bioactivity. This provides openings for increasing CPC-EV production for their therapeutic application. Furthermore, we compared the therapeutic potential of EVs released from different donor cell types and purified with different isolation techniques in myocardial infarction mouse models and demonstrated the influence of these factors on EV functionality. Another important objective before using EVs in clinical trials is determining the biodistribution and retention of EVs after local and systemic administration. We showed that EVs injected in the myocardium displayed no direct flush-out from the myocardium and were retained in the heart up to days after administration. CPC-EVs delivered intravenously were rapidly cleared and mainly distributed to the liver. This is a challenge for the delivery of EVs to the heart. We showed that EV engineering techniques can be used to direct EVs to the endothelium and to achieve simultaneous cargo protein loading inside EVs. These strategies could further improve the targeted delivery of therapeutic EV cargo to the injured heart. Taken together, the results in this thesis highlight the broad applicability and complexity of EVs as therapeutic agents. Further improvements, such as developing standardized EV separation and characterization techniques to safeguard batch-to-batch variations and prevent problems with reproducibility, will lead to the development of EV therapeutics for cardiac repair.