Mesenchymal stem cell extracellular vesicles for ischemic retinal damage

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Central retinal artery occlusion (CRAO) is an ophthalmological emergency with few proven therapies. Stem cell-based retinal cell replacement is a highly encouraging approach to achieve retinal neuroprotection and to save vision in retinal diseases. However, with limitations including few cells integrated, adverse immune responses, and aberrant growth, an alternative cell-free approach is required. EVs are nano-vesicular bodies that, when endocytosed by target cells, trigger specific responses. Here, the microRNA (miRNA) cargo of the EVs plays a key role. This proposal targets restoration of retinal function using engineered MSC-EVs with function-specific miRNA. Our studies indicate that EVs can rescue retinal cells that have been acutely subjected to hypoxia or ischemia, the key mechanism that starts cells dying in CRAO. We also found that hypoxic preconditioning of MSCs resulted in EVs (H-EVs) with enhanced cytoprotective properties including anti-apoptosis and anti-inflammation. A number of miRNAs overexpressed in the H-EVs have cytoprotective properties in retinal cells. Our central hypothesis is that targeted EV-specific expression of key miRNAs in MSC-EVs will re-capitulate the anti-apoptosis and anti-inflammatory actions of H-EVs. We designate such EVs as Functionally Engineered EVs (FEEs). To facilitate clinical translation of MSC-EV therapy, we identified key knowledge gaps: (1) The relationship between EV miRNA and its anti-apoptotic properties; (2) EV miRNA and its role in anti-inflammatory actions of MSC-EVs in retina; and (3) Can MSC-EVs be enhanced for targeted functionality by engineering their miRNA cargo? Aim 1 will produce FEEs overexpressing miR-424 (FEE-424) and 146b (FEE-146b). We will evaluate their mechanisms of action, and their candidacy for generation of FEEs in retinal ganglion cells, microglia, Muller cells, and retinal vascular endothelial cells using loss and gain of function studies in simulated ischemia in vitro. These results will serve as proof-of-principle for development of FEEs for amelioration of cell damage in the retina. In Aim 2, FEEs containing miR-424 and -146b will test specific targeting of anti-apoptotic and inflammatory mechanisms in a rodent model of CRAO. Proposed studies are expected to provide transformative results whereby MSC-EVs are modified and delivered for retinal protective action after the ischemic event to treat CRAO. The Supplement for Diversity will train a graduate student in data collection and analysis, reviewing literature critically, designing experiments, and presenting and writing research reports. The goal is for this experience to increase the student’s skills in performing translational basic science research and to ensure more opportunities for diversification of the scientific workforce.