Exosomal vesicles for neuroprotection and repair after SCI

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An estimated 294,000 people live with spinal cord injury (SCI) in the United States of which over 40,000 are veterans. Though several therapeutic directions have shown promise in experimental paradigms, there does not exist a restorative treatment clinically that can significantly reverse the neurological deficits associated with SCI to improve function. At the forefront of experimental regenerative therapies that are being translated to clinical trials for human SCI is the transplantation of cells, from neural and mesenchymal stem cells to Schwann cells and olfactory ensheathing cells. Though benefits are observed with cell implantation after SCI, critical challenges associated with their use remains, including poor viability within the injured spinal cord, the need for an immunosuppressant when not autologous as well as the possibility of unwanted cell differentiation, proliferation, or migration of the implantation cells leading to various undesirable effects. Whereas combinatory approaches have been demonstrated to overcome some of these deficiencies, an alternate strategy to exogenous cell therapy is to stimulate host repair through exosomal vesicles (EVs). EVs are nanosized endocytic vesicles that cells release into the immediate environment, allowing transfer of biomolecules between them. EVs contain a variety of cargoes from microRNA to proteins and signaling intermediaries that can promote cell survival, differentiation, axon growth and myelination or subdue inflammation and scar formation. There is a growing consensus that EVs play a crucial role in regulating the adult neural stem niche. These EVs also offer the capacity to be engineered to express a fluorescent label, be targeted to a selective cell type, or be loaded with specific cargoes (e.g. small molecules, peptides, and miRNAs) for tissue or targeted cell specific delivery. Recent advances in our understanding of cell derived EVs and realization of their therapeutic potential in conditions such as stroke and cardiovascular disease have expanded the EV field. However, their use as a therapeutic modality after SCI has been limited and remains largely in its infancy. In the proposed studies, we will focus on the comparative assessment of the neuroprotective, neurogenic and the regenerative potential of EVs derived from disparate parental cell populations and under different cell culture conditions. Microglia (MG) and Schwann cells (SCs), immunologically primed or growth-stimulated, will be evaluated for their capacity to promote repair and recovery in murine models of subacute SCI to answer fundamental questions of feasibility, delivery, and efficacy. The goals of the proposed study will be accomplished through two Specific Aims. In Aim 1, the most effective cell-derived EV type will be identified according to their ability to promote neural cell survival and axon growth in vitro. Further, the aim will optimize their in vivo delivery in an experimental SCI mouse model and assess their comparitive effects on ameliorating inflammation, astrogliosis and regeneration associated gene (RAG) expression repression while promoting neurogenesis, axonal growth and functional recovery. The vesicle content of the most efficacious EV after SCI will be characterized with respect to its nucleic acid content to identify specfic microRNA sequences that correlate with their reparative potential. In Aim 2 EVs will be engineered for cell-specific delivery of reparative and neurogenic microRNA. The feasibility and functionality of microglia and Schwann cell derived EV engineering for NSC targeting with a specific microRNA: miRNA-9, that has been demonstrated to alter the neural stem cell fate program, neurogenesis and the restriction of gliogenesis, respectively while promoting angiogenesis. The engineered EV will be tested using in vitro assays and, in vivo experiments for effects on functional efficacy.The overall objective of the proposed studies is to improve our understanding of how cell derived EVs may be involved in neurorepair and whether they can be engineered to further enhance their beneficial effects on host cells and subsequent reparative actions following SCI.