Exosomal vesicles for neuroprotection and repair after SCI

据估计，在美国有294，000人患有脊髓损伤（SCI），其中超过40，000人 老兵虽然几个治疗方向在实验范例中显示出希望，但没有 临床上存在一种恢复性治疗，可以显著逆转与SCI相关的神经功能缺损 改善功能。在实验性再生疗法的最前沿， 人类脊髓损伤的试验是细胞移植，从神经和间充质干细胞到雪旺细胞 和嗅鞘细胞。虽然观察到SCI后细胞植入的益处，但关键挑战 与它们的使用相关的问题仍然存在，包括在受伤的脊髓内的生存能力差， 免疫抑制剂，以及不需要的细胞分化，增殖， 或移植细胞的迁移导致各种不希望的效果。而组合方法 已经证明克服了这些缺陷中的一些，这是外源细胞的替代策略， 治疗是通过外泌体囊泡（EV）刺激宿主修复。EV是纳米大小的内吞囊泡， 细胞释放到周围环境中，允许生物分子在它们之间转移。电动汽车包含一个 从microRNA到蛋白质和信号中介的各种货物可以促进细胞存活， 分化、轴突生长和髓鞘形成或抑制炎症和瘢痕形成。人们越来越 一致认为EV在调节成体神经干生态位中起着至关重要的作用。这些电动汽车还提供 被工程化以表达荧光标记、靶向选择性细胞类型或负载 用于组织或靶向细胞特异性递送的特异性货物（例如小分子、肽和miRNA）。 我们对细胞源性EV的理解及其治疗潜力的认识的最新进展， 诸如中风和心血管疾病的病症已经扩大了EV领域。然而，它们作为 SCI后的治疗方式受到限制，并且大部分仍处于初期阶段。在研究中，我们 将集中在神经保护，神经原性和再生潜力的比较评估 EV来源于不同的亲本细胞群体和在不同的细胞培养条件下。小胶质细胞（MG） 和施旺细胞（SC），免疫致敏或生长刺激，将评估其能力， 促进亚急性SCI小鼠模型的修复和恢复，以回答可行性的基本问题， 递送和功效。拟议研究的目标将通过两个具体目标来实现。在Aim中 1，最有效的细胞来源的EV类型将根据其促进神经细胞存活的能力进行鉴定 和体外轴突生长。此外，该目标将优化它们在实验性SCI小鼠模型中的体内递送 并评估它们在改善炎症、星形胶质细胞增生和再生相关基因方面的比较作用 (RAG)表达抑制，同时促进神经发生、轴突生长和功能恢复。囊泡 SCI后最有效EV的含量将根据其核酸含量来表征， 鉴定与其修复潜力相关的特异性microRNA序列。在Aim 2中， 被设计用于修复性和神经源性microRNA的细胞特异性递送。的可行性和功能 小胶质细胞和雪旺细胞衍生的EV工程，用于用特异性microRNA：miRNA-9靶向NSC， 已被证明可以改变神经干细胞的命运程序、神经发生和胶质细胞生成的限制， 同时促进血管生成。工程EV将使用体外试验和体内试验进行测试。 对功能功效的影响的实验。拟议研究的总体目标是提高 我们对细胞衍生电动汽车如何参与神经修复以及它们是否可以参与神经修复的理解 工程化以进一步增强它们对宿主细胞的有益作用和随后的修复作用 SCI之后。

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.