Mesenchymal stem cell extracellular vesicles for ischemic retinal damage

视网膜中央动脉阻塞是一种眼科急症，目前治疗方法尚不成熟。干 基于细胞的视网膜细胞替代是一种非常令人鼓舞的方法， 挽救视网膜疾病的视力。然而，由于局限性，包括很少的细胞整合，不良的免疫反应， sponses和异常生长，需要一种替代的无细胞方法。EV是纳米囊泡体， 当被靶细胞内吞时，触发特异性反应。在这里，EV的microRNA（miRNA）货物 扮演着关键的角色。该提案的目标是使用工程化的MSC-EV恢复视网膜功能， 功能特异性miRNA。与MSC相比，它们的EV是非免疫原性的，非致瘤性的，并且是可修饰的。 特定的交付模式。这些特性使它们成为理想的仿生剂， 药我们的研究表明，电动汽车可以拯救视网膜细胞已急性缺氧或 缺血是CRAO中细胞死亡的关键机制。我们还发现，低氧预适应， MSC导致EV（H-EV）具有增强的细胞保护特性，包括抗凋亡和抗炎症。 mation。在H-EV中过表达的许多miRNA在视网膜细胞中具有细胞保护特性。 我们的中心假设是，在MSC-EV中靶向EV特异性表达miR-424/其他关键miRNA， 将重新发挥H-EV的抗凋亡和抗炎作用。我们将这种电动汽车命名为Func- 工程电动汽车（FEE）。为了促进MSC-EV治疗的临床转化，我们确定了关键的乐趣- 主要的知识空白：（1）EV miRNA与其抗凋亡特性的关系;（2）EV miRNA与其抗凋亡特性的关系;（3）EV miRNA与其抗凋亡特性的关系;（4）EV miRNA与其抗凋亡特性的关系;（5）EV miRNA与其抗凋亡特性的关系。 miRNA及其在视网膜MSC-EVs抗炎作用中的作用;（3）是否可以增强MSC-EVs， 通过设计他们的miRNA货物来实现靶向功能？在目标1中，我们将产生过表达miR- 424（FEE-424）和146 b（FEE-146 b）。我们将评估FEE的作用机制，以及它们的可靠性- 在视网膜神经节细胞、小胶质细胞、Muller细胞和视网膜血管内皮细胞中产生FEE的时间 使用体外模拟缺血模型中的功能丧失和获得研究。这些结果将作为 用于开发FEE以改善视网膜细胞损伤的原理验证模型。在目标2中， 含有miR-424和-146b的细胞将用于测试抗凋亡和炎症机制的特异性靶向作用。 CRAO啮齿动物模型中的寄生虫。总的来说，拟议的研究预计将提供变革性的结果。 由此MSC-EV被修饰并在缺血事件后递送用于视网膜保护作用以治疗CRAO。 创新是视网膜疾病的无细胞治疗，EV miR介导的应用特异性，以及直接 确定EV对参与视网膜缺血性损伤的特定细胞的影响。翻译意义 是很有可能影响到新的分子疗法。基础研究的根本意义在于， 研究将通过确定EV在视网膜中的作用机制来实现该领域的垂直发展。

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 re- sponses, 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 containing function-specific miRNA. Compared to MSCs, their EVs are non-immunogenic, non-tumorigenic, and modifiable for specific delivery modes. These characteristics render them ideal biomimetic agents fitting precision-based medicine. 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-inflam- mation. 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 miR-424/other key miRNAs in MSC-EVs will re-capitulate the anti-apoptosis and anti-inflammatory actions of H-EVs. We designate such EVs as Func- tionally Engineered EVs (FEEs). To facilitate clinical translation of MSC-EV therapy, we have identified key fun- damental 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? In Aim 1 we will produce FEEs overexpressing miR- 424 (FEE-424) and 146b (FEE-146b). We will evaluate the mechanisms of action of the FEEs, and their candi- dacy for generation of FEEs in retinal ganglion cells, microglia, Muller cells, and retinal vascular endothelial cells using loss and gain of function studies in models of simulated ischemia in vitro. These results will serve as a proof-of-principle model for development of FEEs for amelioration of cell damage in the retina. In Aim 2, FEEs containing miR-424 and -146b will be used to test specific targeting of anti-apoptotic and inflammatory mecha- nisms in a rodent model of CRAO. Overall, the 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. Innovations are cell-free therapy of retinal diseases, EV miR-mediated application-specificity, and direct determination of the impact of EVs on specific cells involved in retinal ischemic injury. Translational significance is the high likelihood of impacting novel molecular therapy. Underlying basic research significance is that the studies will enable vertical advancement of the field by determining mechanisms of actions of EVs in the retina.