Mesenchymal stem cell extracellular vesicles for ischemic retinal damage

视网膜中央动脉阻塞(CRAO)是一种眼科急症，几乎没有得到证实的治疗方法。茎 基于细胞的视网膜细胞替代是一种非常令人鼓舞的方法，以实现视网膜神经保护和 在视网膜疾病中挽救视力。然而，由于整合的细胞很少，不利的免疫重新出现。 自体和异常生长，需要另一种无细胞方法。电动汽车是纳米泡状体， 当被靶细胞吞噬时，会触发特定的反应。在这里，电动汽车的microRNA(MiRNA)货物 起着关键作用。这项建议的目标是使用含有以下物质的工程MSC-EVS恢复视网膜功能 功能特定的miRNA。与MSCs相比，它们的EV是非免疫原性、非致瘤性和可修饰的 适用于特定的交付模式。这些特性使它们成为理想的仿生制剂，适合基于精确度的 医药。我们的研究表明，EVS可以挽救受到急性缺氧或 缺血，启动CRAO中细胞死亡的关键机制。我们还发现，低氧预适应 MSCs诱导的EVS(H-EVS)具有增强的细胞保护特性，包括抗凋亡和抗炎。 信息传递。在H-EV中过度表达的一些miRNAs在视网膜细胞中具有细胞保护特性。 我们的中心假设是MSC-EV中靶向EV特异性的miR-424/其他关键miRNAs的表达 将使H-EVS的抗细胞凋亡和抗炎作用重新降温。我们将这类电动汽车命名为Func- 专门设计的电动汽车(费用)。为了促进MSC-EV疗法的临床翻译，我们确定了关键的乐趣- 基础知识空白：(1)肠道病毒miRNA与其抗细胞凋亡特性的关系；(2)肠道病毒 MiRNA及其在视网膜MSC-EVS抗炎作用中的作用；以及(3)MSC-EVS能否增强 通过设计他们的miRNA货物来实现目标功能？在目标1中，我们将产生过度表达miR-的费用 424(费用-424)和146b(费用-146b)。我们将评估这些费用的作用机制，以及它们的成本- 视网膜神经节细胞、小胶质细胞、穆勒细胞和视网膜血管内皮细胞产生费用的危险性 在体外模拟缺血模型中应用功能损失率和获得率研究。这些结果将作为一种 开发改善视网膜细胞损伤费用的原则证明模型。在目标2中，费用 含有miR-424和-146b的将用于测试抗细胞凋亡和炎症机制的特异性靶向。 CRAO啮齿动物模型中的nisms。总体而言，拟议的研究可望提供变革性的结果。 由此MSC-EVS被修饰并在缺血事件后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EVS对参与视网膜缺血损伤的特定细胞的影响。翻译意义 是影响新的分子疗法的高可能性。潜在的基础研究意义在于 研究将通过确定EVS在视网膜中的作用机制来实现该领域的垂直推进。

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.