Smarter exosomes derived from engineered MSCs promote neo-vascularization

项目摘要/摘要 间充质干细胞(MSC)疗法在实现心脏组织修复方面显示出巨大的前景 缺血性损伤。干细胞的治疗作用是由旁分泌因子介导的。几项研究表明 干细胞来源的Exosome(EXO)在干细胞介导的缺血性治疗中发挥关键作用 心肌通过增加血管生成。从翻译的角度来看，EXO有更大的好处 治疗效果优于全细胞：外源性有较少潜在不良反应，较少免疫排斥反应，较多 易操控，EXO货物可通过预适应或基因操控进行修饰。加塔- 4，一种心脏转录因子，促进心脏形态发生，延长心肌细胞(CM)存活和 通过调节各种生物活性分子和激活心脏保护性MIR来保护心脏功能。 我们已发表的数据表明，MSC过表达GATA-4(MSCGATA-4)可提高MSC的存活率，保护原生细胞 CM和促进血管生成，与载体转染型MSC(MSCNULL)相比。EXO派生自EXO派生 从GATA-4过表达的GATA-4(ExoGATA-4)比从载体转染的MSC获得的EXO更有效 (Exonull)对心肌缺血损伤的保护作用。在我们的初步研究中，ExoGATA-4富含促血管生成 MIRS，调节血管形成。ExoGATA-4下调凝血酶敏感蛋白1的表达 内源性新生血管抑制因子。该项目的总体目标是利用 在ExoGATA-4的修复效果上，GATA-4赋予EXO比普通EXO。我们的中心假设是 ExoGATA-4富含促血管生成的miRs和蛋白质，它们被转移到受体细胞和 活跃的多个信号通路，导致血管生成和心脏修复。三个具体目标是 建议：目的1，确定EXO携带的miRs在GATA-4转染反应中的作用。目标2，至 证明转移的生物活性分子是否在ExoGATA-4介导的血管生成中起关键作用。目标 3.检验GATA-4过表达的MSCs来源的EXO比来自GATA-4的MSCs更有效的假设 载体转染的骨髓间充质干细胞促进血管生成和心肌再生。建议进行的研究包括 创新是因为以前没有研究系统地检查ExoGATA-4的有效性， 特别关注EXO介导的血管生成和全身缺血后心脏修复的效果 行政管理。静脉注射的优点是可以重复治疗，而不会有 反复心肌内注射。拟议的研究具有非常重要的意义，因为它将评估 细胞治疗的有效性可以通过无细胞EXO复制的概念，并将探索这一新的 静脉给药为今后临床应用提供了模式。这个项目将是第一个系统性的努力 旨在开发一种全新的战略，使用EXO管理取代细胞，这将 彻底改变细胞疗法。这一成果将提供一种变革性的验证和指导翻译研究和 基于来自基因组编辑的MSCs的EXO增强的血管生成特性的假定治疗。 1

Project Summary/Abstract Mesenchymal stem cell (MSC) therapy has shown tremendous promise for enabling heart tissue repair after ischemic injury. The therapeutic effects of stem cells are mediated by paracrine factors. Several studies illustrate that exosomes (EXO) derived from stem cells play a critical role in stem cell mediated therapy of ischemic myocardium via increasing angiogenesis. From the translational perspective, EXO have greater salutary therapeutic effects than whole cells: EXO have fewer potential adverse effects, less immune rejection, are more amenable to manipulation, and EXO cargo can be modified by preconditioning or genetic manipulation. GATA- 4, a cardiac transcription factor, promotes cardiac morphogenesis, extends cardiomyocyte (CM) survival and preserves cardiac function via regulating various bioactive molecules and activating cardiac protective miRs. Our published data indicate that MSC overexpressing GATA-4 (MSCGATA-4) increase MSC survival, protect native CM and promote angiogenesis, compared to vector-transfect MSC (MSCnull). EXO derived from EXO derived from GATA-4 overexpressing GATA-4 (ExoGATA-4) are more efficient than EXO from vector-transfected MSC (Exonull) in protecting CM from ischemic injury. In our pilot study, ExoGATA-4 are enriched with pro-angiogenic miRs, which regulate vascularization. ExoGATA-4 downregulate the expression of thrombospondin 1, a well-known endogenous inhibitor of neovascularization. The overarching goal of this project is to exploit effectiveness of GATA-4 conferred EXO than the ordinary EXO in repairable effects of ExoGATA-4. Our central hypothesis is that the ExoGATA-4 are enriched with pro-angiogenic miRs and proteins that are transferred into recipient cells and active multiple signaling pathways, leading to angiogenesis and cardiac repair. Three Specific Aims are proposed: Aim 1, to determine the role of miRs carried by EXO in response to GATA-4 transfection. Aim 2, to demonstrate if the transferred bioactive molecules play a critical role in ExoGATA-4 mediated angiogenesis. Aim 3, to test the hypothesis that EXO derived from GATA-4 overexpressing MSCs are more effective than that from vector-transfected MSCs in promoting angiogenesis and myocardial regeneration. The proposed studies are innovative because no previous study has systematically examined the effectiveness of ExoGATA-4, with particular focus on the efficacy of EXO mediated angiogenesis and ischemic heart repair following systemic administration. Intravenous injection offers the advantage of enabling repeated treatments without the stress of repeated intramyocardial injections. The proposed study is highly significant because it will evaluate the concept that the effectiveness of cellular therapy can be reproduced by cell-free EXO and will explore this new modality for future clinical application by intravenous administration. This project will be the first systematic effort aimed at developing a completely new strategy in use of EXO administration in lieu of cells, which would revolutionize cell therapy. The outcome will offer a transformative validation and guide translational research and putative therapies based on enhanced angiogenic properties of EXO from genome-edited MSCs. 1