Engineered extracellular vesicles as a targeted drug delivery system for multiple sclerosis

摘要 此F31 Ruth L.Kirchstein NRSA的最终目标是请求支持，以解决以下方面的根本差距 进行性多发性硬化症(PMS)有效药物输送载体的知识预防工程。 经前综合征是一种常见的、衰弱的神经退行性疾病，会导致中枢广泛的脱髓鞘。 神经系统。目前还没有可靠的治疗方法来补救经前综合症的进展，但正在出现一种 策略是通过启动少突胶质前体细胞(OPC)的分化来促进恢复 少突胶质细胞是产生髓鞘的细胞，在经前综合征中耗尽。目前的药物递送技术实现 再髓鞘形成要么效果不佳，要么具有高度侵袭性，是临床翻译的主要障碍。 治疗经前综合征的有效再髓鞘疗法必须穿过完整的血脑屏障，然后靶向 OPC。这项建议集中在工程细胞外囊泡(EEVS)的合成和作为药物的测试 运输工具来完成这些壮举。天然的EV已被证明既能靶向特定的细胞/组织 而且还可以跨越内皮屏障。然而，由于它们巨大的功能异构性，这些特性确实 不会出现在相同的电动汽车上。尽管已经确定了一批有效瞄准OPC的电动汽车，但 促进血脑屏障跨越的最佳EV种群和关键蛋白尚不清楚，从而阻止了新的仿生 工程战略。为了解决当前疗法的局限性，我建议识别和融合 有效地跨越血脑屏障和靶向OPC的EV亚群，将microRNA-219 OPC加载到它们身上 差异剂，以设计一种生物可用和选择性的药物输送载体。 在我初步数据的基础上，我将分三步进行这个项目：(1)优化一个 荧光激活囊泡分选法鉴定和分离跨越血脑屏障的EV；(2)阐明 利用功能转井模型研究血脑屏障转运过程中的分子机制和关键蛋白质； 通过融合内源性EV和加载重新髓鞘治疗药物来产生EEVS。我们将量化EEV 在体外和体内跨血脑屏障和靶向OPC启动分化和产生髓鞘的能力。这 该项目的重点是生产一种独特的适用于经前综合症的新疗法，但这条设计电动汽车的管道 具有多个靶向功能的药物可用于解决许多医疗问题的药物传递障碍。 这个项目是与严格的培训计划并行开发的，以增强我的培训和技术技能 神经学、药理学和转化医学领域。这项计划将使我能够过渡到 独立，因为我专注于我的长期目标，即追求发展神经学的学术生涯 治疗学。培训将利用大学的许多资源来促进专业和教育发展。

Abstract The ultimate goal of this F31 Ruth L. Kirchstein NRSA is to request support to address a fundamental gap in knowledge preventing engineering of efficacious drug delivery vehicles for progressive multiple sclerosis (PMS). PMS is a common, debilitating neurodegenerative disease that causes widespread demyelination in the central nervous system. There are currently no therapies that reliably remediate the advance of PMS, but an emerging strategy is to promote recovery by initiating differentiation of oligodendrocyte progenitor cells (OPCs) to oligodendrocytes, the myelin producing cells depleted in PMS. Current drug delivery techniques to achieve remyelination are either poorly efficacious or highly invasive, major impediments to clinical translation. An effective remyelinating therapeutic for PMS must cross the intact blood-brain barrier (BBB) and then target OPCs. This proposal focuses on the synthesis and testing of engineered extracellular vesicles (eEVs) as a drug delivery vehicle to accomplish these feats. Natural EVs have been shown to both target specific cells/tissues and also cross endothelial barriers. However, due to their immense functional heterogeneity, these qualities do not occur in the same EVs. Although a population of EVs that efficiently targets OPC has been identified, the best EV population and key proteins that promote BBB crossing remain unknown, preventing new biomimetic engineering strategies. To address the limitations of current therapeutics, I propose to identify and fuse subpopulations of EVs that efficiently cross the BBB and target OPCs, load them with microRNA-219, an OPC differentiating agent, to engineer a bioavailable and selective drug delivery vehicle. Building on results obtained in my preliminary data, I will carry out this project in three steps: (1) to optimize a method of fluorescence activated vesicle sorting to identify and isolate BBB-crossing EVs; (2) to elucidate the molecular mechanisms and key proteins during BBB transcytosis using a functional transwell model; and (3) to produce eEVs via fusion of endogenous EVs and loading with remyelinating therapeutics. We will quantify eEV ability to cross the BBB and target OPCs to initiate differentiation and produce myelin in vitro and in vivo. This project is focused on producing a novel therapeutic uniquely suited to PMS, but this pipeline to engineer EVs with multiple targeted functions could be applied to address drug delivery barriers for many medical problems. This project was developed in parallel with a rigorous training plan to enhance my training and technical skills in the areas of neurology, pharmacology, and translational medicine. This plan will enable my transition to independence as I focus on my long-term goal of pursuing an academic career developing neurological therapeutics. Training will exploit the university’s many resources for professional and educational development.