Novel Approaches to Capture, Sorting, and Characterization of CNS-Origin Extracellular Vesicles

用于诊断、预后和监测中枢神经系统（CNS）的新工具 疾病是迫切需要的。为了正确看待这个问题，只有一个神经退行性疾病 阿尔茨海默病（AD）是美国第六大死亡原因， 每年给护理人员和纳税人带来近5000亿美元的负担，预计将翻一番。 在接下来的十年里流行起来。可靠和可解释的液体活检测试， 诸如血液的流体在临床上具有很高的价值。 细胞外囊泡（extracellular vesicles，EVs）是近年来发现的一种重要的中枢神经系统病理生理机制 疾病EV包含多种双叶膜结合颗粒， 包括我们在内的几个研究小组报道， 和体内。重要的是，来自中枢神经系统的EV可以在外周中发现， 似乎很容易穿过血脑屏障。这些EV可以提供非侵入性的 这是一个了解CNS健康状况的窗口，可能是特定CNS细胞类型。事实上，我们的合作者 在此应用中，D. Kapogiannis发表了从AD中获得的多种生物标志物发现， 使用沉淀/免疫亲和（PIA）方法富集患者血液中的神经元和星形胶质细胞 电动车尽管取得了这一重大成功，但现在有机会改善现有的 技术，提高灵敏度，特异性和通量，同时减少样本量和动手操作 时间我们计划利用创新的技术、工具和方法来满足这一需求， 来自四种主要CNS细胞类型的CNS-EV的高纯度含抗原群体。 我们首先假设（目标1和2，R21阶段）EV隔离和 表征-1）nanoView的多路复用捕获/干涉仪（ExoView） 诊断和2）下一代、支持多通道的光集成电阻脉冲 技术从电子生物科学-提供实质性的改进，比目前的状态- 现场PIA技术在工作流程的所有步骤，基准与我们的指导， 合作者和PIA共同开发者。第二，在R33阶段（目标3和4），我们希望验证 并优化灵敏度、特异性、检测限和任何样品预处理步骤。 使用精心设计的掺入物和混合物，沿着来自iPSC衍生的细胞培养物的纯EV， 细胞和基因组编辑的细胞作为阴性对照，我们将确认EV的起源细胞， 多种生物来源。每个阶段都提供了具体、详细的里程碑，包括 从R21原理验证阶段过渡到更广泛的R33验证阶段。

Novel tools for diagnosis, prognosis, and monitoring of central nervous system (CNS) diseases are urgently needed. To place the problem in perspective, just one neurodegenerative disease, Alzheimer’s (AD), is now the sixth-leading cause of death in the United States, places a burden of nearly half a trillion dollars per year on caregivers and taxpayers, and is expected to double in prevalence within the next decade. Reliable and interpretable liquid biopsy tests of easily accessed fluids such as blood would be of high value in the clinic. Extracellular vesicles (EVs) have recently emerged as important players in pathophysiology of CNS diseases. Comprising a diversity of double-leaflet membrane-bound particles, EVs have been reported by several groups including ours to spread proteins implicated in pathogenesis both in vitro and in vivo. Importantly, EVs from the central nervous system can be found in the periphery, appearing to transgress the blood brain barrier quite readily. These EVs may provide a non-invasive window into the health of the CNS and, potentially, specific CNS cell types. Indeed, our collaborator on this application, D. Kapogiannis, has published multiple biomarker findings obtained from AD patient blood using a precipitation/immunoaffinity (PIA) approach to enrich neuronal and astrocytic EVs. Despite this important success, there is now an opportunity to improve on the existing technique, increasing sensitivity, specificity, and throughput while reducing sample size and hands-on time. We plan to address this need, using innovative techniques, tools, and approaches to obtain highly pure antigen-containing populations of CNS-EVs from four main CNS cell types. We hypothesize firstly (Aims 1 and 2, R21 Phase) that novel developments in EV isolation and characterization—1) a multiplexed capture/interferometry instrument (ExoView) of nanoView Diagnostics and 2) a next-generation, multipass-enabled, optically integrated resistive pulse technology from Electronic BioSciences—offer substantial improvements over the current state-of- the-field PIA technique at all steps of the workflow, benchmarked with the guidance of our collaborator and PIA co-developer. Secondly, in the R33 Phase (Aims 3 and 4), we expect to verify and optimize the sensitivity, specificity, limits of detection, and any sample pre-processing steps. Using carefully designed spike-ins and mixtures, along with pure EVs from culture of iPSC-derived cells and cells genomically edited as negative controls, we will confirm the cell of origin of EVs from multiple biological sources. Specific, detailed milestones are offered for each phase, including the transition from the proof-of-principle R21 phase into the more expansive R33 validation phase.