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

中枢神经系统(CNS)诊断、预后和监测的新工具 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 阿尔茨海默氏症(AD)现在是美国第六大死亡原因， 每年给照顾者和纳税人带来近5000亿美元的负担，预计还会翻一番 在接下来的十年里流行起来。可靠且易于理解的液体活组织检查 血液等液体在临床上将具有很高的价值。 近年来，细胞外小泡(EVS)已成为中枢神经系统病理生理学中的重要角色 疾病。EVS由多种双叶膜结合粒子组成，已被 包括我们在内的几个小组报道了在体外传播与发病有关的蛋白质 在活体内。重要的是，来自中枢神经系统的电动汽车可以在外围找到， 看起来很容易越过血脑屏障。这些电动汽车可以提供一种非侵入性 了解中枢神经系统健康状况的窗口，以及潜在的特定中枢神经系统细胞类型。事实上，我们的合作者 在这项申请中，D.Kapogiannis发表了从AD获得的多个生物标记物发现 使用沉淀/免疫亲和(PIA)方法丰富神经元和星形胶质细胞的患者血液 电动汽车。尽管取得了这一重大成功，但现在有机会改进现有的 技术，提高了灵敏度、特异度和吞吐量，同时减少了样本量和可操作性 时间到了。我们计划通过使用创新的技术、工具和方法来满足这一需求 来自四种主要CNS细胞类型的高纯度包含抗原的CNS-EV群体。 我们首先假设(目标1和2，R21阶段)EV隔离和 表征-1)NanView的多路捕获/干涉测量仪(ExoView) 诊断和2)下一代、支持多通道、光学集成的阻性脉冲 来自电子生物科学的技术-提供对当前状态的实质性改进- 工作流程所有步骤的现场PIA技术，在我们的指导下进行了基准测试 合作者和PIA共同开发人员。其次，在R33阶段(目标3和4)，我们希望验证 并优化了灵敏度、特异度、检出限和任何样品前处理步骤。 使用精心设计的插入物和混合物，以及来自IPSC培养的纯电动汽车 细胞和基因组编辑的细胞作为阴性对照，我们将从 多种生物来源。每个阶段都提供了具体、详细的里程碑，包括 从原则验证阶段R21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.