Molecular dissection and imaging of extracellular vesicles to define their origin and targets

细胞外小泡(EV)和其中包含的RNA(EV-RNAs)由每个细胞分泌到生物体液中 单元类型。EV-RNA已成为一系列疾病的潜在预后或预测生物标志物， 提供疾病状态的有针对性的、准确的实时表示。然而，电动汽车的进步-- 临床上作为疾病生物标志物的RNA因其分离和鉴定方面的挑战而受到阻碍 人物刻画。最值得注意的是，缺乏工具和技术来i)隔离和准确地描述 组织特异性EV-RNA群体；ii)定义组织中表面标记和RNA含量的异质性- 以及iii)确定与疾病状态相关的EV-RNAs的变化。在这个多PI中 提案中，我们将使用协作和创新的方法来推进技术，从而允许隔离 以及来自造血细胞、脑和心脏的EV种群的颗粒状特征。 我们在UG3阶段的目标是识别用于分离EVS的细胞/组织特异性标记 遗传小鼠模型中的计算分析、转录和EV跟踪；这种方法将允许 用于以细胞特异性方式基于荧光/抗体的EVS识别。还将获得信息 用一种新的定量单分子定位显微镜(QSMLM)方法对单个电动汽车进行了研究。QSMLM， 一种敏感的基于荧光的成像方法将被用来量化亲和分离的电动汽车的数量，它们的 大小和使用分子信标的关键RNA含量。已鉴定的组织特异性EV标志物和EV-RNA 将在UH3阶段用于在各种人体模型中进行验证。我们在UH3阶段的目标是 确定组织芯片流出物对EV-RNAs的细胞/组织贡献；并评估 来自急性疾病(冠脉缺血或脑血管意外)的人血浆中的EVS或 生理过程(运动)。我们将验证关键的EV-RNAs(使用纳米流式细胞仪和分子 信标)，并使用具有分子信标的qSMLM来提供单个囊泡的EVS的定量概况 决议。值得注意的是，拟议的实验将有助于确定不同组织对血浆的贡献。 生物流体RNA景观的基线，以及对生理或疾病应激源的反应。 总而言之，在普遍定期审议阶段开发并在普遍定期审议阶段得到验证的工具和技术将有助于 作为发现和发展肠道病毒标志物和其他组织特有的肠道病毒RNA的路线图。 最终，使用组织特异性EV-RNA来探测疾病状态将提供一个动态窗口 与当前可用的相比，疾病进展或回归具有更高的敏感度和保真度 技术。

Extracellular vesicles (EVs) and the RNAs contained within them (EV-RNAs) are secreted into biofluids by every cell type. EV-RNAs have emerged as potential prognostic or predictive biomarkers of a wide range of diseases, providing a targetable, accurate real-time representation of the disease state. However, advancement of EV- RNAs in the clinic as biomarkers of disease has been impeded by challenges in their isolation and characterization. Most notably, there is a lack of tools and techniques to i) isolate and precisely characterize tissue-specific EV-RNA populations; ii) define heterogeneity in surface markers and RNA content in tissue- specific EV populations; and iii) determine changes in EV-RNAs associated with disease state. In this multi-PI proposal, we will use a collaborative and innovative approach to advance technology that would allow isolation and granular characterization of EV populations from hematopoietic cells, brain, and heart. Our objective in the UG3 phase is to identify cell/tissue specific markers for isolation of EVs using computational analysis, transcriptomics, and EV-tracking in genetic mouse models; this approach would allow for fluorescence/antibody-based identification of EVs in a cell-specific manner. Information will also be obtained on individual EVs with a novel quantitative single molecule localization microscopy (qSMLM) approach. qSMLM, a sensitive fluorescence-based imaging method, will be used to quantify the number of affinity isolated EVs, their size, and key RNA content using molecular beacons. The identified tissue-specific EV-markers and EV-RNAs will be used in the UH3 phase for validation in a variety of human models. Our objective in the UH3 phase is to determine cellular/tissue contribution to EV-RNAs from Tissue-Chip effluents; and assess dynamic changes in EVs from human plasma from subjects with acute disease (coronary ischemia or cerebrovascular accident) or physiological processes (exercise). We will validate key EV-RNAs (using nano-flow cytometry and molecular beacons) and use qSMLM with molecular beacons to provide a quantitative profile of EVs at a single vesicle resolution. Notably, proposed experiments will help determine contribution of different tissues to the plasma biofluid RNA landscape at baseline, and in response to physiological or disease stressors. Together, the tools and techniques developed in the UG3 phase, and validated in the UH3 phase would serve as a road-map for the discovery and development of EV markers and EV-RNAs specific to other tissues. Ultimately, the use of tissue-specific EV-RNAs to probe disease state would provide a dynamic window into disease progression or regression with higher sensitivity and fidelity compared to currently available technologies.