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

细胞外囊泡（EVs）和其中包含的RNA（EV-RNA）由每个细胞分泌到生物流体中。 细胞类型。EV-RNA已成为多种疾病的潜在预后或预测生物标志物， 提供疾病状态的可定向的、准确的实时表示。然而，EV- RNA在临床上作为疾病的生物标志物受到其分离和鉴定方面的挑战的阻碍。 特征化最值得注意的是，缺乏工具和技术来i）隔离和精确表征 组织特异性EV-RNA群体; ii）定义组织中表面标志物和RNA含量的异质性- 特定EV群体;和iii）确定与疾病状态相关的EV-RNA的变化。在这个多PI 根据这项提议，我们将采用合作和创新的方法来推进技术， 以及来自造血细胞、脑和心脏的EV群体的颗粒表征。 我们在UG 3阶段的目标是鉴定细胞/组织特异性标志物，用于使用 计算分析，转录组学和遗传小鼠模型中的EV跟踪;这种方法将允许 用于以细胞特异性方式进行基于荧光/抗体的EV鉴定。还将获得信息 用一种新的定量单分子定位显微镜（qSMLM）方法对个体EV进行检测。qSMLM， 一种灵敏的基于荧光的成像方法，将用于定量亲和分离的EV的数量， 大小和关键RNA含量。鉴定的组织特异性EV标记物和EV-RNA 将在UH 3阶段用于各种人体模型的确认。我们在UH 3阶段的目标是 确定细胞/组织对来自组织芯片流出物的EV-RNA的贡献; 来自急性疾病（冠状动脉缺血或脑血管意外）受试者的人血浆的EV，或 生理过程（运动）。我们将验证关键的EV-RNA（使用纳米流式细胞术和分子生物学技术）。 信标），并使用具有分子信标的qSMLM来提供单个囊泡处EV的定量概况 分辨率值得注意的是，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景观在基线，并响应于生理或疾病应激。 在UG 3阶段开发并在UH 3阶段验证的工具和技术将共同服务于 作为发现和开发EV标志物和其他组织特异性EV-RNA的路线图。 最终，使用组织特异性EV-RNA探测疾病状态将提供一个动态窗口， 疾病进展或消退的灵敏度和保真度高于目前可用的 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.