mRNA Expression Profiling from Extracellular Vesicles (EVs): Generating a Rapid Diagnostic for Stroke

细胞外囊泡 (EV) 的 mRNA 表达谱分析：快速诊断中风

• 批准号: 10647755
• 负责人: Alison E Baird
• 金额: $ 58.58万
• 依托单位: UNIVERSITY OF KANSAS LAWRENCE
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-07-01 至 2026-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10647755
• 关键词: Acute; Affect; Affinity; Alteplase; Appearance; Biological Assay; Biological Markers; Biotinylation; Blood Tests; Blood flow; Brain; Brain Diseases; Brain hemorrhage; CD8B1 gene; Cause of Death; Cells; Cessation of life; Clinical; Clinical Sensitivity; Collaborations; Complementary DNA; Coulter counter; Data; Detection; Development; Devices; Diagnosis; Diagnostic; Diagnostic tests; Emergency Situation; Expression Profiling; Fluorescence; Genetic Transcription; Harvest; Hospitals; Image; In Vitro; Injections; Ischemic Stroke; Label; Lasers; Length; Leukocytes; Ligase; Light; Magnetic Resonance Imaging; Measurement; Membrane; Messenger RNA; Molds; Molecular; Oligonucleotide Primers; Output; Patients; Peripheral; Phase; Plasma; Plastics; Process; Productivity; RNA; RNA marker; Rapid diagnostics; Reaction; Recovery; Research; Reverse Transcription; Sampling; Secure; Sensitivity and Specificity; Solid; Source; Stroke; Structure; Surface; Syndrome; System; Technology; Testing; Therapeutic; Time; Transcript; Validation; Vascular blood supply; Work; X-Ray Computed Tomography; blood-based biomarker; circulating biomarkers; clinically relevant; design; diagnostic platform; extracellular vesicles; in-vitro diagnostics; innovation; innovative technologies; mRNA Expression; meter; multidisciplinary; nano; nanopore; nanosensors; novel; operation; peripheral blood; point of care testing; single molecule; stroke event; stroke model; stroke patient; stroke therapy; success; tool; transcriptome sequencing; vesicular release

Project Summary/Abstract Stroke is a disorder of the brain by which a part loses its blood supply and the affected region rapidly progresses to death if blood flow is not restored in time. Treatments to restore blood flow after acute ischemic stroke (AIS) are effective if administered <4.5 h from the onset of the stroke event. But, due in part to the lack of an in vitro diagnostic test for AIS, imaging (CT – clinical sensitivity ~26%) at the attending hospital is required for diagnosis. As a result, <7% of AIS patients receive treatment. Therefore, a critical need exists to develop strategies for diagnosing stroke in near real time that potentially can allow for point-of-care testing (POCT). One approach is a peripheral blood test using markers that quickly respond to changes in the brain induced by stroke. The proposed project will develop innovative technologies that uses peripheral blood markers for diagnosing stroke syndromes (AIS and hemorrhagic stroke) in near real time (~31 min for sample-to-answer) with an LOD of ~0.03 ng (total RNA). The research team has found that alterations in mRNA expression secured from white blood cell subsets can be used for stroke diagnosis and appear rapidly in peripheral blood following a stroke event. Resulting from a prior R01, CD15+ and CD8+ leukocytes were discovered as a predominant source of stroke- related mRNA biomarkers. This project seeks to realize the development of an innovative fluidic cartridge and the associated assay for the measurement of stroke-related RNA markers sourced from CD15 and CD8 expressing extracellular vesicles (EVs). The utility of EVs as a source of stroke-related RNA biomarkers is based on their rapid appearance and high abundance in plasma, potentially providing even faster stroke diagnosis compared to the cells from which they originate. This project will discover EV-RNA markers with high clinical sensitivity and specificity (>80%) for diagnosing ischemic and hemorrhagic stroke in <3 h from stroke onset. The cartridge, which consists of task-specific modules made from plastics via replication (i.e., injection molding) connected to a fluidic motherboard, will use the EV-RNA markers emanating from this project. Plasma will serve as the input from which surface-affinity selection of CD8 and CD15 EVs will occur using a specifically designed module. Following EV release from the capture surface via a photocleavable linker (cleaved using a blue-light LED), the cartridge will quantify the number of EVs selected using a label-free readout strategy. The fluidic cartridge also consists of a mixed-scale (nm → µm) module to read electrically copy numbers of stroke EV-RNA markers in a highly multiplexed fashion (>24 targets). This module will consist of in-plane nanopores made in a plastic via nano-injection molding and can identify RNAs using a distinct oligonucleotide primer pair querying a specific RNA using a solid-phase ligase detection reaction (spLDR). Single-molecule readout will allow for high analytical sensitivity to observe subtle changes in EV-RNA marker copy numbers. The utility of this cartridge will be evaluated in a clinical setting. Success of the project is leveraged by a strong multidisciplinary team, whom have a productive record of collaboration in developing stroke markers and diagnostic platforms for stroke.

项目摘要/摘要 中风是大脑的一种疾病，零件失去血液供应，受影响的地区迅速发展 如果没有及时恢复血流，请致死。急性缺血性中风（AIS）后恢复血流的治疗 如果从中风事件开始时给予<4.5小时，则有效。但是，部分原因是缺乏体外 诊断需要进行AIS的诊断测试，成像（CT - 临床敏感性〜26％）。 结果，<7％的AIS患者接受治疗。因此，存在着制定策略的关键需求 近乎实时诊断中风可能允许允许护理点测试（POCT）。一种方法是 使用标记的外围血液测试，该标记迅速响应中风引起的大脑变化。这 拟议的项目将开发创新的技术，该技术使用外围血液标记来诊断性中风 综合征（AIS和出血性中风）几乎实时（样品到撤离者约31分钟），LOD为〜0.03 NG（总RNA）。研究小组发现，从白血细胞中获得的mRNA表达的改变 子集可用于中风诊断，并在中风事件发生后迅速出现在外周血中。 由先前的R01产生的CD15+和CD8+白细胞作为中风的主要来源。 相关的mRNA生物标志物。该项目旨在实现创新的液体墨盒的发展和 与CD15和CD8相关的中风相关RNA标记的测量评估 表达细胞外蔬菜（EV）。电动汽车作为中风相关的RNA生物标志物的效用是基于 关于它们的快速外观和血浆中的高抽象，有可能提供更快的中风诊断 与它们起源的细胞相比。该项目将发现具有高临床的EV-RNA标记 敏感性和特异性（> 80％）对诊断性缺血性和出血性中风的敏感性和特异性（> 80％）。这 弹药筒由由塑料制成的特定于任务的模块组成（即注射成型） 连接到流体主板，将使用该项目发出的EV-RNA标记。血浆将服务 作为使用专门设计的CD8和CD15 EV的表面亲和力选择的输入 模块。在通过可光电连接器从捕获表面释放EV后（使用蓝光裂解 LED），墨盒将使用无标签读数策略量化选择的电动汽车数量。流体 墨盒还由混合尺度（NM→µM）模块组成，用于读取中风EV-RNA的电拷贝数 标记以高度多路复用的方式（> 24个目标）。该模块将由平面内纳米孔组成 通过纳米注射成型塑料，可以使用独特的寡核苷酸引物对查询A的RNA识别RNA 使用固相连接酶检测反应（SPLDR）的特异性RNA。单分子读数将允许高 分析灵敏度观察EV-RNA标记拷贝数的细微变化。该墨盒的效用将 在临床环境中进行评估。该项目的成功是由一个强大的多学科团队利用的，他们 在开发中风和中风诊断平台方面的协作有效记录。