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: 10445743
• 负责人: Alison E Baird
• 金额: $ 61.54万
• 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/10445743
• 关键词: Acute; Affect; Affinity; Alteplase; Appearance; Biological Assay; Biological Markers; 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; Liquid substance; Magnetic Resonance Imaging; Measurement; Membrane; Messenger RNA; Molds; Molecular; Oligonucleotide Primers; Output; Patients; Peripheral; Phase; Plasma; Plastics; Process; RNA; RNA analysis; RNA marker; Rapid diagnostics; Reaction; Recovery; Research; Reverse Transcription; Sampling; Secure; Sensitivity and Specificity; Solid; Source; Stroke; Structure; Surface; Syndrome; System; Testing; Therapeutic; Time; Transcript; Validation; Vascular blood supply; Work; X-Ray Computed Tomography; base; blood-based biomarker; circulating biomarkers; clinically relevant; design; diagnostic platform; extracellular vesicles; in-vitro diagnostics; innovation; innovative technologies; mRNA Expression; multidisciplinary; nano; nanocolumn; nanopore; nanosensors; novel; operation; peripheral blood; point of care testing; single molecule; stroke event; stroke model; stroke patient; stroke therapy; success; time use; 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 中风是一种脑部疾病，其中一部分失去了血液供应，受影响的区域迅速发展 如果血液循环不能及时恢复，会导致死亡。急性缺血性卒中(AIS)后恢复血流的治疗 如果从中风事件发生之日起4.5小时开始服用，则有效。但是，部分原因是缺乏体外培养的 需要在主治医院进行AIS、影像诊断试验(CT-临床敏感度~26%)才能确诊。 因此，7%的AIS患者接受了治疗。因此，迫切需要制定战略，以便 近乎实时地诊断中风，这可能允许进行护理点测试(POCT)。一种方法是 一种外周血液测试，使用能对中风引起的大脑变化做出快速反应的标记物。这个 拟议的项目将开发使用外周血液标志物诊断中风的创新技术 近乎实时的症状(AIS和出血性中风)(样本到答案约为31分钟)，LOD约为0.03 NG(总RNA)。研究小组发现，基因表达的变化与白细胞有关 亚群可用于中风诊断，并在中风事件发生后迅速出现在外周血中。 由于先前的R01，CD15+和CD8+白细胞被发现是中风的主要来源- 相关信使核糖核酸生物标志物。该项目旨在实现一种创新的射流药筒的开发和 CD15和CD8来源的卒中相关RNA标志物的联合检测 表达胞外小泡(EVS)。EVS作为中风相关RNA生物标记物来源的效用是基于 基于它们的快速出现和血浆中的高丰度，潜在地提供了更快的中风诊断 与它们起源的细胞相比。本项目将发现具有较高临床应用价值的EV-RNA标记物 在卒中起病3小时内诊断缺血性和出血性卒中的敏感度和特异度为80%。这个 墨盒，由通过复制(即注塑)由塑料制成的特定任务模块组成 连接到流体主板，将使用来自该项目的EV-RNA标记。血浆将提供服务 作为CD8和CD15电动汽车的表面亲和力选择的输入，将使用专门设计的 模块。在EV通过可光解连接体(使用蓝光裂解)从捕获表面释放之后 LED)，墨盒将使用无标签读出策略来量化选择的电动汽车的数量。《流体学》 盒还包括一个混合刻度(nm→？m)模块，用于读取笔划EV-RNA的电子拷贝数 以高度多元化的方式标记(&gt；24个目标)。该模块将由平面内纳米孔组成，这些纳米孔在 通过纳米注射成型塑料，并可以使用不同的寡核苷酸引物对查询 用固相连接酶检测反应(SpLDR)检测特定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。此墨盒的使用将 在临床环境中进行评估。该项目的成功得益于一个强大的多学科团队，他们 在开发中风标志物和中风诊断平台方面有卓有成效的合作记录。