Investigation of brain-originating circRNAs as targets in blood-based stroke triage diagnostics

研究脑源性 circRNA 作为基于血液的中风分类诊断的靶标

• 批准号: 10563706
• 负责人: Grant C O'Connell
• 金额: $ 60.92万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-03-15 至 2028-02-29
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10563706
• 关键词: Accident and Emergency department; Address; Admission activity; Ambulances; Area; Back; Biological Assay; Biological Markers; Blood; Blood Tests; Blood Volume; Blood specimen; Brain; Brain Injuries; Brain hemorrhage; Caring; Cause of Death; Cell Differentiation process; Cells; Clinical; Clinical Research; Country; Custom; Data; Detection; Development; Diagnosis; Diagnostic; Diagnostic Specificity; Diagnostic tests; Emergency Medicine; Exhibits; Family; Foundations; Hospitalization; Immunoassay; Informatics; Intervention; Investigation; Ischemic Stroke; Laboratories; Laboratory Research; Measures; Mediating; Methods; Modeling; Molecular; Molecular Biology; Morbidity - disease rate; Myocardial Infarction; Nature; Nervous System Trauma; Neurologic Symptoms; Neurons; Nucleic Acids; Organ; Outcome; Patient-Focused Outcomes; Patients; Performance; Process; Proteins; Quantitative Reverse Transcriptase PCR; RNA; Resistance; Ribonucleases; Sampling; Screening procedure; Sensitivity and Specificity; Serum; Source; Stroke; Surrogate Markers; Symptoms; Techniques; Testing; Tissues; Transcript; Triage; Troponin; UCHL1 gene; United States; Untranslated RNA; Variant; Work; amplification detection; brain tissue; cerebrovascular; circular RNA; cohort; diagnostic tool; digital; disability; improved; innovation; interest; isothermal amplification; mortality; nervous system disorder; neural; novel; point of care; random forest; rapid detection; stroke patient; stroke-like outcome; tau Proteins

PROJECT SUMMARY Stroke is currently the leading cause of permanent disability and fifth leading cause of death in the United States. Early and accurate recognition reduces mortality and morbidity by expediting access to neurocritical care. Unfortunately, recognition of stroke during triage is dependent on symptom-based assessments that are often unreliable, and up to 35% of patients are misdiagnosed at initial clinician contact. Thus, the development of accurate biomarker-based screening tools that could be used to rapidly detect stroke in emergency medicine settings could substantially reduce rates of mistriage, enable earlier access to intervention, and improve patient outcomes. Numerous prior investigations have attempted to use blood measures of various proteins released from damaged neural tissue as an indicator of stroke. Unfortunately, it is now known that many of the proteins targeted in these prior studies exhibit a limited degree of enrichment in brain tissue, curtailing their diagnostic specificity. Furthermore, they typically circulate at too low of levels to enable reliable detection using the immunoassay platforms available for rapid blood testing in emergency medicine settings, blocking their path to clinical use. Circular RNAs (circRNAs) are a family of circularly continuous non-coding transcripts that are resistant to RNase degradation; they accumulate in long-lived cells such as neurons, and recent work has demonstrated that there are variants which are truly found only in the brain. In addition to the potential for improved diagnostic specificity, targeting brain-specific circRNAs released from damaged neural tissue could have significant translational advantages over targeting the proteins studied for blood-based stroke recognition in the past. The amplification methods used to measure nucleic acids are thousands of times more sensitive than the immunoassay techniques used to measure proteins, which could dramatically increase the odds of detection. Furthermore, because these circRNAs would be cell-free, they could be directly assayed in serum without upstream RNA isolation; this could allow for rapid direct detection using qRT-PCR on existing hardware found in clinical labs, or even methods such as loop-mediated isothermal amplification (LAMP) at the point-of-care. Despite these tremendous advantages, brain-originating circRNAs have never been investigated as a potential source of biomarkers for stroke recognition. In the work proposed here, we aim to use digital droplet PCR to evaluate the absolute circulating levels of five brain-specific circRNAs in 500 patients with various cerebrovascular and non-cerebrovascular pathologies who present with stroke-like symptoms at hospital admission, as well as develop a set of clinically viable custom rapid qRT-PCR and LAMP assays targeting said circRNAs and evaluate their sensitivity and specificity for stroke. This novel, innovative, and highly translational project addresses an area of dire clinical need; we fully expect the assays generated as part of this work to establish the immediate technical and molecular foundation for the downstream development of a pair of rapid stroke triage diagnostics: one that could be deployed for stat testing in the clinical laboratory on existing hardware, and another that could be deployed at the point-of-care.

项目摘要 目前，中风是美国永久残疾和第五大死亡原因的主要原因。 早期，准确的识别通过加快获得神经关怀的机会来降低死亡率和发病率。 不幸的是，分类过程中对中风的识别取决于基于症状的评估，这些评估通常是 不可靠，在最初的临床医生接触时，多达35％的患者被误诊。因此，发展 基于生物标志物的准确筛选工具，可用于快速检测急诊医学中的中风 设置可能会大大降低误解率，使早期接受干预并改善患者 结果。许多先前的研究试图使用从释放的各种蛋白质的血液测量 受损的神经组织作为中风的指标。不幸的是，现在知道许多蛋白质针对 在这些先前的研究中，在脑组织中的富集程度有限，从而减少了它们的诊断特异性。 此外，它们通常以太低的水平循环，无法使用免疫测定 可在急诊医学环境中快速进行血液测试的平台，阻止了其临床使用的途径。 圆形RNA（CIRCRNA）是一个循环连续的非编码转录本，对RNase具有抗性 降解;它们积聚在长寿细胞（例如神经元）中，最近的工作表明那里 是仅在大脑中才能找到的变体。除了提高诊断特异性的潜力外， 靶向由受损神经组织释放的大脑特异性circRNA可能具有明显的翻译 过去，靶向研究血液中风识别的蛋白质的优势。放大 用于测量核酸的方法比免疫测定技术高数千倍 用于测量蛋白质，这可能会大大增加检测的几率。此外，因为这些 Circrnas无细胞，可以直接在血清中直接测定，而无需上游RNA分离。可以 使用QRT-PCR快速直接检测到临床实验室中发现的现有硬件，甚至是这样的方法 作为循环介导的等温扩增（LAMP），处于护理点。尽管有这些巨大的优势， 从未研究过脑部渗透性circrnas作为中风识别的潜在生物标志物来源。 在此处提出的工作中，我们旨在使用数字液滴PCR评估五个的绝对循环水平 500例患有各种脑血管和非脑血管病理的患者的大脑特异性circrnas 在住院时出现类似中风的症状，并发展一组临床可行的自定义快速 QRT-PCR和LAMP分析靶向circrnas，并评估其对中风的敏感性和特异性。这 新颖，创新且高度转化的项目涉及一个可怕的临床需求领域；我们完全期望 作为这项工作的一部分生成的测定，以建立直接的技术和分子基础 一对快速中风分诊诊断的下游开发：一个可以在统计测试中部署的 现有硬件的临床实验室，另一个可以在护理点部署。