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.

项目概要 目前，中风是美国永久残疾的主要原因和第五大死亡原因。 早期准确的识别可以加快获得神经重症监护的速度，从而降低死亡率和发病率。 不幸的是，在分诊过程中对中风的识别取决于基于症状的评估，而这些评估通常是基于症状的评估。 不可靠，高达 35% 的患者在初次接触临床医生时被误诊。因此，发展 基于生物标志物的准确筛查工具，可用于在急诊医学中快速检测中风 设置可以大大降低误诊率，能够更早获得干预，并改善患者的状况 结果。许多先前的研究尝试使用血液测量从 神经组织受损作为中风的指标。不幸的是，现在已知许多目标蛋白质 在这些先前的研究中，脑组织的富集程度有限，从而降低了其诊断特异性。 此外，它们的循环水平通常太低，无法使用免疫分析进行可靠检测 可在急诊医学环境中进行快速血液检测的平台，阻碍了其临床应用。 环状 RNA (circRNA) 是一类对 RNase 具有抗性的环状连续非编码转录本 降解;它们在神经元等长寿细胞中积累，最近的研究表明， 是真正只在大脑中发现的变异。除了提高诊断特异性的潜力之外， 针对受损神经组织释放的大脑特异性 circRNA 可能具有显着的转化作用 与过去针对基于血液的中风识别研究的蛋白质相比具有优势。放大倍数 用于测量核酸的方法比免疫测定技术灵敏数千倍 用于测量蛋白质，这可以大大增加检测的几率。此外，由于这些 circRNA 是无细胞的，可以直接在血清中进行检测，无需分离上游 RNA；这可以 允许在临床实验室现有硬件上使用 qRT-PCR 进行快速直接检测，甚至可以使用诸如此类的方法 作为现场护理的环介导等温扩增 (LAMP)。尽管有这些巨大的优势， 大脑起源的 circRNA 从未被研究为中风识别生物标志物的潜在来源。 在这里提出的工作中，我们的目标是使用数字液滴 PCR 来评估五种病毒的绝对循环水平。 500 名患有各种脑血管和非脑血管病变的患者的脑特异性 circRNA 入院时出现中风样症状，并开发一套临床上可行的定制快速治疗方案 针对所述 circRNA 的 qRT-PCR 和 LAMP 检测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解决了临床迫切需要的领域；我们充分期待 作为这项工作的一部分产生的测定为建立直接的技术和分子基础 一对快速中风分类诊断的下游开发：可以部署用于统计测试 现有硬件上的临床实验室，以及另一个可以部署在护理点的实验室。