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 中风目前是美国永久性残疾的主要原因和第五大死亡原因。 早期和准确的识别通过加快获得神经重症监护来降低死亡率和发病率。 不幸的是，在分诊过程中识别卒中依赖于基于脑卒中的评估， 不可靠，高达35%的患者在初次接触临床医生时被误诊。因此， 基于生物标志物的精确筛查工具，可用于在急诊医学中快速检测卒中 设置可以大大降低误治率，使早期获得干预，并改善患者 结果。许多先前的研究已经尝试使用血液测量从血液中释放的各种蛋白质。 神经组织受损是中风的征兆不幸的是，现在已知许多靶向的蛋白质 在这些先前的研究中显示出在脑组织中有限程度的富集，削弱了它们的诊断特异性。 此外，它们通常以太低的水平循环而不能使用免疫测定进行可靠的检测 在紧急医疗环境中，可用于快速血液检测的平台阻碍了其临床应用。 环状RNA（CircRNA）是一类环状连续的非编码转录物，对RNA酶具有抗性 降解;它们积累在长寿命的细胞，如神经元，最近的工作表明， 只有在大脑中才能找到。除了提高诊断特异性的潜力外， 靶向从受损神经组织释放的脑特异性circRNA可能具有显著的翻译活性， 与过去研究用于基于血液的中风识别的靶向蛋白质相比，扩增 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将是无细胞的，它们可以在血清中直接测定而无需上游RNA分离;这可以 允许在临床实验室中发现的现有硬件上使用qRT-PCR进行快速直接检测，或者甚至使用诸如 如在床旁的环介导等温扩增（LAMP）。尽管有这些巨大的优势， 脑源性circRNA从未被研究作为中风识别的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来评估五种血吸虫的绝对循环水平。 脑特异性circRNA在500名患有各种脑血管和非脑血管病变的患者中， 在入院时出现中风样症状，并开发一套临床可行的定制快速 靶向所述circRNA的qRT-PCR和LAMP测定，并评估它们对中风的敏感性和特异性。这 新颖，创新和高度转化的项目解决了一个迫切的临床需求领域;我们完全期待 作为这项工作的一部分产生的测定，以建立直接的技术和分子基础， 下游开发一对快速中风分类诊断：一个可以部署在统计测试， 一个是现有硬件上的临床实验室，另一个是可以在护理点部署的。