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

项目总结 在美国，中风目前是导致永久性残疾的首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危重护理来减少死亡率和发病率。 不幸的是，在分诊期间对中风的识别依赖于基于症状的评估，这些评估通常 不可靠，高达35%的患者在最初的临床医生接触时被误诊。因此，发展 基于生物标志物的准确筛查工具，可用于急诊医学中快速检测中风 设置环境可以显著降低失婚率，使患者能够更早地获得干预，并改善患者 结果。许多先前的研究试图使用血液中各种蛋白质的测量方法 受损的神经组织作为中风的指标。不幸的是，现在已经知道许多蛋白质是靶向的 在这些先前的研究中，脑组织中的浓缩程度有限，降低了它们的诊断特异性。 此外，它们通常以太低的水平循环，无法使用免疫分析进行可靠的检测 在急诊医疗环境中可用于快速血液测试的平台，阻碍了它们进入临床使用的道路。 环状RNAs(CircRNAs)是一类对RNase具有抗性的环状连续的非编码转录本 降解；它们在神经元等长寿命细胞中积累，最近的研究表明，在 是真正只在大脑中发现的变体。除了提高诊断特异性的可能性外， 靶向受损神经组织释放的脑特异性CircRNA可能具有重要的翻译作用 相对于过去研究的基于血液的中风识别的靶向蛋白质的优势。扩大化 用来测量核酸的方法比免疫分析技术灵敏数千倍 用于测量蛋白质，这可能会极大地增加检测的几率。此外，因为这些 CircRNA将是无细胞的，它们可以在血清中直接检测，而不需要上游RNA分离；这可能 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，甚至是这样的方法 作为环介导的等温扩增(LAMP)在护理点。尽管有这些巨大的优势， 脑源性CircRNA作为中风识别生物标志物的潜在来源从未被研究过。 在这里提出的工作中，我们的目标是使用数字液滴聚合酶链式反应来评估五种病毒的绝对循环水平。 500例各种脑血管疾病和非脑血管疾病患者的脑特异性CircRNA检测 入院时出现中风样症状，以及快速制定一套临床可行的习惯 QRT-PCR和LAMP检测针对上述CircRNA，并评估其对中风的敏感性和特异性。这 新颖、创新和高度翻译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化验，以建立直接的技术和分子基础 两个快速中风分类诊断的下游开发：一个可以部署用于统计测试的 现有硬件上的临床实验室，以及可以部署在护理地点的另一个。

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.