Liposome fusion enabled extracellular vesicle detection for COVID-19

摘要 新冠肺炎可引起在最初的SARS-CoV-2消退后很长一段时间仍持续存在的病变 感染。需要新的、更灵敏的检测方法来更好地了解潜在的机制 参与这一过程并提高对有症状和无症状新冠肺炎病例的检测， 包括长期感染，这可能会被忽略，因为黄金的显著假阴性率- 标准新冠肺炎检测、逆转录定量聚合酶链式反应(RT-qPCR)。呼吸性 样本似乎是检测和监测SARS-CoV-2感染的糟糕选择，超出了相对狭窄的范围 病毒暴露后的窗口，因为在上呼吸道有一过性和间歇性的病毒产生 在感染之后。有证据表明，SARS-CoV-2可能通过循环系统传播，这表明 这种血液是均匀的，定期采集，不适和暴露风险最小，可以起到 作为替代的诊断和监测标本。然而，RT-qPCR显示出较差的诊断敏感性 新冠肺炎在分析血样时。我们团队开发了一种快速、超灵敏的新冠肺炎检测方法 (CRISPR-FDS)，其中CRISPR活性与扩增的DNA靶标成比例地切割猝灭的探针 将检测灵敏度提高20倍。这种检测方法现在被用作调查测试，可以检测出新冠肺炎病例 不需要昂贵的鼻咽拭子样本重复RT-qPCR检测而漏掉 设备、重要的技术专长或防护设备。我们最近的初步数据表明， CRISPR-FDS分析分离血浆RNA罐检测循环中无细胞病毒RNA 无论感染部位(S)或病程长短，均可确诊新冠肺炎病例。因此，我们建议调整我们的 CRISPR-FDS方法允许直接定量血浆中的病毒RNA。在这项试验中，细胞外小泡 直接从血浆中捕获的(EV)被诱导与负载CRISPR-FDS的合成脂质体融合 使用标准的酶联免疫吸附试验，在小体积内检测试剂，以有效地扩增和定量目标RNA 工作流程。我们选择了肠道病毒进行检测，因为这些囊泡由感染的细胞大量分泌， 将病毒RNA保存在其管腔内，并可被抗体特异性地从血浆/血清中捕获 以它们的表面蛋白为靶点，减少无细胞核酸的背景。目标1将优化CRISPR- FDS检测程序(例如，EV捕获、脂质体包装和融合以及试剂滴定步骤) 最大限度地提高反应灵敏度，并评估如何用靶向性修饰脂质体表面 抗体会影响分析性能。目标2将对优化后的分析性能进行评估 与血浆样本的RT-qPCR相比，进行脂质体分析并进行分析验证，并使用以下方法 直接定量分析纵向血浆队列中EVS中SARS-CoV-2RNA的方法 肠病毒介导的新冠肺炎的血清或血浆诊断，并定量评估其动态变化 新冠肺炎患者纵向血样中的SARS-CoV-2RNA检测。

Abstract COVID-19 can cause pathological changes that persist long after the resolution of the initial SARS-CoV-2 infection. New, more sensitive detection approaches are needed to better understand the potential mechanisms involved in this process and to improve the detection of both symptomatic and asymptomatic COVID-19 cases, including long-term infections, that may be missed by due to the significant false negative rate of the gold- standard COVID-19 test, reverse transcriptase quantitative polymerase chain reaction (RT-qPCR). Respiratory samples appear to be a poor choice to detect and monitor SARS-CoV-2 infections beyond a relatively narrow window after virus exposure, as there is transient and intermittent viral production in the upper respiratory tract after infection. Evidence indicates that SARS-CoV-2 may spread systemically through the circulation, suggesting that blood, which is homogeneous and routinely collected with minimal discomfort and exposure risk, could serve as an alternate diagnostic and monitoring specimen. RT-qPCR, however, exhibits poor diagnostic sensitivity for COVID-19 when analyzing blood samples. Our team has developed a rapid, ultrasensitive COVID-19 assay (CRISPR-FDS) where CRISPR activity cleaves a quenched probe in proportion to an amplified DNA target to increase assay sensitivity 20-fold. This assay, now used as an investigational test, can detect COVID-19 cases missed by repeated RT-qPCR testing of nasopharyngeal swab samples, and does not require expensive equipment, significant technical expertise, or protective equipment. Our recent preliminary data suggest that detection of circulating cell-free viral RNA by a CRISPR-FDS assay that analyzes isolated plasma RNA can diagnose COVID-19 cases regardless of infection site(s) or duration. We therefore propose to adapt our CRISPR-FDS method to allow direct quantification of viral RNA in plasma. In this assay, extracellular vesicles (EVs) captured directly from plasma are induced to fuse with synthetic liposomes loaded with CRISPR-FDS assay reagents within a small volume to amplify and quantify target RNA efficiently, using a standard ELISA workflow. We selected EVs for this assay, since these vesicles are abundantly secreted by infected cells and preserve viral RNA within their lumen, and can be specifically captured from plasma/serum by antibodies targeting their surface protein to reduce background from cell-free nucleic acid. Aim 1 will optimize CRISPR- FDS assay procedures (e.g., EV capture, liposome packaging and fusion, and reagent titration steps) to maximize reaction sensitivity, and evaluate how modification of the liposome surface with target-specific antibodies influences assay performance. Aim 2 will evaluate the analytical performance of the optimized assay versus RT-qPCR for plasma samples, conduct and analytical validation of the liposome assay, and employ this assay for quantitative analysis of SARS-CoV-2 RNA in plasma EVs present in longitudinal plasma cohort direct EV-mediated COVID-19 diagnosis using serum or plasma, and quantitatively evaluate dynamic changes in SARS-CoV-2 RNA in longitudinal blood samples from a cohort of COVID-19 patients.