Organ-on-Chip Approach for Assessing Tissue-specific SARS-CoV-2 Infection and Response to Antiviral Therapy

用于评估组织特异性 SARS-CoV-2 感染和抗病毒治疗反应的器官芯片方法

• 批准号: 10171540
• 负责人: Thomas Neumann
• 金额: $ 25.57万
• 依托单位: NORTIS, INC.
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10171540
• 关键词: 2019-nCoV; Address; Administrative Supplement; Affect; Agonist; Alveolus; Animals; Antibodies; Antimalarials; Antiviral Agents; Antiviral Therapy; Apical; Award; Binding; Biological Assay; Blood Vessels; COVID-19; COVID-19 pandemic; COVID-19 vaccine; Cell Culture System; Cell Culture Techniques; Cells; ChIP-on-chip; Chloroquine; Clinical; Communicable Diseases; Communities; Data; Development; Diabetes Mellitus; Drug Kinetics; Drug Transport; Drug or chemical Tissue Distribution; Drug toxicity; Electron Microscopy; Endocytosis; Engineering; Environment; Gold; Harvest; Health; Human; Hypertension; In Vitro; Infection; Interferon Inducers; Interferon-beta; Intestinal Mucosa; Invaded; Kidney; Kidney Diseases; Kidney Glomerulus; Lung; Measures; Methods; Modeling; Molecular; Monkeys; Mutation; Myocardium; Organ; Outcome; Parents; Performance; Plaque Assay; Play; Proteins; Protocols documentation; Proximal Kidney Tubules; Pulmonary alveolar structure; RNA; RNA Polymerase Inhibitor; Reporter Genes; Research; SARS coronavirus; Severe Acute Respiratory Syndrome; Severities; Side; Structure; Subfamily lentivirinae; Technology; Testing; Time; TimeLine; Tissue Extracts; Tissues; Vascular Endothelium; Vero Cells; Viral; Viral Load result; Viral load measurement; Virulence; Virus; Virus Diseases; Virus Replication; animal data; base; chromatin immunoprecipitation; clinical predictors; coronavirus disease; experience; experimental study; fluid flow; human tissue; in vitro testing; kidney epithelial cell; older patient; organ on a chip; parent grant; receptor; remdesivir; response; screening; shear stress; success; translational impact; two-dimensional; vaccine candidate; virus host interaction

The current COVID-19 pandemic is a worldwide, rapidly developing, health crisis caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). As of May 18, 2020, over 4.7 million infections are confirmed globally and over 315,000 people have died from COVID-19 related complications. Efforts to develop and test COVID-19 vaccines are in high gear. In the meantime, there is a dire need for fast and robust in-vitro tests that can be used to study the mechanisms of host-virus interactions and help assess whether existing antivirals could be used against for SARS-CoV-2. Current static 2D cell culture systems and animal-based models are of limited use for these purposes. To address this gap, the proposed project aims to develop organ- on-chip (OOC)-based assays for quantifying SARS-CoV-2 inoculation and replication in three human tissues that have been shown to be severely affected by SARS-CoV-2. In order to enable an immediate start, a fast timeline, and milestones with translational impact, the approach of this supplement will mainly repurpose already existing, validated, and commercialized OOC models that were developed under the parent grant. AIM1 is to develop SARS-CoV-2 assays for kidney proximal tubule and vascular endothelium, models that were initially developed for assessing drug toxicity and drug transport. In addition, an OOC model of the lung alveolus will be developed. SARS-CoV-2 Wuhan Reference Strain, the SARS-CoV-2 Spike Mutation D614G Strain, as well as a Spike-pseudotyped lentivirus will be tested and compared for differences in inoculation rate and replication rate (AIM2). The assay protocols will include introducing the viruses via the perfusate to the lumen of the tissue structures in order to bring the virus in contact with the ACE2 and CD 147 receptors that reside on the apical side of the cell and are responsible for virus binding and subsequent endocytosis. To quantify viral inoculation, the tissues will be removed after a short but adequate incubation period. The viruses will be extracted from the tissues, serially diluted and quantified using plaque assays. In order to assess viral replication, tissues will be harvested from the chips after a pre-determined, longer, incubation period that gives the cells enough time for viral replication. Viral load will be quantified with plaque assays. AIM3 is to use the OOC-based assays for testing a number of candidate antivirals and compare their effect against baseline SARS-COVID-19 virus load. The list of antivirals to be tested includes antibodies against ACE2 and CD147 receptors; RNA polymerase inhibitor Remdesivir; PAMP RNA, a RIG-agonist and interferon inducer; and the antimalarial chloroquine. The data obtained from the OOC assays will be correlated with pre-existing in-vitro data, animal data, and clinical findings. The technology can be quickly made available to the research community. Models of other tissue structures affected by SARS-COVID-19, such as myocardium, intestinal mucosa, and kidney glomerulus can be subsequently added to the portfolio. Further, the models can be tailored to include cells from elderly patients or mimic conditions connected with severe outcomes, such as diabetes, hypertension, or kidney disease.

当前的新冠肺炎大流行是一种世界性的、迅速发展的、由严重急性呼吸综合征引起的健康危机。 呼吸综合征冠状病毒2(SARS-CoV-2)。截至2020年5月18日，超过470万人感染 在全球范围内得到确认，已有超过315,000人死于新冠肺炎相关并发症。努力发展 而测试新冠肺炎的疫苗也在如火如荼地进行。与此同时，迫切需要快速和强大的体外培养。 可用于研究主机-病毒相互作用的机制并帮助评估是否存在 抗病毒药物可以用来对抗SARS-CoV-2。当前静态2D细胞培养系统和动物基础 模型对于这些目的的使用是有限的。为了解决这一差距，拟议的项目旨在开发器官- 基于芯片(OOC)检测SARS-CoV-2在三种人体组织中的接种和复制 已被证明受到SARS-CoV-2严重影响的国家。为了能够立即启动，快速启动 时间表和具有翻译影响的里程碑，本附录的方法将主要重新调整用途 在母公司资助下开发的现有的、经过验证的和商业化的OOC模型。AIM1是TO 建立肾近端小管和血管内皮细胞的SARS-CoV-2检测方法 为评估药物毒性和药物转运而开发。此外，一个肺泡的OOC模型将被 发展起来的。SARS-CoV-2武汉参考毒株、SARS-CoV-2尖峰突变株D614G以及 将测试和比较一种尖峰假型慢病毒在接种率和复制方面的差异 速率(AIM2)。检测方案将包括通过灌流液将病毒引入组织的管腔。 结构，以使病毒与驻留在顶端的ACE2和CD147受体接触 在细胞的一侧，负责病毒结合和随后的内吞作用。为了量化病毒接种， 这些组织将在一段短暂但足够的潜伏期后被移除。病毒将从 组织，连续稀释，并使用斑块分析进行定量。为了评估病毒复制，将对组织进行 在预先确定的、更长的孵化期后从芯片中收获，使细胞有足够的时间 病毒复制。病毒载量将通过空斑分析进行量化。AIM3将使用基于OOC的检测方法进行测试 一些候选的抗病毒药物，并比较它们与基线SARS-新冠肺炎病毒载量的效果。这份名单 待检测的抗病毒药物包括针对ACE2和CD147受体的抗体；RNA聚合酶抑制剂 雷米西韦；PAMP RNA，一种RIG激动剂和干扰素诱导剂；以及抗疟疾药物氯喹。数据 从OOC检测中获得的数据将与先前存在的体外数据、动物数据和临床结果相关联。 这项技术可以迅速提供给研究界。其他组织结构的模型 受SARS-新冠肺炎影响，如心肌、肠粘膜、肾小球等均可 随后添加到投资组合中。此外，这些模型可以被定制为包括来自老年患者或 模拟与严重后果相关的情况，如糖尿病、高血压或肾脏疾病。