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

当前的COVID-19大流行是由严重急性呼吸道感染引起的全球性、迅速发展的健康危机， 呼吸道综合征冠状病毒2（SARS-CoV-2）。截至2020年5月18日，超过470万例感染 全球确诊，超过315，000人死于COVID-19相关并发症。努力发展 新型冠状病毒疫苗的测试工作正在紧锣密鼓地进行。与此同时，迫切需要快速和强大的体外 可用于研究宿主-病毒相互作用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。目前的静态2D细胞培养系统和基于动物的 模型对于这些目的的用途有限。为了弥补这一差距，拟议的项目旨在开发器官- 用于定量SARS-CoV-2在三种人体组织中的接种和复制的基于芯片（OOC）的检测 严重感染了SARS-CoV-2。为了能够立即启动， 时间轴和具有转化影响的里程碑，本补充材料的方法将主要重新调整用途 现有的，经过验证的和商业化的OOC模型是在母基金下开发的。AIM 1是 开发SARS-CoV-2检测肾脏近端小管和血管内皮的模型，这些模型最初是 用于评估药物毒性和药物转运。此外，还将建立肺泡的OOC模型， 开发SARS-CoV-2武汉参考株、SARS-CoV-2刺突突变D 614 G株，以及 a将检测刺突假型慢病毒，并比较接种率和复制的差异 速率（AIM 2）。测定方案将包括通过灌注液将病毒引入组织腔 为了使病毒与位于顶端的ACE 2和CD 147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， 在短暂但足够的孵育期后将组织取出。这些病毒将从 组织，连续稀释并使用噬斑测定定量。为了评估病毒复制，将组织 在预先确定的、更长的孵育期后从芯片上收获，该孵育期给予细胞足够的时间， 病毒复制将采用空斑试验定量病毒载量。AIM 3将使用基于OOC的检测试剂盒进行检测 一些候选抗病毒药物，并将其效果与基线SARS-COVID-19病毒载量进行比较。名单 包括抗ACE 2和CD 147受体的抗体; RNA聚合酶抑制剂 Remdesivir; PAMP RNA，一种RIG激动剂和干扰素诱导剂;以及抗疟疾氯喹。数据 将从OOC测定中获得的结果与预先存在的体外数据、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。其他组织结构模型 受SARS-COVID-19影响的心肌、肠粘膜和肾小球， 后来又加入了投资组合。此外，可以定制模型以包括来自老年患者或老年人的细胞。 模拟与严重后果相关的情况，如糖尿病、高血压或肾脏疾病。

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.