Developing and Applying a Safe, Tractable Derivative of SARS-CoV-2

开发和应用安全、易处理的 SARS-CoV-2 衍生物

基本信息

批准号：
10173035
负责人：
PAUL F. LAMBERT
金额：
$ 7.59万
依托单位：
UNIVERSITY OF WISCONSIN-MADISON
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-07-01 至 2021-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10173035
关键词：
2019-nCoV Affect Antibodies Antibody titer measurement Antiviral Therapy Binding Biological Assay COVID-19 COVID-19 pandemic CRISPR screen CRISPR/Cas technology Cancer Center Cancer Patient Cell Line Cells Containment Critical Pathways Dependence Doxycycline Engineering Ensure Evaluation Fostering Gene Library Genes Genetic Recombination Genome Immune response Immunoglobulin A Infection Laboratories Libraries Life Cycle Stages Measurement Measures Messenger RNA Nonstructural Protein Open Reading Frames Pathway interactions Patient-Focused Outcomes Patients Pharmaceutical Preparations Phase Plasma Plasmid Cloning Vector Plasmids Population Proliferating Property Proteins RNA replication Replicon Research Research Personnel Role Safety Sampling Scientist Services Structural Protein Supporting Cell Testing Tetanus Helper Peptide Therapeutic Transfection Translational Research Universities Variant Viral Viral Genes Viral Genome Virion Virus Wisconsin Work adaptive immune response biobank biosafety level 2 facility biosafety level 3 facility design experimental study extracellular improved knockout gene neutralizing antibody pandemic disease particle pathogen therapy development tool viral RNA viral genomics

项目摘要

Infectious stocks of SARS-CoV-2 are now generally studied under BSL3 containment limiting the number of researchers who can work with it and increasing the difficulties of performing some of their experiments. We shall develop a derivative of SARS-CoV-2 that is replication-competent, but propagationdefective, supporting only a single round of infection, and therefore safe to examine under BSL2 containment. Many more scientists will then be able to study this pathogen. This derivative, termed CoV-2.def, will be carried in cells such that its expression is repressed and can only be transcribed upon treatment with an inducer, doxycycline. It will also have two deletions of the structural proteins encoded by genes, E and M, so it will not be infectious in their absence. Neither of these engineered viral genes will have homology to CoV-2.def thus minimizing the chance of their recombining with CoV-2.def. M will be supplied in trans and can be expressed only upon induction. E will be supplied only by transfection of either an mRNA or the protein itself. These latter properties are designed to ensure that the cells that carry CoV-2.def do not accumulate viral RNAs during their passage that could contribute to recombination and that the derivative can infect cells for only a single round. The derivative CoV-2.def will be constructed in multiple phases in order to ensure its safety and functioning at each step. The first two orfs, 1A and 1B, which encode non-structural proteins of SARS-CoV-2, will be introduced into a plasmid vector derived from an Epstein-Barr Viral plasmid replicon. These orfs comprise the first 2/3 of the viral genome and will be regulated by the binding of a Tet-KRAB repressor so that they can be expressed only following induction by treatment with doxycycline. This construction will be examined for its conditional expression and for its dependence on M and E and perhaps on the N gene too for its release in extracellular particles. Only when these properties are established as being effective and safe will the intact CoV-2.def be constructed and tested under BSL3 containment. After CoV-2.def is found to be replication-competent, propagation-defective, and support only a single round of infection, it can be examined safely in BSL2 labs. Two sets of experiments with CoV-2.def will be conducted to improve treatment of patients with COVID19. Because CoV-2.def supports one round of infection, it can and will be used to measure titers of neutralizing antibodies in the plasma of patients. Neutralizing antibodies can only be measured with infectivity assays so that CoV-2.def is a powerful, safe tool with which to evaluate this facet of the adaptive immune response and correlate it with patient outcomes. We shall measure these titers in samples provided by the Translational Science BioCore (TSB) BioBank, which is a shared service at the University of Wisconsin Carbone Cancer Center, and be able to assess how being a cancer patient may affect this immune response to COVID-19. It is also clear that an effective, safe assay for the titers of neutralizing antibodies can be used to identify samples of plasma that can be provided therapeutically to patients with COVID-19. In the second set of experiments, engineered derivatives of CoV-2.def will be used in two complementary CRISPR/Cas9 screens to identify cell-dependencies of SARS-CoV-2. We shall identify these cellular dependencies by establishing a library of gene knockouts with CRISPR/Cas9, infecting this library with two engineered derivatives of CoV-2.def to select for and against the cells that support infection, and determining the responsible genes by sequencing the sgRNAs in the selected populations. Inactivating these genes in our confirmatory experiments should block infection by CoV-2.def and thereby highlight cellular genes and pathways which are targets for anti-viral therapies.

现在，在BSL3遏制下，通常对SARS-COV-2的传染股进行了研究，从而限制了可以与之合作的研究人员的数量，并增加了执行某些实验的困难。我们将开发一种具有复制能力的SARS-COV-2的衍生物，但传播作用仅支持一轮感染，因此可以安全地检查BSL2遏制。然后，更多的科学家将能够研究这种病原体。该衍生物称为COV-2.DEF，将在细胞中携带，以使其表达受到抑制，并且只能在用诱导剂强力霉素治疗后转录。它还将有两种由基因，E和M编码的结构蛋白的缺失，因此它在不存在的情况下不会具有感染性。这些工程的病毒基因都不具有与COV-2的同源性。 M将在反式中提供，并且只能在诱导后表达。 E仅通过转染mRNA或蛋白质本身提供。这些后一种特性旨在确保携带COV-2.DEF的细胞在通过期间不会积聚病毒RNA，这可能导致重组，并且衍生物只能感染单个回合的细胞。派生cov-2.def将以多个阶段构建，以确保其在每个步骤中的安全性和功能。编码SARS-COV-2的非结构蛋白的前两个ORF 1A和1B将被引入源自Epstein-Barr病毒质粒复制子的质粒载体。这些ORF构成了病毒基因组的前2/3，将由TET-KRAB阻遏物的结合来调节，因此仅通过用多西环素处理在诱导后才能表达它们。该结构将检查其条件表达及其对M和E的依赖，也许对N基因也可能释放到细胞外颗粒中。只有在将这些属性确定为有效且安全的情况下，才能在BSL3遏制下构建和测试完整的COV-2。发现COV-2.DEF具有复制能力，传播缺陷和仅支持一轮感染后，可以在BSL2 Labs中对其进行安全检查。将进行两组COV-2.DEF实验，以改善COVID19患者的治疗。由于COV-2.DEF支持一轮感染，因此它可以并且将被用来测量患者血浆中中和抗体的滴度。中和抗体只能通过感染性测定来测量，因此COV-2.DEF是一种强大，安全的工具，可以评估自适应免疫反应的这一方面，并将其与患者结局相关联。我们将在威斯康星大学Carbone Cancer Center提供的转化科学生物库（TSB）Biobank提供的样品中测量这些滴度，并能够评估癌症患者如何影响对Covid-19的这种免疫反应。同样很明显，可以使用中和抗体的有效，安全的测定方法来鉴定可以为Covid-19患者提供治疗的血浆样品。在第二组实验中，COV-2.DEF的工程衍生物将在两个互补的CRISPR/CAS9屏幕中使用，以识别SARS-COV-2的细胞依赖性。我们将通过建立具有CRISPR/CAS9的基因敲除库来确定这些细胞依赖性，并用Cov-2.def的两个工程衍生物感染该文库，以选择和反对支持感染的细胞，并通过对所选人群中的SGRNA进行测序来确定负责的基因。在我们的验证性实验中灭活这些基因应通过COV-2.DEF阻断感染，从而突出显示抗病毒疗法靶标的细胞基因和途径。