Core A - Administrative/Statistics

核心 A - 行政/统计

• 批准号: 10268311
• 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
• 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/10268311
• 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; statistics; therapy development; tool; viral RNA; viral genomics

PROJECT SUMMARY 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 experi- ments. We shall develop a derivative of SARS-CoV-2 that is replication-competent, but propagation- defective, supporting only a single round of infection, and therefore safe to examine under BSL2 contain- ment. 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 recombina- tion 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 function- ing 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 COVID- 19. 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 adap- tive 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 Univer- sity 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 neutraliz- ing 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.

项目摘要 SARS-COV-2的传染性股票现在通常在BSL3遏制限制下研究 可以与之合作并增加执行某些实验的困难的研究人员 ments。我们将开发一种具有复制能力的SARS-COV-2衍生物，但传播 - 有缺陷，仅支持一轮感染，因此可以安全检查BSL2包含 - 精神。然后，更多的科学家将能够研究这种病原体。这个衍生物称为cov-2.def， 将在细胞中携带，以使其表达受到抑制，并且只能在治疗后转录 与诱导剂强力霉素。它还将具有通过基因编码的结构蛋白的两种缺失， E和M，因此在他们缺席的情况下不会传染。这些工程的病毒基因都不会 cov-2.def的同源性，从而最大程度地减少了它们与cov-2.def重组的机会。 M会 在trans中提供，只能在诱导后表达。 E仅通过转染而提供 mRNA或蛋白质本身。这些后一种特性旨在确保单元格 携带COV-2.DEF在通过期间不会积聚病毒RNA Tion和衍生物只能在单个弹药中感染细胞。 派生cov-2.def将在多个阶段构建，以确保其安全性和功能 - 在每个步骤中。编码SARS-COV-2的非结构性蛋白质的前两个ORF，1A和1B将 被引入来自爱泼斯坦 - 巴尔病毒质粒复制子的质粒载体。这些orf 包括病毒基因组的前2/3，将由TET-KRAB阻遏物的结合来调节 因此，只能通过强力霉素治疗诱导后才表达它们。这个结构 将检查其条件表达及其对M和E的依赖以及可能对N的依赖 基因也可以释放在细胞外颗粒中。仅当这些属性被确定为 在BSL3遏制下构建和测试完整的COV-2.DEF将有效且安全。后 发现COV-2.DEF具有复制能力，传播缺陷，并且仅支撑一轮 感染，可以在BSL2实验室中对其进行安全检查。 将进行两组COV-2.DEF的实验，以改善Covid-患者的治疗 19。因为COV-2.DEF支持一轮感染，因此可以并且将用于测量 中和患者血浆中的抗体。中和抗体只能通过 感染性测定使COV-2.DEF是一种强大，安全的工具，可以评估ADAP的这一方面 免疫反应并将其与患者结局相关。我们将在样品中测量这些滴度 由翻译科学生物谷（TSB）生物库提供，该银行是Univer的共享服务 威斯康星州碳纤维癌中心的性，并能够评估癌症患者如何影响 这种对COVID-19的免疫反应。同样很明显，对中和的有效，安全的测定 ING抗体可用于识别可以在患者治疗的血浆样本 与Covid-19。 在第二组实验中，COV-2的工程衍生物将在两个互补中使用 CRISPR/CAS9筛选以识别SARS-COV-2的细胞依赖性。我们将确定这些细胞 依赖性通过建立一个用CRISPR/CAS9建立基因淘汰的库，以感染该库 COV-2.DEF的两个工程衍生物，以选择和反对支持感染的细胞，以及 通过对选定人群中的SGRNA进行测序来确定负责的基因。灭活 这些基因在我们的确认实验中应通过COV-2.DEF阻断感染，从而突出显示 细胞基因和途径是抗病毒疗法的靶标。