Core A - Administrative/Statistics

项目总结 SARS-CoV-2的传染性储存现在一般在BSL3遏制下进行研究，限制数量 可以使用它的研究人员，并增加了执行他们的一些实验的难度- 曼茨。我们将开发一种SARS-CoV-2的衍生品，它具有复制能力，但传播- 有缺陷的，仅支持单轮感染，因此在BSL2下进行检查是安全的，包含- 门槛。到那时，更多的科学家将能够研究这种病原体。这种被称为CoV-2.def的衍生品， 将在细胞中携带，使其表达受到抑制，只有在治疗后才能转录 使用诱导剂多西环素。它还将有两个由基因编码的结构蛋白的缺失， E和M，所以在他们不在的情况下不会有传染性。这些经过改造的病毒基因都不会有 与CoV-2.def同源，从而最大限度地减少了它们与CoV-2.def重组的机会。M将会是 以反式形式提供的，只有在诱导时才能表达。E将仅通过转染法获得 要么是信使核糖核酸，要么是蛋白质本身。后一种属性旨在确保单元格 携带CoV-2.def不会在传代过程中积累有助于重组的病毒RNA- 而且这种衍生物只能感染一轮细胞。 派生的CoV-2.def将分多阶段建造，以确保其安全和功能- 一步一个脚印。编码SARS-CoV-2非结构蛋白的前两个ORF，1A和1B，将 被引入到由Epstein-Barr病毒质粒复制子衍生的质粒载体中。这些ORF 包括病毒基因组的前2/3，并将受Tet-KRAb抑制子的结合调节 因此，它们只有在用多西环素诱导后才能表达。这一结构 将检查它的条件表达式以及它对M和E的依赖，可能还会检查N 基因对于它在胞外颗粒中的释放也是如此。仅当这些属性被确定为 有效和安全的完整CoV-2.def将在BSL3安全壳下建造和测试。之后 CoV-2.def被发现是复制能力强、传播有缺陷的，并且仅支持单轮 感染，它可以在BSL2实验室进行安全检测。 将对CoV-2.def进行两组实验，以改进对COVID患者的治疗。 19.由于CoV-2.def支持一轮感染，它可以并将被用于测量 患者血浆中的中和抗体。中和抗体只能用 传染性分析，因此CoV-2.def是一种强大、安全的工具，可以用来评估ADAP的这一方面。 积极的免疫反应，并与患者的预后相关。我们将在样品中测量这些滴度。 由翻译科学生物核心(TSB)生物库提供，这是一项在Univer- 威斯康星州碳骨癌中心，并能够评估作为癌症患者可能会产生的影响 这种对新冠肺炎的免疫反应。同样清楚的是，一种有效、安全的中和剂滴度测定方法- ING抗体可用于识别可用于治疗的血浆样本 还有新冠肺炎。 在第二组实验中，CoV-2.def的工程衍生品将用于两个互补的 CRISPR/Cas9筛查以确定SARS-CoV-2的细胞依赖性。我们将鉴定这些细胞 依赖关系：用CRISPR/Cas9建立基因敲除文库，感染该文库 CoV-2.def的两个工程衍生物，用于选择支持感染的细胞和针对支持感染的细胞，以及 通过对所选群体中的sgRNAs进行测序来确定责任基因。失活 在我们的验证性实验中，这些基因应该可以阻止CoV-2.def的感染，从而突出 作为抗病毒治疗靶点的细胞基因和途径。

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.