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

目前，SARS-CoV-2的感染性种群通常在BSL 3控制下进行研究，这限制了能够使用它的研究人员的数量，并增加了进行一些实验的难度。我们将开发一种SARS-CoV-2的衍生物，它具有复制能力，但繁殖缺陷，只支持单轮感染，因此在BSL 2控制下可以安全地进行检查。更多的科学家将能够研究这种病原体。这种衍生物，称为CoV-2.def，将携带在细胞中，使其表达受到抑制，只能在用诱导剂多西环素处理后转录。它也会有两个由基因E和M编码的结构蛋白缺失，所以在它们缺失的情况下它不会有传染性。这些工程改造的病毒基因都不与CoV-2.def具有同源性，因此使它们与CoV-2.def重组的机会最小化。M将以反式提供，并且仅在诱导时才能表达。E将仅通过mRNA或蛋白质本身的转染来提供。这些后一种性质旨在确保携带CoV-2.def的细胞在其传代过程中不会积累可能有助于重组的病毒RNA，并且该衍生物只能感染细胞一轮。衍生型CoV-2.def将分多个阶段构建，以确保其在每个步骤的安全性和功能。将编码SARS-CoV-2的非结构蛋白的前两个orfs 1A和1B引入源自Epstein-Barr病毒质粒复制子的质粒载体中。这些orf包含病毒基因组的前2/3，并且将通过Tet-KRAB阻遏物的结合来调节，使得它们仅在用多西环素处理诱导后才能表达。将检查该构建体的条件表达及其对M和E的依赖性，可能还检查N基因在细胞外颗粒中的释放。只有当这些特性被确定为有效和安全时，才能在BSL 3控制下构建和测试完整的CoV-2.def。在发现CoV-2.def具有复制能力、传播缺陷并且仅支持单轮感染后，可以在BSL 2实验室中安全地进行检查。将进行两组CoV-2.def实验，以改善COVID患者的治疗19。由于CoV-2.def支持一轮感染，因此它可以并将用于测量患者血浆中中和抗体的滴度。中和抗体只能用感染性检测来测量，因此CoV-2.def是一种强大、安全的工具，可以用来评估适应性免疫反应的这一方面，并将其与患者结局相关联。我们将测量转化科学生物核心（TSB）生物库提供的样本中的这些滴度，这是威斯康星州大学Carbone癌症中心的一项共享服务，并能够评估癌症患者如何影响这种对COVID-19的免疫反应。同样清楚的是，中和抗体滴度的有效、安全测定可用于鉴定可向COVID-19患者提供治疗的血浆样品。在第二组实验中，CoV-2.def的工程衍生物将用于两个互补的CRISPR/Cas9筛选，以鉴定SARS-CoV-2的细胞依赖性。我们将通过建立CRISPR/Cas9基因敲除文库来鉴定这些细胞依赖性，用两种CoV-2.def的工程衍生物感染该文库以选择支持感染的细胞和对抗支持感染的细胞，并通过对所选群体中的sgRNA进行测序来确定负责基因。在我们的验证性实验中灭活这些基因应该可以阻断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。

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.