Project 2: Use of 2D cultures and 3D organoids to identify candidate antiviral compounds; to use genetic approaches to identify host genes that promote or protect against flavivirus infection

为了满足对改进的人体组织模型来研究传染病的迫切需求，我们建议建立 麻省理工学院人类组织和传染病中心(MIT.HTMID)。MIT.HTMID中心将研究 利用生物学上相关的二维技术对人脑和中枢神经系统的病毒感染 (2D)人类神经细胞和三维(3D)人脑器官。细胞是同质的。 人神经前体细胞、神经元、少突胶质细胞、星形胶质细胞和小胶质细胞的制备 来自胚胎干细胞(ES)或诱导多能干细胞(iPS细胞)，并将在 人体细胞和组织核心。先前的工作已经证明，这些细胞和有机体 形成的生物功能接近生理功能，从而代表了一种实验系统，即 优于啮齿动物模型。MIT.HTMID项目2将侧重于2D和3D组织模型的应用 为了a)测试一组已发表的抗病毒化合物在阻断病毒感染方面的有效性， 和b)通过进行CRISPR-Cas9基因敲除来识别宿主，研究传染病的遗传学 调节病毒感染或受病毒感染调节的依赖基因。项目2 Aim 2A将进行测试 一组已发表的有效阻断病毒感染的抗病毒化合物。这些方法将结合在一起 用两种病毒感染的细胞和细胞器，产生于MIT.HTMID病毒学核心。 首先，我们将研究黄病毒，包括登革热病毒、西尼罗河病毒和寨卡病毒。事实上，一个 研究人脑组织和病毒的重要动机是了解寨卡病毒的发病机制 病毒感染，在世界范围内导致小头畸形和格林-巴利综合征。第二，我们将使用 假型水泡性口炎病毒(VSV)研究几种脑炎病毒的入侵，包括东方人 马脑炎病毒(EEEV)、西部马脑炎病毒(Weev)和委内瑞拉马 脑炎病毒(VEEV)。通过用EEEV、WeEV或VEEV替换VSV病毒包膜，我们 能够研究选择的(即高度危险的，可能在生物武器中应用的)制剂在 2D细胞和3D有机体。使用伪型选择病毒和多种相关细胞类型将使我们能够 研究病毒的嗜性、病理学和潜在的治疗靶点。项目2目标2a不是高吞吐量 筛查；相反，目标是测试一组已发表的抗病毒药物在阻断2D感染方面的有效性 神经细胞和3D有机体。项目2目标2B.i和2B.ii将侧重于执行和验证CRISPR-- Cas9筛查将神经细胞类型与寨卡病毒或假型VSV的感染结合在一起。这些 筛查将识别病毒复制周期所需的宿主依赖因子(HDF)，并且可以 确定治疗干预的目标。抗病毒测试和基因筛查方法都将 评价人2D神经细胞和人脑3D器官作为人体组织的应用 传染病模型。

To address a critical need for improved human tissue models to study infectious diseases, we propose to form the MIT Center for Human Tissues and Infectious Diseases (MIT.HTMID). The MIT.HTMID Center will study viral infections of the human brain and central nervous system by using biologically relevant two dimensional (2D) human neural cells and three dimensional (3D) human cerebral organoids. The cells are homogeneous preparations of human neuronal progenitors, neurons, oligodendrocytes, astrocytes, and microglia, derived from embryonic stem (ES) cells or induced pluripotent stem cells (iPS cells), and will be produced in the MIT.HTMID Human Cell and Tissue Core. Prior work has demonstrated that these cells and the organoids formed are near-physiological in their biological functions, thereby representing an experimental system that is superior to rodent models. MIT.HTMID Project 2 will focus on the applied use of the 2D and 3D tissue models for the purposes of a) testing a panel of published antiviral compounds for efficacy in blocking virus infections, and b) studying the genetics of infectious disease by performing CRISPR-Cas9 gene knockouts to identify host dependency genes that regulate virus infections or are regulated by virus infections. Project 2 Aim 2A will test a panel of published antiviral compounds for efficacy in blocking virus infections. The approaches will couple the cells and organoids with infections using two types of viruses, produced in the MIT.HTMID Virology Core. First, we will study flaviviruses, including Dengue Fever Virus, West Nile Virus, and Zika Virus. Indeed, an important motivation for studying human brain tissue and viruses is to understand the pathogenesis of Zika virus infections, which are causing microcephaly and Guillain-Barre syndrome worldwide. Second, we will use pseudotyped vesicular stomatitis viruses (VSV) to study entry of several encephalitic viruses, including Eastern Equine Encephalitis Virus (EEEV), Western Equine Encephalitis Virus (WEEV) and Venezuelan Equine Encephalitis Virus (VEEV). By replacing the VSV viral envelope with that of EEEV, WEEV, or VEEV, we are able to study virus entry of select (i.e. highly dangerous with potential application in biowarfare) agents in the 2D cells and 3D organoids. Using pseudotyped select viruses and multiple relevant cell types will allow us to study virus tropism, pathology, and potential therapeutic targets. Project 2 Aim 2A is not a high throughput screen; rather, the goal is to test the efficacy of a panel of published antivirals in blocking infections of the 2D neural cells and 3D organoids. Project 2 Aim 2B.i and 2B.ii will focus on performing and validating CRISPR- Cas9 screens that couple the neural cell types with infections by Zika virus or pseudotyped VSV. These screens will identify host dependency factors (HDF) that are required for the viral replication cycle, and may identify targets for therapeutic intervention. Both the antiviral testing and the genetic screening approaches will critically evaluate the human 2D neural cells and human 3D cerebral organoids for use as a human tissue model for infectious disease.