Human 3D neuro-muscular assembloids to study cell tropism and host factor utilization of divergent neuropathogenic enteroviruses

人类 3D 神经肌肉组合体用于研究不同神经致病性肠道病毒的细胞向性和宿主因子利用

• 批准号: 10595022
• 负责人: Jan E Carette
• 金额: $ 76.23万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-03-21 至 2027-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10595022
• 关键词: 3-Dimensional; Address; Affect; Antiviral Therapy; Astrocytes; Biological Assay; Biology; Cell Communication; Cell Line; Cell Lineage; Cells; Central Nervous System; Cerebral cortex; Child; Clustered Regularly Interspaced Short Palindromic Repeats; Cortical Cord; Cytoprotection; Cytosol; Disease; Disease Outbreaks; Disease model; Encephalitis; Endocytosis; Enterovirus; Enterovirus 68; Enterovirus 71; Enterovirus Infections; Enzymes; Foot Diseases; Foundations; Genes; Genetic; Genetic Screening; Genome; Goals; Hand' s disease; Human; Human poliovirus; Huntington gene; Immune response; In Vitro; Individual; Infection; Infectious Agent; Integration Host Factors; Knock-out; Knowledge; Laboratories; Learning; Life; Mediating; Medical; Meningitis; Modeling; Molecular; Motor; Motor Neurons; Mouth Diseases; Movement; Mus; Muscle; Muscle Contraction; Muscle Fibers; Myocarditis; Names; Nervous System; Neurologic; Neuromuscular Junction; Neurons; Neuropathogenesis; Neurosciences; Neurotropism; Oligodendroglia; Organoids; Paralysed; Pathogenesis; Phospholipase; Physiological; Pluripotent Stem Cells; Poliomyelitis; Preparation; Process; Proteins; Publishing; Regulation; Respiratory Disease; Rodent; Role; Series; Site; Skeletal Muscle; Spinal Cord; Synapses; System; Therapeutic Intervention; Tissues; Tropism; Viral; Viral meningitis; Virus; Virus Diseases; Virus Receptors; Work; acute flaccid myelitis; cell type; engineered stem cells; genome-wide; hindbrain; human disease; human stem cells; in vivo; insight; mouse model; neonatal infection; neural; neuromuscular; neuropathology; neurotropic; new outbreak; new therapeutic target; novel; novel strategies; overexpression; pathogen; receptor; stem cell biology; transcriptomics; translational potential; virus host interaction

PROJECT SUMMARY Enteroviruses are the leading cause of viral meningitis in children and recent outbreaks of emerging non-polio enteroviruses (NPEVs) have been associated with a polio-like paralysis named acute flaccid myelitis (AFM). Discovery and characterization of cellular components that are critical for neuropathogenesis hold promise for revealing new approaches to treat enterovirus disease. In recent years, multiple receptors have been identified for EV-A71 and EV-D68, NPEVs, which are most commonly associated with AFM. Using unbiased genome- scale screens, we have identified the phospholipase PLA2G16 as an entry factor acting immediately downstream of receptor engagement following NPEV infection. How the multiple receptors and PLA2G16 work together to enable infection in cell types relevant for neuropathogenesis is, however, largely unknown. Infection of cell types present in the central nervous system is critical for developing severe neurological forms of disease following infection with NPEVs. Although mouse models have been widely used to gain insights into enterovirus infection processes, genetic and physiological differences between human and rodents limit their translational potential. Moreover, species incompatibilities in host factor interactions of these human enteroviruses necessitate overexpression of human receptors, mouse-adapted strains or neonatal infections. In work that forms a foundation for this proposal, we have developed from pluripotent stem cells human spinal cord organoids that recapitulate some of the cell diversity of the human spinal cord. Importantly, we have pioneered an approach to functionally connect motor neurons in spinal cord organoids with human skeletal muscle and cortical neurons in a preparation we named assembloids. These motor assembloids form functional neuro-muscular junctions and can control muscle contraction. Here, we propose to systematically study the role of known host factors in cell lines derived from neural tissue on EV-A71 and EV-D68, discover novel host factors by performing unbiased genome-scale genetic screens in neural cell lines, and compare cell lineage tropism and effect on neuronal function during enterovirus infections of cortico-motor assembloids. Our results will reveal the role and relative contribution of a distinct set of critical receptors and broad-acting host factors to infection by multiple enteroviruses, discover and provide details on the molecular mechanism of novel host factors in neural cell types, and leverage a unique neural organoid system to uncover the specific tropism and functional effect on human neural-muscular circuits during infections with the paralytic enteroviruses EV-D68 and EV-A71.

项目摘要 肠病毒是儿童病毒性脑膜炎的主要原因，最近出现的非polio爆发 肠病毒（NPEVS）与称为急性松弛脊髓炎（AFM）的脊髓灰质炎类麻痹有关。 对神经病生成至关重要的细胞成分的发现和表征对 揭示了治疗肠病毒疾病的新方法。近年来，已经确定了多个受体 对于EV-A71和EV-D68，NPEVS最常见于AFM。使用公正的基因组 - 比例屏幕，我们已经将磷脂酶PLA2G16确定为作用的入口因子 NPEV感染后受体参与的下游。多个受体和PLA2G16如何工作 然而，共同使与神经病发生相关的细胞类型感染是未知的。感染 中枢神经系统中存在的细胞类型对于发展严重的神经系统形式至关重要 NPEV感染后的疾病。尽管鼠标模型已被广泛用于洞悉 肠病毒感染过程，人与啮齿动物之间的遗传和生理差异限制 翻译潜力。此外，这些人类的宿主因子相互作用中的物种不相容性 肠病毒需要过度表达人体受体，小鼠适应性菌株或新生儿感染。 在构成该建议基础的工作中，我们从多能干细胞人类脊柱中发展出来 概括了人脊髓的某些细胞多样性的脐带官。重要的是，我们有 开创了一种将脊髓器官中运动神经元与人骨骼连接的方法连接运动神经元的方法 在制剂中，肌肉和皮质神经元命名为组合。这些运动组合形式 功能性神经肌肉连接，可以控制肌肉收缩。在这里，我们建议系统地 研究已知宿主因子在EV-A71和EV-D68上衍生自神经组织的细胞系中的作用，发现 通过在神经细胞系中执行公正的基因组尺度遗传筛选，并比较细胞来进行新型宿主因素 皮质运动组合体的肠病毒感染期间的谱系对流及其对神经元功能的影响。 我们的结果将揭示一组不同的关键受体和广泛作用的作用和相对贡献 多种肠病毒感染的宿主因素，发现并提供有关分子机制的细节 神经细胞类型中的新型宿主因素，并利用独特的神经器官系统来揭示特定 瘫痪期间对人类神经肌肉回路的向流和功能作用 肠病毒EV-D68和EV-A71。