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: 10450520
• 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/10450520
• 关键词: 3-Dimensional; Address; Affect; Antiviral Therapy; Astrocytes; Biological Assay; Biology; Cell Communication; Cell Line; Cell Lineage; Cells; Cerebral cortex; Child; Clustered Regularly Interspaced Short Palindromic Repeats; Cytosol; Disease; Disease Outbreaks; Disease model; Encephalitis; Endocytosis; Enterovirus; Enterovirus 68; Enterovirus Infections; Enzymes; Foundations; Genes; Genetic; Genetic Screening; Genome; Goals; Hand, Foot and Mouth Disease; Human; Human poliovirus; Huntington gene; Immune response; In Vitro; Individual; Infection; Infectious Agent; Integration Host Factors; Knock-out; Knowledge; Laboratories; Life; Mediating; Medical; Meningitis; Modeling; Molecular; Motor; Motor Neurons; Movement; Mus; Muscle; Muscle Contraction; Muscle Fibers; Myocarditis; Names; Nervous system structure; Neuraxis; Neurologic; Neuromuscular Junction; Neurons; Neuropathogenesis; Neurosciences; Neurotropism; Oligodendroglia; Organoids; Paralysed; Pathogenesis; Phospholipase; Physiological; 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 pluripotent stem cell; in vivo; insight; mouse model; neonatal infection; neuromuscular; neuropathology; neurotropic; new therapeutic target; novel; novel strategies; overexpression; pathogen; receptor; relating to nervous system; 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.

项目概要 肠道病毒是儿童病毒性脑膜炎和最近爆发的新发非脊髓灰质炎的主要原因 肠道病毒（NPEV）与一种名为急性弛缓性脊髓炎（AFM）的脊髓灰质炎样麻痹有关。 对神经发病机制至关重要的细胞成分的发现和表征有望为 揭示治疗肠道病毒疾病的新方法。近年来，多种受体被发现 对于 EV-A71 和 EV-D68，NPEV，最常与 AFM 相关。使用公正的基因组- 通过规模筛选，我们已确定磷脂酶 PLA2G16 作为立即起作用的进入因子 NPEV 感染后受体参与的下游。多重受体和 PLA2G16 如何发挥作用 然而，它们共同作用以实现与神经发病机制相关的细胞类型的感染在很大程度上是未知的。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 感染 NPEV 后发生的疾病。尽管小鼠模型已被广泛用于深入了解 肠道病毒感染过程、人类和啮齿动物之间的遗传和生理差异限制了它们 翻译潜力。此外，这些人类宿主因素相互作用中的物种不相容性 肠道病毒需要人类受体、小鼠适应株或新生儿感染的过度表达。 在为该提案奠定基础的工作中，我们利用多能干细胞开发了人类脊髓 脊髓类器官再现了人类脊髓的一些细胞多样性。重要的是，我们有 开创了一种将脊髓类器官中的运动神经元与人类骨骼进行功能连接的方法 我们将其命名为“集合体”的制剂中的肌肉和皮质神经元。这些电机组件形成 功能性神经肌肉接头，可以控制肌肉收缩。在此，我们建议系统地 研究已知宿主因子在 EV-A71 和 EV-D68 神经组织来源的细胞系中的作用，发现 通过在神经细胞系中进行无偏基因组规模的遗传筛选来发现新的宿主因素，并比较细胞 皮质运动组合体肠道病毒感染期间的谱系向性及其对神经元功能的影响。 我们的结果将揭示一组独特的关键受体和广泛作用的作用和相对贡献 多种肠道病毒感染的宿主因素，发现并提供详细的分子机制 神经细胞类型中的新宿主因子，并利用独特的神经类器官系统来揭示特定的 麻痹症感染期间对人类神经肌肉回路的向性和功能影响 肠道病毒 EV-D68 和 EV-A71。