Proteins to Cell Systems

蛋白质到细胞系统

• 批准号: 10670368
• 负责人: Jack M Parent
• 金额: $ 45.6万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-15 至 2025-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10670368
• 关键词: 3-Dimensional; Acceleration; Acute; Address; Affect; Biological Assay; Biological Models; CRISPR interference; Calcium; Cell Line; Cell physiology; Cells; Colony-Forming Units Assay; Complex; Computer Analysis; DNA sequencing; Development; Diagnosis; Disease; Electrophysiology (science); Epilepsy; Future; Gene Expression; Gene Mutation; Genes; Genetic; Genome; Glass; Goals; Human; Image; In Vitro; Ion Channel; Knock-out; Microelectrodes; Modeling; Morphology; Neuroglia; Neurons; Organoids; Parents; Pathogenicity; Patients; Phenotype; Precision therapeutics; Proteins; Publishing; Reagent; Rodent; Slice; System; Testing; Therapeutic Studies; Variant; Work; Zebrafish; clinically relevant; exome; experience; expression vector; gamma-Aminobutyric Acid; gene discovery; genetic disorder diagnosis; genetic variant; human pluripotent stem cell; in vitro testing; in vivo; in vivo evaluation; inhibitory neuron; knock-down; loss of function; multi-electrode arrays; next generation; patch clamp; small molecule; tool; transcription factor; variant of unknown significance

PROJECT SUMMARY – PROJECT 2 Next generation DNA sequencing (NGS) has led to the rapid discovery of large numbers of epilepsy genes, and the list of epilepsy genes has grown well beyond ion channels to those that affect a wide array of cellular functions. Our understanding of how any specific gene mutation leads to epilepsy, however, increasingly lags behind gene discovery. Moreover, NGS has led to increased numbers of genetic variants of uncertain significance (VUS) that are difficult to interpret diagnostically. We lack the tools to assay VUS effects or effectively study pathogenic mechanisms for these epilepsy genes. To address these shortfalls, the EpiMVP will optimize cutting-edge multiplatform assays for epilepsy genes that include cell lines (Project 1), human pluripotent stem cells (hPSCs; Projects 1 and 2), human cortical organoids (hCOs; Project 2), and in vivo rodent and zebrafish models (Project 3). The Gene and Variant Curation Core (GVCC) will interact with the projects to select and refine specific genes and variants for testing as the VUS list is streamlined from Projects 1 to 2 to 3. Key to this effort is the Human Epilepsy Tools Core (HETC) which will provide cell lines (for Projects 1 and 2) and variant expression vectors (for all 3 projects). The long-term goal of our work is to deliver an in vitro testing pipeline in human neuronal models to assay clinically relevant VUS for all non-ion channel epilepsy genes. We have identified relevant morphological/functional 2-D or hCO phenotypes for 6 genes in the top 10 most commonly diagnosed non-ion channel genetic epilepsies, as well as reagents for several others, using: 1) 2-D hPSC cultures, including small molecule differentiation into excitatory or inhibitory cortical neurons, excitatory induced neurons and induced GABA neurons (iNeurons/iGNs) generated by transcription factor expression, and mixed cultures (iNeurons, iGNs and glia); and 2) 3-D hCO cultures, including multi-rosette, single rosette, excitatory, inhibitory and fusion hCOs. We will use these assays to test the hypothesis that our platforms will predict VUS pathogenicity and effectively prioritize variants for in vivo testing in Project 3. Our immediate goals are to optimize assays for 1-2 genes per year, determine VUS pathogenicity in vitro for these genes and, in concert with the VGCC, refine the VUS list for further in vivo testing in Project 3. The goals will be accomplished using 2-D hPSCs for Milestone 1 and 3-D hCOs in Milestone 2, and will include structural and functional assays for each model system. These studies will provide the following deliverables: 1) multiple optimized, cross-validated (between Parent and Ross labs) hPSC platforms to interrogate epilepsy genes; 2) determination of in vitro human neuronal VUS pathogenicity for at least 5 non-ion channel epilepsy genes; 3) human neuronal models for each epilepsy gene; and 4) optimized platforms for future mechanistic and precision therapeutic studies.

项目摘要 - 项目2 下一代DNA测序（NGS）导致了大量癫痫基因的快速发现，并且 癫痫基因的列表已超出离子通道超出了影响各种细胞的癫痫通道 功能。但是，我们对任何特定基因突变如何导致癫痫的理解，越来越滞后 基因发现背后。此外，NGS导致不确定的遗传变异数量增加 意义（VU）难以诊断。我们缺乏分析VUS效应或有效的工具 研究这些癫痫基因的致病机制。为了解决这些缺口，EPIMVP将优化 包括细胞系（项目1），人多能茎的癫痫基因的尖端乘数测定 细胞（HPSC；项目1和2），人皮质器官（HCO；项目2）以及体内啮齿动物和斑马鱼 模型（项目3）。基因和变体策划核心（GVCC）将与选择和选择项目相互作用 精炼特定的基因和用于测试的变体，因为VUS列表从项目1到2到3简化了。 努力是人类癫痫工具核心（HETC），它将提供细胞系（项目1和2）和变体 表达向量（所有3个项目）。我们工作的长期目标是在 人神经元模型，用于分析所有非离子通道癫痫基因的临床相关VU。我们有 在最常见的十大基因中确定了相关的形态/功能2-D或HCO表型 诊断为非离子通道遗传癫痫以及其他几种试剂，使用：1）2-D HPSC 培养物，包括小分子分化为兴奋或抑制性皮质神经元，兴奋引起的 通过转录因子表达产生的神经元和诱导的GABA神经元（INERORS/IGN），混合 培养物（INERORS，IGNS和GLIA）； 2）3-D HCO培养物，包括多杆，单杆，兴奋性， 抑制性和融合HCO。我们将使用这些测定法来检验我们平台将预测VUS的假设 致病性，并有效地优先考虑项目3中体内测试的变体。我们的直接目标是 优化每年1-2个基因的测定法，确定这些基因的VUS致病性，并在协调一致 使用VGCC，请完善项目3中进一步体内测试的VUS列表。 里程碑2中的2-D HPSC用于里程碑1和3-D HCO，并将包括用于结构和功能测定 每个模型系统。这些研究将提供以下可交付成果：1）多次优化，交叉验证 （在父母和罗斯实验室之间）HPSC平台询问癫痫基因； 2）确定体外人 至少5个非离子通道癫痫基因的神经元VUS致病性； 3）每个人的人类神经元模型 癫痫基因； 4）优化的平台，用于未来的机械和精确治疗研究。