Proteins to Cell Systems

蛋白质到细胞系统

• 批准号: 10265442
• 负责人: Jack M Parent
• 金额: $ 44.74万
• 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/10265442
• 关键词: 3-Dimensional; Acute; Address; Affect; Biological Assay; Biological Models; CRISPR interference; Calcium; Cell Line; Cell physiology; Cells; Complex; Computer Analysis; DNA sequencing; Development; Diagnosis; Diagnostic; 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; Structure; System; Testing; Therapeutic Studies; Variant; Work; Zebrafish; base; 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) 已导致大量癫痫基因的快速发现，并且 癫痫基因的列表已经远远超出了离子通道，扩展到了影响广泛细胞的基因 然而，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。 此外，NGS 导致了不确定性基因变异数量的增加。 显着性（VUS）难以进行诊断解释，我们缺乏分析 VUS 效果或有效的工具。 研究这些癫痫基因的致病机制 为了解决这些缺陷，EpiMVP 将进行优化。 针对癫痫基因的尖端多平台检测，包括细胞系（项目 1）、人类多能干细胞 细胞（hPSC；项目 1 和 2）、人类皮质类器官（hCO；项目 2）以及体内啮齿动物和斑马鱼 模型（项目 3）。基因和变体管理核心（GVCC）将与项目互动以选择和 随着 VUS 列表从项目 1 到项目 2 到项目 3 的精简，完善用于测试的特定基因和变体。这一点的关键 人类癫痫工具核心 (HETC) 的工作将提供细胞系（用于项目 1 和 2）和变体 表达载体（适用于所有 3 个项目）。我们工作的长期目标是提供体外测试管道。 我们拥有用于分析所有非离子通道癫痫基因的临床相关 VUS 的人类神经元模型。 确定了前 10 个最常见基因中 6 个基因的相关形态/功能 2-D 或 hCO 表型 诊断非离子通道遗传性癫痫，以及其他几种的试剂，使用：1) 2-D hPSC 培养物，包括小分子分化为兴奋性或抑制性皮质神经元，兴奋性诱导 神经元和由转录因子表达产生的诱导 GABA 神经元（iNeurons/iGN），以及混合 培养物（iNeurons、iGN 和神经胶质细胞）；和 2) 3-D hCO 培养物，包括多玫瑰花结、单玫瑰花结、兴奋性、 我们将使用这些测定来测试我们的平台将预测 VUS 的假设。 致病性和有效变异优先考虑项目 3 中的体内测试。我们的近期目标是 每年优化 1-2 个基因的检测，确定这些基因的体外 VUS 致病性，并一致 与 VGCC 合作，完善项目 3 中进一步体内测试的 VUS 列表。目标将通过使用来实现 里程碑 1 的 2-D hPSC 和里程碑 2 的 3-D hCO，并将包括结构和功能测定 这些研究将提供以下成果：1）多重优化、交叉验证。 （Parent 和 Ross 实验室之间）用于询问癫痫基因的 hPSC 平台；2) 体外人类的测定； 神经元 VUS 对至少 5 个非离子通道癫痫基因的致病性；3) 每个人类神经元模型 癫痫基因；4）用于未来机制和精准治疗研究的优化平台。