Proteins to Cell Systems

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，以及 癫痫的基因清单已经远远超出了离子通道，扩展到了影响多种细胞的基因。 功能。然而，我们对任何特定基因突变如何导致癫痫的理解却越来越滞后。 基因发现的背后。此外，NGS导致了不确定的遗传变异的数量增加 诊断难以解释的重要性(VU)。我们缺乏工具来分析VUS的影响或有效 研究这些癫痫基因的致病机制。为了解决这些不足，EpiMVP将优化 尖端多平台癫痫基因分析，包括细胞系(项目1)，人类多能干细胞 细胞(hPSCs；项目1和2)、人类皮质类器官(HCOs；项目2)以及活体啮齿动物和斑马鱼 模型(项目3)。基因和变种修复核心(GVCC)将与项目相互作用，以选择和 随着VU列表从项目1精简到项目2到项目3，细化特定的基因和变种以进行测试。 努力是人类癫痫工具核心(HETC)，它将提供细胞系(用于项目1和2)和变种 表达载体(用于所有3个项目)。我们工作的长期目标是在 用于检测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。我们有 确定了最常见的前10个基因中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，以及其他几种药物，使用：1)2-D hPSC 兴奋性诱导的培养，包括向兴奋性或抑制性皮质神经元的小分子分化 神经元和诱导的GABA神经元(iNeurons/IGN)由转录因子表达产生，并混合 培养(iNeurons，IGN和Gliar)；以及2)3D HCO培养，包括多玫瑰花环，单玫瑰花环，兴奋性， 抑制和融合HCOs。我们将使用这些分析来测试我们的平台将预测VU的假设 在项目3中，有效地确定体内测试的变种的致病性和优先级。我们的近期目标是 优化每年1-2个基因的检测，确定这些基因的体外致病性，并协调 使用VGCC，细化VUS列表，以便在项目3中进行进一步的体内测试。 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分析 每个模型系统。这些研究将提供以下交付成果：1)多个优化、交叉验证 (父母和罗斯实验室之间)用于询问癫痫基因的hPSC平台；2)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)每个基因的人类神经元模型 癫痫基因；4)优化平台，为未来的机械性和精确化治疗研究。

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.