Proteins to Cell Systems

项目概要-项目2 下一代DNA测序（NGS）已经导致了大量癫痫基因的快速发现， 癫痫基因的名单已经远远超出了离子通道， 功能协调发展的然而，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 基因发现的背后此外，NGS还导致了不确定的遗传变异数量的增加。 显著性（VUS），难以诊断解释。我们缺乏工具来分析VUS效应或有效地 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 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）。基因和变异管理核心（GVCC）将与项目互动， 随着VUS列表从项目1到项目2再到项目3的精简，我们将细化特定的基因和变体以进行测试。的关键 人类癫痫工具核心（HETC）将提供细胞系（用于项目1和2）和变体 表达载体（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， 人神经元模型，以测定所有非离子通道癫痫基因的临床相关VUS。我们有 确定了前10个最常见的6个基因的相关形态/功能2-D或hCO表型 诊断的非离子通道遗传性癫痫，以及其他几种试剂，使用：1）2-D hPSC 培养，包括小分子分化成兴奋性或抑制性皮质神经元，兴奋性诱导 通过转录因子表达产生的神经元和诱导的GABA神经元（iNeurons/iGN），以及混合 培养物（iNeurons，iGN和神经胶质）;和2）3-D hCO培养物，包括多玫瑰花结，单玫瑰花结，兴奋性， 抑制和融合hCO。我们将使用这些测定来检验我们的平台将预测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。我们的近期目标是 每年优化1-2个基因的检测，确定这些基因的VUS体外致病性， 使用VGCC，完善VUS列表，以便在项目3中进行进一步体内试验。这些目标将通过使用 里程碑1中的2-D hPSC和里程碑2中的3-D hCO，并且将包括用于以下的结构和功能测定： 每个模型系统。这些研究将提供以下可交付成果：1）多重优化、交叉验证 （在Parent和Ross实验室之间）用于询问癫痫基因的hPSC平台; 2）测定体外人癫痫基因; 至少5个非离子通道癫痫基因的神经元VUS致病性; 3）每个基因的人神经元模型 癫痫基因;和4）为未来的机制和精确的治疗研究优化平台。

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.