Cell Systems to Pre-Clinical Models

细胞系统到临床前模型

• 批准号: 10670374
• 负责人: Yu Wang
• 金额: $ 57.78万
• 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/10670374
• 关键词: 3-Dimensional; Behavioral; Benign; Biochemical; Biological; Biological Assay; Biological Models; Brain; Calcium; Cells; Classification; Clustered Regularly Interspaced Short Palindromic Repeats; Complex; Cortical Dysplasia; D Cells; DNA sequencing; Data; Diagnosis; Disease Outcome; Electrophysiology (science); Electroporation; Embryo; Epilepsy; Exhibits; Explosion; Fishes; Future; Gene Mutation; Genes; Genetic; Genetic Counseling; Goals; Human; Image; In Vitro; Intellectual functioning disability; Knowledge; Laboratories; Mediating; Methods; Modeling; Mutagenesis; Mutation; Nature; Partial Epilepsies; Pathogenicity; Phenotype; Pre-Clinical Model; Probability; Proteins; Rattus; Recommendation; Rodent; Rodent Model; Seizures; Syndrome; System; Testing; Uncertainty; Variant; Zebrafish; childhood epilepsy; clinically relevant; drug discovery; gene discovery; genetic variant; human disease; in silico; in utero; in vitro Model; in vivo; in vivo Model; in vivo evaluation; loss of function; machine learning model; model organism; mutant; next generation; syntaxin binding protein 1; treatment strategy; two-dimensional; variant of unknown significance

PROJECT SUMMARY Knowledge of epilepsy genes has exploded with the advent of next generation DNA sequencing (NGS), yet our understanding of how any specific gene mutation leads to epilepsy increasingly lags behind gene discovery. With the expanding list of potential epilepsy genes comes the problem of genetic variants of uncertain significance (VUS), confounding definitive diagnosis, genetic counselling and treatment strategies. The ultimate goal of the EpiMVP team is to develop a highly integrated and effective multiplatform pipeline to determine the functional impact of VUS for non-ion channel epilepsy genes. To this end, the Gene and Variant Curation Core (GVCC) provides in silico prediction of VUSs to prioritize and refine specific genes and VUS for study as Project 1 (Genes to Proteins) begins in vitro biochemical and cell biological assessment and Project 2 (Proteins to Cell Systems) further explores the prioritized VUS in two dimensional (2-D) and 3-D cell systems. However, due to the complex nature of epilepsy and the brain, uncertainty and even conflicting interpretation will at times arise from in vitro biochemical and cell-based functional assays. Thus, it is ultimately critical to apply refined VUS list for in vivo functional assessment in model organisms. The long-term goal of project 3 (Cell Systems to Preclinical Models) is to incorporate in vivo functional assays into EpiMVP pipeline in which the functional and genetic data of a specific VUS are merged to arrive at a decisive probability of pathogenicity. Project 3 will take advantage of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) gene editing methods to generate well-defined loss-of-function (LOF) epilepsy models in rodent and zebrafish as a null background on which to evaluate human VUS. Data generated in Project 3 will be shared with Projects 1 and 2 and the GVCC in an iterative fashion with an overall deliverable to develop EpiPred (Epilepsy Variant Prediction), a machine learning model that will allow accurate classification of missense epilepsy gene variants as likely pathogenic or benign. With the prioritized and refined VUS list, we will test 3-4 VUS each for 1-2 genes per year and complete testing of at least 5 genes in both fish and rodent model systems. Our first-year goal is to examine functional rescue of 3-4 VUS each for STXBP1 and DEPDC5. Deliverables include: 1) Cross-validated in vivo models to interrogate epilepsy genes; 2) Determination of VUS pathogenicity for at least 5 non-ion channel epilepsy genes using 2 in vivo models; 3) Assessment of in silico and in vitro models for VUS pathogenicity prediction, and whether in vivo models are required for certain genes and VUS; and 4) Optimized in vivo models for each epilepsy gene that will be powerful platforms for future drug discovery and for exploring underlying mechanisms in these genetic epilepsies.

项目摘要 随着下一代DNA测序（NGS）的出现，癫痫基因的知识已经爆炸，但 我们对任何特定基因突变如何导致癫痫的理解越来越落后于基因突变。 的发现随着潜在癫痫基因的名单不断扩大， 不确定的意义（VUS），混淆明确的诊断，遗传咨询和治疗策略。 EpiMVP团队的最终目标是开发一个高度集成和有效的多平台管道 以确定VUS对非离子通道癫痫基因的功能影响。为此，基因和 变体管理核心（GVCC）提供VUS的计算机预测，以优先考虑和细化特定基因 和VUS研究项目1（基因到蛋白质）开始在体外生物化学和细胞生物学 评估和项目2（蛋白质到细胞系统）进一步探讨了两个优先VUS 三维（2-D）和3-D细胞系统。然而，由于癫痫和大脑的复杂性， 不确定性和甚至相互矛盾的解释有时会产生于体外生物化学和基于细胞的 功能测定因此，最终关键是应用改进的VUS列表进行体内功能评估， 模式生物项目3（细胞系统到临床前模型）的长期目标是将 将体内功能测定引入EpiMVP管道，其中特定VUS的功能和遗传数据 融合在一起得出了致病性的决定性概率。项目3将利用 定期间隔短回文重复序列（CRISPR）基因编辑方法，以产生定义明确的 啮齿动物和斑马鱼的功能丧失（LOF）癫痫模型作为评价的零背景 人类VUS。项目3中生成的数据将与项目1和2以及GVCC以迭代方式共享。 时尚的整体可交付成果，用于开发机器学习EpiPred（癫痫变异预测） 该模型将允许将错义癫痫基因变异准确分类为可能的致病性或 良性的有了优先和完善的VUS名单，我们将测试3-4 VUS每1-2基因每年， 在鱼类和啮齿动物模型系统中完成至少5个基因的测试。我们第一年的目标是 STXBP 1和DEPDC 5各3-4个VUS的功能拯救。可验证内容包括：1）交叉验证， 2）测定至少5种非离子型VUS的致病性; 3）VUS的计算机模拟和体外模型评估 致病性预测，以及某些基因和VUS是否需要体内模型;以及4） 针对每个癫痫基因的优化体内模型，将成为未来药物发现的强大平台， 来探索这些遗传性癫痫的潜在机制