Human Induced Pluripotent Stem Cells As Models for Inherited Developmental Disorders

项目摘要 许多神经发育障碍中神经功能障碍的机制具有挑战性， 地址有很多原因。人类的大脑是相对难以直接评估，人体解剖 成像研究提供了有限的机会来研究机制，动物模型有时不 复制关键特征，永生化的神经元样细胞模型对于研究正常 发展过程。诱导性多能干细胞（iPSC）提供了一种强大的新实验工具， 解决这些限制，因为它们可以从具有定义突变的患者中创建，并且它们可以 在体外分化成特定的细胞谱系，以提供物种特异性和谱系特异性的细胞模型。在 目前的建议，我们描述了iPSC资源的开发和表征， Lesch-Nyhan病（LND）及其减毒变体的基于细胞的实验模型。这些疾病 是由HPRT 1基因突变引起的，导致嘌呤补救酶缺乏， 次黄嘌呤-鸟嘌呤磷酸核糖基转移酶（HGprt）。LND提供了一个非常容易处理的孟德尔 用于iPSC建模的障碍。因为这项技术还没有被系统地用于这种疾病， 目前提案的主要重点是开发和初步表征iPSC资源， 发现新的生物过程和治疗干预。具体来说，我们将建立一个良好的- 来自具有疾病严重程度谱的患者的经表征和临床注释的iPSC库 作为其中通过基因编辑方法引入了特定突变的等基因系（目的1）; 表征与这些iPSC分化成神经元相关的形态学缺陷（Aim 2）; 确定这些细胞中受影响的分子和生化途径， 转录组学、蛋白质组学和代谢组学（目标3）;并确定在发育过程中 必须恢复HGprt以纠正神经元缺陷（目的4）。这个项目将产生一个新颖的和批判性的 为今后研究LND的发病机制和今后的药理学或免疫学研究提供了重要资源。 遗传治疗干预。

PROJECT SUMMARY The mechanisms responsible for neural dysfunction in many neurodevelopmental disorders are challenging to address for many reasons. The human brain is relatively inaccessible for direct evaluation, human autopsy and imaging studies provide limited opportunities to study mechanism, animal models sometimes do not replicate key features, and immortalized neuron-like cell models have uncertain value for studying normal developmental processes. Induced pluripotent stem cells (iPSCs) provide a powerful new experimental tool to address these limitations, because they can be created from patients with defined mutations, and they can be differentiated in vitro into specific cell lineages to provide species-specific and lineage-specific cell models. In the current proposal we describe the development and characterization of a resource of iPSCs to serve as experimental cell-based models for Lesch-Nyhan disease (LND) and its attenuated variants. These disorders are caused by mutations in the HPRT1 gene, resulting in deficiency of the purine salvage enzyme, hypoxanthine-guanine phosphoribosyltransferase (HGprt). LND provides an exceptionally tractable Mendelian disorder for iPSC modeling. Because this technology has not yet been methodically exploited for this disorder, the main focus of the current proposal is the development and initial characterization of a resource of iPSCs for discovery of novel biological processes and therapeutic interventions. Specifically, we will establish a well- characterized and clinically annotated bank of iPSCs from patients with a spectrum of disease severity as well as isogenic lines in which specific mutations have been introduced via gene editing methods (Aim 1); characterize morphological defects associated with differentiation of these iPSCs into neurons (Aim 2); determine the molecular and biochemical pathways affected in these cells using comprehensive transcriptomics, proteomics and metabolomics (Aim 3); and determine how early in the developmental process HGprt must be restored to correct neuronal defects (Aim 4). This project will result in a novel and critically important resource for future studies of the pathogenesis of LND and future studies of pharmacological or genetic therapeutic interventions.