Generation of DYT1 dystonia-specific iPS cells with isogenic controls

描述(由申请人提供)：了解和治疗肌张力障碍的一个主要挑战是目前研究这种疾病的模型的局限性。肌张力障碍通常被认为代表控制运动的大脑区域内神经元的慢性信号功能障碍，尽管在分子水平上表征这些细胞中与肌张力障碍相关的微妙障碍一直是困难的。建立疾病模型的传统方法依赖于(1)外周患者细胞(成纤维细胞或淋巴母细胞)；(2)突变小鼠；和/或(3)培养细胞，其携带与疾病过程有关的蛋白质的操纵。虽然这些策略有价值，但它们所揭示的与肌张力障碍相关的基因突变对人类神经元的影响是有限的。将患者体细胞重新编程为可分化为神经元的诱导多能干细胞(IPSCs)已成为建模中枢神经系统疾病的一种越来越常见的方法。然而，随着时间的推移，很明显，IPSCs可以表现出很大的个体间变异性，这可能会混淆疾病特定表型的识别。在这里，我们提出了一种新的人类DYT1肌张力障碍神经元培养模型，该模型结合了iPSCs重编程和使用转录激活物样效应核酸酶(TALENS)的基因打靶。通过使用工程TALEN来纠正患者IPSCs中的DYT1基因突变，我们将从相同的表观遗传背景中产生匹配的等基因对照品系。作为该模型的第一个应用，我们将通过途径分析进行转录图谱分析，以确定与匹配的对照细胞相比，DYT1 iPSC来源神经元中受干扰的功能网络。这项工作的结果将成为探索DYT1肌张力障碍神经元特异性缺陷的强大新资源，详细的表达谱将为该模型系统的未来研究提供信息。 公共卫生相关性：早发性(DYT1)肌张力障碍是一种严重的神经运动障碍。研究DYT1的一个主要挑战是，据信受疾病突变影响最大的细胞是神经元，这可能在功能上与经常用作实验模型的细胞系不同。在这里，我们建议建立一个新的模型系统，由DYT1患者特异性的诱导多能干细胞组成，可以分化为神经元，从而促进对可能导致这种疾病的神经元特异性功能缺陷的新分析。

DESCRIPTION (provided by applicant): A major challenge in understanding and treating dystonia is the limitation of current models for studying the disease. The dystonias are generally believed to represent chronic signaling dysfunctions in neurons within brain regions controlling movement, although characterizing subtle, dystonia-related disturbances in these cells at the molecular level has been difficult. Traditional approaches to model the disorder have relied on (1) peripheral patient cells (fibroblasts or lymphoblasts); (2) mutant mice; and/or (3) cultured cells bearing manipulations in proteins implicated in the disease process. While these strategies have value, there are limits to what they can reveal about effects of dystonia-related gene mutations on human neurons. Reprogramming patient somatic cells to induced pluripotent stem cells (iPSCs) which can be differentiated into neurons has become an increasingly common approach to modeling CNS disorders. Yet over time it has become clear that iPSCs can display substantial inter-individual variability which may confound identification of disease-specific phenotypes. Here we propose a strategy to develop a new human neuronal culture model of DYT1 dystonia that combines iPSCs reprogramming with gene targeting using transcription activator-like effector nucleases (TALENs). By using engineered TALENs to correct the DYT1 gene mutation in patient iPSCs, we will generate matched isogenic control lines from the same epigenetic background. As a first application of this model, we will perform transcriptional profiling with pathway analyses to identify functional networks that are perturbed in DYT1 iPSC-derived neurons compared to matched control cells. The outcome of this work will be a powerful new resource for probing neuron-specific deficits in DYT1 dystonia, with detailed expression profiling that will inform future studies of this model system. PUBLIC HEALTH RELEVANCE: Early onset (DYT1) dystonia is a crippling neurologic movement disorder. A major challenge in studying DYT1 is that the cells believed to be most affected by the disease mutation are neurons, which may be functionally distinct from the cell lines frequently used as experimental models. Here we propose to establish a new model system consisting of DYT1 patient-specific, induced pluripotent stem cells which can be differentiated into neurons, thereby facilitating new analyses of neuron-specific functional defects that may underlie this disease.