Elucidating the neuropathophysiology of TSC using genetically engineered human neurons

项目摘要 多发性硬化症（TSC）是一种神经发育障碍，由TSC 1基因突变引起。 或TSC 2基因。TSC 1和TSC 2的蛋白产物形成复合物，该复合物是mTOR的关键负调节物 信号TSC与一系列神经和精神问题有关，包括癫痫， 自闭症谱系障碍和智力残疾。TSC的神经系统表现是其中之一， 虽然TSC对患者的影响最大，但我们对TSC的神经病理生理学的了解有限。动物模型 TSC的研究对于解决基础生物学问题很有价值，揭示了神经元的变化， 发育、形态学和突触通讯。下一个主要挑战是翻译这些 人类的发现。这将需要定义TSC突变在人类遗传学中的后果， 发展背景。为了实现这一目标，我们将建立一个新的人类神经元模型的TSC的基础上， Cas9介导的人类胚胎干细胞（hESC）的基因编辑。为此，我们已经生成了一个 hESC中具有杂合、纯合和条件性功能丧失突变的同基因组， TSC 1或TSC 2基因。这些细胞将分化成神经祖细胞、神经元和脑神经元。 当TSC相关表型首次出现时，类器官模拟人类皮质发育的早期阶段。 我们将确定TSC 1或2的突变如何影响人类神经元的发育和功能， 生物化学、全基因组分析、成像和电生理学方法。我们的发现将回答 与TSC的基因型-表型关系和TSC的发育起源有关的几个关键问题 皮质畸形是这种疾病的标志此外，我们产生的细胞系将是一个 研究界研究疾病机制和测试潜力的宝贵资源 用于直接在原代人细胞中治疗TSC和其它“mTOR-病变”的药物。

PROJECT SUMMARY Tuberous Sclerosis Complex (TSC) is a neurodevelopmental disorder caused by mutations in the TSC1 or TSC2 genes. The protein products of TSC1 and 2 form a complex that is a key negative regulator of mTOR signaling. TSC is associated with an array of neurological and psychiatric problems that can include epilepsy, autism spectrum disorder, and intellectual disability. The neurological manifestations of TSC are amongst the most debilitating to patients, yet our knowledge of the neuropathophysiology of TSC is limited. Animal models of TSC have been valuable to address questions of basic biology, revealing alterations in neuronal development, morphology, and synaptic communication. The next major challenge is to translate these findings to humans. This will require defining the consequences of TSC mutations in a human genetic and developmental context. To achieve this we will establish a novel human neuronal model for TSC based on Cas9-mediated gene editing of human embryonic stem cells (hESCs). To this end we have generated an isogenic panel of hESCs with heterozygous, homozygous, and conditional loss of function mutations in the TSC1 or TSC2 genes. These cells will be differentiated into neural progenitors, neurons, and cerebral organoids to model the early stages of human cortical development when TSC-related phenotypes first arise. We will determine how mutations in TSC1 or 2 affect the development and function of human neurons using biochemical, genome-wide profiling, imaging, and electrophysiological approaches. Our findings will answer several key questions related to genotype-phenotype relationships in TSC and the developmental origin of the cortical malformations that are a hallmark of this disorder. In addition, the cell lines we generate will be a valuable resource for the research community to investigate disease mechanisms and test potential therapeutics for TSC and other “mTOR-opathies” directly in primary human cells.