Tuberous Sclerosis Complex Patients iPSC-derived NPCs and NCCs as Human Model Systems to Identify Novel Targets

结节性硬化症 (TSC) 是一种单基因疾病，癫痫发作、智力障碍的发生率增加 残疾（ID）和自闭症谱系障碍（ASD）。尽管在理解方面已经取得了重大进展 TSC，充分理解 TSC 作为一种神经发育障碍的能力，以及共同的分子机制 这可能解释了 TSC 和 ASD 之间重叠表型的原因是缺乏合适的人类 神经元细胞系以及建立疾病相关的人类同基因细胞模型的挑战。的 将体细胞重编程为诱导多能干细胞 (iPSC) 的能力，以及基因组学的最新进展 编辑技术提供了一个及时的机会来建立不同基因组的基因匹配的人类 iPSC 系。 专门针对引起疾病的基因改变。最近，我们利用 CRISPR/Cas9 基因组编辑技术 从两个不相关的 TSC 患者中产生了这样的同基因 iPSC 系，具有明确的杂合失活 TSC1 或 TSC2 分别发生突变。此外，我们还从另外三个不相关的 TSC2 获得了 iPSC 系 个人，他们将作为验证队列，确保我们数据的稳健性和可重复性。这些 iPSC 细胞系使我们能够衍生出神经祖细胞（NPC）的谱系，神经祖细胞是中枢神经系统的起源细胞 TSC 和神经嵴细胞 (NCC) 的表现，负责 TSC 的非 CNS 方面。初始 使用基因匹配的 TSC1-NPC（Het、Null 和 Corrected-WT）组进行的研究证实 TSC1 Het 和 Null 细胞中细胞大小的增加和 mTORC1 的激活。我们观察到明显的激活 用雷帕霉素处理 NPC 后，ERK1/2 信号传导和 MNK-eIF4E 增加。有趣的是，TSC1 Het 与匹配的 WT 对照相比，空 NPC 揭示了 NPC 增殖以及 神经突的数量和长度，这是与 ASD 相关的早期神经发育表型。作为 mTORC1、MEK-ERK a 以及 s MNK-eIF4E 信号调节翻译，我们建议生成 TSC1/2 Het、Null 和 Corrected-WT NPC 中的全面转录组和翻译组概况 NCC，它将定义 TSC1/2 丢失时潜在的分子变化。最后，我们将进行一次 在 NPC 的 TSC1/2 同基因组中进行无偏见的高通量单一和组合药物筛选， 与 NIH-NCATS 合作，确定对 TSC1/2 Het 和 Null 产生优先影响的潜在药物 细胞。顶级单药和组合药物将在多个 TSC 的 Ramesh 实验室进行独立验证 源自患者的 NPC 系。对 TSC1/2 Het 或 Null 细胞表现出选择性偏向的化合物的效果将是 在二次测定中进一步进行测试，以评估它们标准化转录组和翻译组的能力 签名。使用患者特异性、iPSC 衍生的 NPC 和 NCC 作为遗传精确的人类细胞 理解疾病和药物筛选的模型将提供对病理生理学和新颖的见解 治疗开发的目标，从而对 TSC 研究和患者护理产生直接影响，以及 最终将导致更好地理解 TSC、ASD 和 ID 之间共享的分子机制。

Tuberous Sclerosis Complex (TSC) is a monogenic disorder with increased incidence of seizures, intellectual disability (ID), and autism spectrum disorder (ASD). Although significant progress has been achieved in understanding TSC, the ability to fully comprehend TSC as a neurodevelopmental disorder, and the shared molecular mechanisms that may explain the overlapping phenotypes between TSC and ASD is hampered by the lack of suitable human neuronal cell lines as well as challenges in establishing disease-relevant human isogenic cellular models. The capability to reprogram somatic cells into induced pluripotent stem cells (iPSCs), and the recent advances in genome editing technologies provide a timely opportunity to establish genetically matched sets of human iPSC lines that differ exclusively at the disease-causing genetic alteration. Employing CRISPR/Cas9 genome editing, we have recently generated such isogenic iPSC lines from two unrelated TSC patients, with a defined heterozygous inactivating mutation in TSC1 or TSC2, respectively. Further, we have obtained iPSC lines from three additional unrelated TSC2 individuals, which will serve as a validation cohort ensuring robustness and reproducibility of our data. These iPSC lines have allowed us to derive lineages of neural progenitor cells (NPCs), the cell of origin for the CNS manifestations of TSC, and neural crest cells (NCCs), responsible for the non-CNS aspects of TSC. Initial studies carried out with the genetically matched sets of TSC1-NPCs (Het, Null and Corrected-WT) confirm an increase in cell size and activation of mTORC1 in TSC1 Het and Null cells. We observe distinct activation of ERK1/2 signaling and an increase in MNK-eIF4E after treating NPCs with rapamycin. Interestingly, TSC1 Het and Null NPCs when compared with the matched WT control reveal an increase in NPC proliferation as well as neurite number and length, which are early-stage neurodevelopmental phenotypes linked to ASD. As mTORC1, MEK-ERK a as well s MNK-eIF4E signaling regulate translation, we propose to generate comprehensive transcriptome and translatome profiles in TSC1/2 Het, Null and Corrected-WT NPCs and NCCs, which will define the underlying molecular changes upon TSC1/2 loss. Finally, we will undertake an unbiased high-throughput single and combination drug screen in TSC1/2 isogenic sets of NPCs, in collaboration with NIH-NCATS, to identify potential drugs that exert preferential impact on TSC1/2 Het and Null cells. The top single and combination drugs will be independently validated in the Ramesh lab in multiple TSC patient-derived NPC lines. The effects of compounds that show selective bias toward TSC1/2 Het or Null cells will be further tested in secondary assays that will assess their ability to normalize transcriptome and translatome signatures. The use of patient-specific, iPSC-derived NPCs and NCCs as genetically accurate human cellular models for understanding the disease and for drug screening will provide insights into pathophysiology and novel targets for therapeutic development, thus having a direct impact on TSC research as well as patient care, and ultimately will lead to a better understanding of the shared molecular mechanisms between TSC, ASD, and ID.