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

多发性硬化症（TSC）是一种单基因疾病， 残疾（ID）和自闭症谱系障碍（ASD）。尽管在理解上取得了重大进展， TSC，充分理解TSC作为神经发育障碍的能力，以及共享的分子机制 这可能解释TSC和ASD之间的重叠表型是由于缺乏合适的人 神经元细胞系以及在建立疾病相关的人类同基因细胞模型中的挑战。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，建立基因匹配的人类iPSC系， 专门针对致病基因的改变。利用CRISPR/Cas9基因组编辑，我们最近 从两名不相关的TSC患者中产生了这样的同基因iPSC系， TSC 1或TSC 2突变。此外，我们已经从另外三个不相关的TSC 2获得了iPSC系。 个人，这将作为一个验证队列，确保我们的数据的鲁棒性和可重复性。这些iPSC 细胞系使我们能够获得神经祖细胞（NPC）的谱系，NPC是CNS的起源细胞 TSC的表现和神经嵴细胞（NCC），负责TSC的非CNS方面。初始 用TSC 1-NPC的遗传匹配组（Het、Het和校正的WT）进行的研究证实， TSC 1 Het和TSC 2细胞中细胞大小增加和mTORC 1活化。我们观察到 用雷帕霉素处理NPC后ERK 1/2信号传导和MNK-eIF 4 E的增加。TSC1 Het 与匹配的WT对照相比， 神经突数量和长度，这是与ASD相关的早期神经发育表型。作为 mTORC 1、MEK-ERK α以及MNK-eIF 4 E信号转导调节翻译，我们建议产生 TSC 1/2 Het、TSC 2和校正WT NPC中的全面转录组和翻译组谱， NCC，这将定义TSC 1/2丢失后的潜在分子变化。最后，我们将进行一项 在TSC 1/2等基因NPC组中进行无偏高通量单一和组合药物筛选， 与NIH-NCATS合作，以确定对TSC 1/2 Het和TSC 2产生优先影响的潜在药物 细胞顶级单药和复方药物将在Ramesh实验室的多个TSC中进行独立验证 患者来源的NPC细胞系。显示出对TSC 1/2 Het或HSC细胞的选择性偏好的化合物的作用将是 在二级检测中进一步测试，以评估其使转录组和翻译组正常化的能力。 签名.使用患者特异性iPSC衍生的NPC和NCC作为遗传上准确的人细胞免疫调节剂。 用于理解疾病和药物筛选的模型将提供对病理生理学和新的 治疗开发的目标，从而对TSC研究以及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.