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，癫痫发生率增加 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，完全理解TSC是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之间重叠的表型因缺乏合适的人而受到阻碍 神经细胞系以及在建立与疾病相关的人类等基因细胞模型方面的挑战。这个 体细胞重新编程为诱导多能干细胞的能力和基因组研究的最新进展 编辑技术为建立基因匹配的不同的人类IPSC株系提供了一个及时的机会 专门针对致病的基因改变。利用CRISPR/Cas9基因组编辑，我们最近 从两个不相关的TSC患者中产生了这样的等基因iPSC系，具有明确的杂合子失活 TSC1和TSC2分别发生突变。此外，我们还从另外三个不相关的TSC2中获得了IPSC基因 个人，这将作为一个验证队列，确保我们数据的稳健性和可重复性。这些iPSC 细胞系允许我们衍生出神经前体细胞(NPC)的谱系，这是中枢神经系统的起源细胞 TSC的表现，以及神经脊细胞(NCC)，负责TSC的非中枢方面。首字母 用基因匹配的TSC1-NPC集(Het，Null和校正后的WT)进行的研究证实了一种 TSC1Het和Null细胞的细胞大小增加和mTORC1的激活。我们观察到明显的激活 雷帕霉素作用于鼻咽癌后，ERK1/2信号转导通路和MNK-eIF4E表达增加。有趣的是，TSC1 Het 与WT对照组相比，Null NPC显示鼻咽癌增殖和 轴突数量和长度，这是与ASD相关的早期神经发育表型。AS MTORC1MEK-ERKa以及S MNK-eIF4E信号调节翻译，我们建议生成 TSC1/2 Het，Null和校正-WT鼻咽癌和TSC1/2 Het，Null和校正-WT的全面转录组和翻译组图谱 NCCS，它将定义TSC1/2丢失后潜在的分子变化。最后，我们将进行一项 在鼻咽癌TSC1/2等基因组中进行无偏高通量单一药物和联合药物筛选 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。顶级单一药物和联合药物将在Ramesh实验室的多个TSC中进行独立验证 患者派生的鼻咽癌细胞系。对TSC1/2 Het或Null细胞表现出选择性偏向的化合物的影响将是 在二次化验中进一步测试，以评估他们将转录组和翻译组正常化的能力 签名。使用患者特有的IPSC来源的NPC和NCC作为遗传学上准确的人类细胞 了解疾病和药物筛选的模型将提供对病理生理学的洞察和新的 治疗发展的目标，因此对TSC研究和患者护理有直接影响；以及 最终将有助于更好地理解TSC、ASD和ID之间的共同分子机制。

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.