Drug development for tuberous sclerosis complex and other pediatric epileptogenic diseases using neurovascular and cardiac microphysiological models

这项提议的目标是建立密切相关的神经疾病的体外组织芯片模型。 结节性硬化症(TSC)癫痫、DEPDC5相关癫痫及其相关心脏 功能障碍。这项拟议的研究利用新兴的生物工程技术进行微生理学 范德比尔特综合生物系统研究和教育研究所(VIIBRE)开发的系统 范德比尔特大学医学中心常规使用的人类诱导多能干细胞工具询问 探索困扰心脏和大脑的遗传疾病以及治疗它们的药物的问题。 VIIBRE神经血管单元(NVU)/血脑屏障和心脏I-Wire器官芯片模型将测试 假设mTORC1和mTORC2信号不同地影响TSC的神经和心脏功能障碍- 和DEPDC5相关的癫痫。TSC和DEPDC5患者的原发和共有异常 相关的癫痫是mTOR激酶复合体1(MTORC1)信号通路的失调。台积电也 在DEPDC5相关性癫痫中未见mTORC2信号的异常。关注mTOR信令 在这些人类疾病中，多发性疾病有几个优点。首先，雷帕霉素和相关化合物是FDA- 批准的mTORC1抑制剂，并已被证明在疾病的某些方面有效 TSC的临床表现。第二，与TSC和DEPDC5相关的癫痫都与神经性和 心脏功能不全。第三，代偿性或差异性mTORC2活动的作用尚不清楚 有争议的。对于TSC患者，靶向mTORC1信号通路的药物已被关联 脑瘤缩小，癫痫发作减少，心功能改善。因此，药物开发用于 这组疾病非常适合使用NVU和I-Wire心脏组织芯片进行研究。在前两部中 几年后，该项目将开发NVU和I-Wire疾病模型，旨在完善TSC和DEPDC5 NVU模型；将I-Wire模型应用于TSC和DEPDC5心肌细胞；并验证结果 控制和患者派生的NVU和I-Wire芯片的方法学。未来三年的目标是评估，为 对照、TSC和DEPDC5 NVU和I-Wire芯片中的生物标记物识别，mTORC1和 MTORC2信号，细胞健康和毒性的蛋白质标记物，代谢物，功能测量和 电生理活性；并使用离子迁移率-质谱仪评估NVU和I-Wire的结果 措施加上mTORC1抑制剂雷帕霉素、癫痫药物Vigabatrin治疗后的药物代谢产物， 和新的临床前mTOR候选药物。NVU和I-Wire将评估这些药物的疗效和毒性 并定义了TSC/DEPDC5共有的效应与疾病特有的效应。通过这种芯片上的器官/人类诱导 多能干细胞平台，将有可能解决目前混乱的机制 发病机制，识别新的疾病生物标记物，量化药物如何通过正常和疾病的血脑 这是一种障碍，并最终开发出有效的治疗方法，从而实现从板凳到床边的转换。

The goal of this proposal is to establish in vitro tissue chip models of the closely related neurological disorders tuberous sclerosis complex (TSC) epilepsy, DEPDC5-associated epilepsy, and their associated cardiac dysfunction. The proposed research leverages emerging bioengineering technology for microphysiological systems developed at the Vanderbilt Institute for Integrative Biosystems Research and Education (VIIBRE) with human induced pluripotent stem cell tools in regular use at Vanderbilt University Medical Center to ask probing questions about genetic disorders that afflict the heart and brain and about the drugs to treat them. The VIIBRE neurovascular unit (NVU)/blood-brain barrier and cardiac I-Wire organ-on-chip models will test the hypothesis that mTORC1 and mTORC2 signaling differentially affect neural and cardiac dysfunction in TSC- and DEPDC5-associated epilepsy. The primary and shared abnormality in patients with TSC and DEPDC5- associated epilepsy is dysregulation of the mTOR kinase complex 1 (mTORC1) signaling pathway. TSC also has abnormalities in mTORC2 signaling not seen in DEPDC5-associated epilepsy. A focus on mTOR signaling in these human mTORopathies has several advantages. First, rapamycin and related compounds are FDA- approved mTORC1 inhibitors and have been shown to have efficacy in some aspects of the disease manifestations of TSC. Second, TSC- and DEPDC5-associated epilepsy are both associated with neural and cardiac dysfunction. Third, the role for compensatory or differential mTORC2 activity is unclear and controversial. For patients with TSC, drugs targeting the mTORC1 signaling pathway have been associated with shrinkage of brain tumors, reduced seizures, and improved cardiac function. Thus, drug development for this group of diseases is well suited for study using both the NVU and I-Wire cardiac-tissue chips. In its first two years, the project will develop the NVU and I-Wire disease models, aimed at refining the TSC and DEPDC5 NVU model; applying the I-Wire model to TSC and DEPDC5 cardiomyocytes; and validating outcome methodologies in control and patient-derived NVU and I-Wire chips. The next three years aim to evaluate, for biomarker identification in control, TSC, and DEPDC5 NVU and I-Wire chips, changes in mTORC1 and mTORC2 signaling, protein markers of cellular health and toxicity, metabolites, functional measures and electrophysiological activity; and, use ion mobility-mass spectrometry to evaluate NVU and I-Wire outcome measures plus drug metabolites after treatment with mTORC1 inhibitor rapamycin, the seizure drug vigabatrin, and novel pre-clinical mTOR drug candidates. The NVU and I-Wire will assess the efficacy and toxicity of these agents and define TSC/DEPDC5 shared vs disease-specific effects. With this organ-on-chip/human induced pluripotent stem cell platform, it will be possible to address currently confounding mechanisms of pathogenesis, identify new disease biomarkers, quantify how drugs cross the normal and diseased blood-brain barrier, and ultimately develop effective therapies and hence enable bench-to-bedside translation.