Human-iPSC derived neuromuscular junctions as a model for neuromuscular diseases.

人 iPSC 衍生的神经肌肉接头作为神经肌肉疾病的模型。

基本信息

批准号：
10727888
负责人：
Helen C Miranda
金额：
$ 39万
依托单位：
CASE WESTERN RESERVE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-09-08 至 2024-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10727888
关键词：
Accounting Acetylcholine Address Adult Amyotrophic Lateral Sclerosis Animal Disease Models Animal Model Biological Biological Assay Biological Models Brain C9ORF72 CRISPR correction Cell Line Cells Central Nervous System Characteristics Chromosome 9 Clinical Trials Communication Complex Denervation Development Disease Disease model Drug Screening Evaluation Foundations Functional disorder Genes Genetic Genotype Goals Human In Vitro Individual Introns Investigation Measures Modeling Molecular Morphology Motor Neurons Mus Muscle Muscle Contraction Mutation Neuromuscular Diseases Neuromuscular Junction Organism Pathway interactions Patients Pharmaceutical Preparations Phase Phased Innovation Awards Phenotype Physiological Positioning Attribute Predisposition Prevention Process Protocols documentation Reporting Reproducibility Signal Transduction Skeletal Muscle Spinal Cord Stem Cell Research Synaptic Cleft System Testing Therapeutic Validation axion disease phenotype effective therapy experience experimental study familial amyotrophic lateral sclerosis frontotemporal lobar dementia amyotrophic lateral sclerosis high throughput screening induced pluripotent stem cell multi-electrode arrays neuronal survival novel optogenetics prevent reduce symptoms reinnervation spinal and bulbar muscular atrophy stem cell model stem cells

项目摘要

Motor neurons carry electrical signals from the brain through the spinal cord to ultimately generate muscle contraction via the neuromuscular junction (NMJ). The mechanisms involved in initiating and maintaining proper communication between the central nervous system and muscles are incredibly complex, and damage in this communication is the cause of neuromuscular diseases (NMD), such as Amyotrophic Lateral Sclerosis (ALS) and Spinal and Bulbar Muscular Atrophy (SBMA). While these disorders are among the most common NMDs, there is currently no cure or effective treatment for them. The NMD field acknowledges that a substantial number of drugs found to alleviate symptoms in animal models have failed in clinical trials. Even though this highlights the importance of the development of humanized models, a caveat of converting studies into iPSC models is the focus on single cells in detriment of the complex systems of the adult organism. In the NMD field specifically, iPSC investigations have largely focused on addressing motor neuron phenotypes that would prevent their degeneration. Unfortunately, this approach is no longer sufficient, as prolonging motor neuron survival does not assure re-innervation, nor does it guarantee prevention of denervation. Therefore, it is crucial for therapeutic advancement in the NMD field that stem cell research needs to focus not only on identifying cell-specific targets but also on testing those targets on functional NMJ systems that comprise both iPSC-derived motor neurons and skeletal muscles. To achieve this goal, we have recently developed a 2D functional human NMJ system comprised of both iPSC-derived motor neurons and skeletal muscles. Our human iPSC-NMJ model is responsive to optogenetics and we are able to quantitatively measure NMJ function in a multi-electrode array system. Hence, in this R61/R33 IGNITE Phased Innovation Award System, we propose to leverage our newly developed NMJ system to scale functional and morphological assessment (Aims 1 and 2) and validate the system by assaying NMJ-specific dysfunction using two NMDs: SBMA and ALS (Aims 3 and 4). Our lab has previously established an iPSC model for SBMA (R01NS121374-01, K01NS116119-01) and has extensive experience modeling this disease. Additionally, we selected the iPSCs harboring G4C2 hexanucleotide repeat expansion on chromosome 9 within the first intron of C9ORF72, as it represents is the most common genetic contributor to frontotemporal dementia (FTD) and ALS, accounting for ~10% of all cases of those diseases. Thus, successful completion of this R61/R33 will establish and validate a novel model system to facilitate therapeutic discovery for NMDs.

运动神经元通过脊髓携带来自大脑的电信号，最终产生肌肉通过神经肌肉接头（NMJ）进行收缩。参与启动和维持的机制中枢神经系统和肌肉之间的正常沟通非常复杂，并且损伤此通讯中的内容是神经肌肉疾病 (NMD) 的病因，例如肌萎缩侧索硬化症（ALS）和脊髓和延髓肌萎缩症（SBMA）。虽然这些疾病是最常见的 NMD，目前尚无治愈或有效的治疗方法。 NMD 领域承认在动物模型中发现的大量缓解症状的药物在临床试验中都失败了。甚至尽管这凸显了开发人性化模型的重要性，但转换研究的一个警告 iPSC 模型的重点是单细胞，而不利于成体有机体的复杂系统。在具体来说，在 NMD 领域，iPSC 研究主要集中于解决运动神经元表型，可以防止它们的退化。不幸的是，这种方法已经不够了，因为延长了运动时间神经元存活并不能保证重新神经支配，也不能保证防止去神经支配。因此，它是对于 NMD 领域的治疗进展至关重要，干细胞研究不仅需要关注识别细胞特异性靶点，同时也在包含两者的功能性 NMJ 系统上测试这些靶点 iPSC 衍生的运动神经元和骨骼肌。为了实现这一目标，我们最近开发了一个 2D 功能性人类 NMJ 系统由 iPSC 衍生的运动神经元和骨骼肌组成。我们的人类 iPSC-NMJ 模型对光遗传学有反应，我们能够定量测量 NMJ 功能在多电极阵列系统中。因此，在这个R61/R33 IGNITE阶段性创新奖励体系中，我们建议利用我们新开发的 NMJ 系统来扩展功能和形态评估（目标 1 和 2）并通过使用两种 NMD 分析 NMJ 特异性功能障碍来验证系统：SBMA 和 ALS（目标 3 和 4）。我们实验室之前已经为SBMA建立了iPSC模型（R01NS121374-01， K01NS116119-01），并且在模拟这种疾病方面拥有丰富的经验。此外，我们选择了 iPSC 在 C9ORF72 的第一个内含子内的 9 号染色体上包含 G4C2 六核苷酸重复扩增，因为它代表是额颞叶痴呆 (FTD) 和 ALS 最常见的遗传因素，占约占这些疾病所有病例的 10%。因此，成功完成 R61/R33 将建立并验证促进 NMD 治疗发现的新型模型系统。