Immortalized Striatal Precursor Neurons as a Screenable Model of HD

永生化纹状体前体神经元作为 HD 的筛选模型

• 批准号: 10550333
• 负责人: Christopher A Ross
• 金额: $ 40.94万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10550333
• 关键词: Adherent Culture; Adopted; Alleles; Biochemical; Biological Assay; Brain-Derived Neurotrophic Factor; CAG repeat; Cell Differentiation process; Cell Line; Cell model; Cells; Chemicals; Collection; Complex; Corpus striatum structure; Data; Development; Disadvantaged; Disease; Future Generations; Generations; Huntington Disease; Huntington gene; Investigational Therapies; Length; Libraries; Modeling; National Institute of Neurological Disorders and Stroke; Neurons; Nuclear; Pathogenesis; Patients; Pharmacology; Phase; Phenotype; Physical condensation; Post-Translational Protein Processing; Production; Proteomics; Series; Therapeutic; Toxic effect; Western Blotting; differentiation protocol; drug discovery; induced pluripotent stem cell; kinase inhibitor; protein kinase inhibitor; screening; small molecule; small molecule libraries; transcriptome sequencing

Patient-derived Huntington's disease iPS cell models represent a substantial advance over previous cell models of HD, but they have a number of disadvantages – a long, complex and expensive differentiation protocol, heterogeneous differentiated cell phenotypes, variability of experimental results, and difficulty in production of sufficient amounts of differentiated cells for biochemical assays or pharmacological screens. We have therefore developed a strategy to differentiate iPS cells to a striatal precursor stage, and immortalize them, to derive homogeneous clonal lines. These cells can be maintained as routine monolayer cultures and differentiated to a medium-spiny neuron phenotype in only two weeks (versus 3 months for iPSCs). We hypothesize that differentiated immortalized Striatal Precursor Neurons (SPNs) will recapitulate the CAG-repeat-expansion-associated phenotypes of the HD iPSCs, and should have advantages in being more homogenous, and more suitable for development of screenable assays to identify HD therapeutics. In the R21 phase, we will generate an allelic series of SPNs, perform omics analysis of the cell lines, and validate their suitability for screening in a 24-well plate format. In the R33 phase, we will format the assay for 96-well plates and screen with libraries of small molecules including a library of inhibitors of protein kinases for targets identified in our NINDS/CHDI supported project to study posttranslational modifications (PTMs) of huntingtin. We believe these cells will be highly amenable for development into a screenable cell model for drug discovery efforts.

患者来源的亨廷顿舞蹈病iPS细胞模型代表了比以前的亨廷顿舞蹈病细胞模型的重大进步，但它们有许多缺点-长，复杂和昂贵的分化方案，异质分化细胞表型，实验结果的可变性，以及难以生产足够数量的分化细胞进行生化分析或药理筛选。因此，我们开发了一种策略，将iPS细胞分化到纹状体前体阶段，并使它们永生化，以获得同质无性系。这些细胞可以维持为常规的单层培养，并在两周内分化为中等棘神经元表型（而iPSCs则为3个月）。我们假设分化的永活纹状体前体神经元（SPNs）将重现HD iPSCs的cag -repeat-扩增相关表型，并且应该具有更均匀的优势，更适合开发可筛选的检测方法来识别HD治疗方法。在R21期，我们将生成一系列spn等位基因，对细胞系进行组学分析，并验证其在24孔板格式下筛选的适用性。在R33阶段，我们将在96孔板上进行分析，并使用小分子文库进行筛选，其中包括一个蛋白激酶抑制剂文库，用于我们在NINDS/CHDI支持的研究亨廷顿蛋白翻译后修饰（PTMs）的项目中确定的靶标。我们相信这些细胞将非常适合开发成可筛选的细胞模型，用于药物发现工作。