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.

患者来源的亨廷顿病 iPS 细胞模型比以前的 HD 细胞模型取得了实质性进展，但它们也有许多缺点——分化方案长、复杂且昂贵、异质分化细胞表型、实验结果的可变性以及生产困难因此，我们开发了一种策略，将 iPS 细胞分化为纹状体前体阶段，并使它们永生化，以获得可按常规维持的同质克隆系。我们发现，分化的永生化纹状体前体神经元 (SPN) 将重现 HD iPSC 的 CAG 重复扩增相关表型，并且应该具有更均一性的优势，并且更适合开发可筛选的测定法来识别 HD 疗法。在 R21 阶段，我们将生成等位基因。系列 SPN，对细胞系进行组学分析，并验证其在 24 孔板格式中筛选的适用性 在 R33 阶段，我们将格式化 96 孔板的测定并使用包括小分子的文库进行筛选。我们的 NINDS/CHDI 支持的研究亨廷顿蛋白翻译后修饰 (PTM) 项目中确定的蛋白激酶抑制剂库非常适合开发成可筛选的药物细胞模型。发现努力。