Immortalized Striatal Precursor Neurons as a Screenable Model of HD

患者来源的亨廷顿氏病iPS细胞模型代表了相对于先前的HD细胞模型的实质性进步，但它们具有许多缺点-长、复杂和昂贵的分化方案、异质的分化细胞表型、实验结果的可变性以及难以产生足够量的分化细胞用于生物化学测定或药理学筛选。 因此，我们开发了一种策略，使iPS细胞分化为纹状体前体阶段，并使其永生化，以获得同质克隆系。这些细胞可以维持为常规单层培养物，并在仅两周内分化为中等多刺神经元表型（而iPSC为3个月）。 我们假设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，并且应该具有更同质的优势，并且更适合于开发可筛选的测定以鉴定HD治疗剂。在R21阶段，我们将产生一系列等位基因的SPN，对细胞系进行组学分析，并验证其在24孔板中筛选的适用性。在R33阶段，我们将为96孔板设计检测方法，并使用小分子文库进行筛选，包括NINDS/CHDI支持的研究亨廷顿蛋白翻译后修饰（PTM）项目中确定的靶点的蛋白激酶抑制剂文库。 我们相信这些细胞将非常适合发展成为药物发现工作的可筛选细胞模型。

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.