A genome-wide phenotypic screen for modifiers of SMN expression and function

SMN 表达和功能修饰因子的全基因组表型筛选

• 批准号: 8702410
• 负责人: Livio Pellizzoni
• 金额: $ 24万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-02-01 至 2016-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8702410
• 关键词: Address; Bioinformatics; Biological Assay; Biological Models; Biology; Candidate Disease Gene; Cause of Death; Cell Line; Cell Proliferation; Cell model; Cells; Chemicals; Childhood; Complementary DNA; Defect; Development; Disease; Embryo; Event; Fibroblasts; Future; Gene Expression; Genes; Genetic; Genetic Screening; Goals; Growth; Housing; Human; In Vitro; Knowledge; Lead; Libraries; Mammalian Cell; Measures; Motor Neuron Disease; Motor Neurons; Mus; Neurodegenerative Disorders; Pathology; Patients; Phenotype; Play; Proteins; RNA Processing; Research; Research Design; Research Project Grants; SMN protein (spinal muscular atrophy); SMN1 gene; SMN2 gene; Severity of illness; Specificity; Spinal Muscular Atrophy; System; Testing; Therapeutic; Therapeutic Intervention; Werdnig-Hoffmann Disease; base; candidate validation; chemical genetics; effective therapy; embryonic stem cell; functional genomics; gene discovery; genetic analysis; genome-wide; improved; infancy; insight; lentiviral-mediated; motor neuron degeneration; motor neuron function; mouse model; next generation sequencing; novel; novel strategies; novel therapeutic intervention; novel therapeutics; protein function; public health relevance; research study; screening; skeletal muscle wasting; small molecule; stem; therapeutic target; tool; validation studies

DESCRIPTION (provided by applicant): Spinal muscular atrophy (SMA) - the most common genetic cause of death in infancy - is a motor neuron disease that is caused by reduced levels of the survival motor neuron (SMN) protein and for which no effective therapy is currently available. Therefore, there is an urgent need to identify treatments that can restore SMN levels or can correct the deficits downstream of SMN depletion. SMN is an essential protein that is ubiquitously expressed and functions in RNA processing. SMA patients have homozygous loss of the SMN1 gene and retain at least one copy of the nearly identical SMN2 gene. The SMN2 gene produces low levels of the SMN protein leading to motor neuron degeneration and skeletal muscle wasting. Since higher copy numbers of the hypomorphic SMN2 gene reduce disease severity, to date most therapeutic discovery efforts for SMA have focused on increasing expression of SMN. Enhancing SMN function or correcting SMN-dependent downstream events may represent additional therapeutic avenues. However, a current limitation to the development of a treatment for SMA is the limited knowledge of suitable therapeutic targets. This research project aims to identify and characterize cellular factors that control the expression and function of the SMN protein with the ultimate goal of identifying novel avenues of therapeutic intervention for SMA. To reach this objective, we will employ a newly developed discovery platform that uses cell proliferation defects triggered by SMN deficiency as a robust phenotypic readout of functional SMN levels produced from the human SMN2 gene in NIH3T3 fibroblasts with regulated knockdown of endogenous mouse Smn. Our preliminary studies indicate that this system recapitulates, at least in part, mechanisms at play in disease. In Aim 1, we will perform a genome-wide cDNA screen in order to identify genes whose over-expression improves the cell proliferation phenotype of Smn-deficient NIH3T3 fibroblasts. A unique advantage of the phenotypic screening strategy we propose is the potential to identify modifier genes that act on SMN biology through multiple mechanisms of action. In Aim 2, we will use a panel of orthogonal assays to determine whether candidate modifiers revealed by the genetic screen as well as any chemical compounds known to mimic their effects i) increase SMN2 gene expression, ii) enhance SMN function, or iii) influence SMN-dependent downstream events. Importantly, we will also test the ability of these modifiers to improve survival of SMA motor neurons differentiated from mouse embryonic stem cells in order to establish their disease relevance and therapeutic potential. Collectively, our studies are designed to identify genetic modifiers of SMN expression and function as candidate targets for developing novel therapeutic approaches to SMA. They may also lead to the discovery of genes and small molecules as new research tools to study fundamental aspects of SMN biology and SMA disease mechanisms.

描述（由申请人提供）：脊髓性肌萎缩症（SMA）——婴儿期最常见的遗传性死亡原因——是一种运动神经元疾病，由运动神经元存活蛋白（SMN）水平降低引起，目前尚无有效治疗方法目前可用。因此，迫切需要找到能够恢复 SMN 水平或纠正 SMN 耗竭下游缺陷的治疗方法。 SMN 是一种普遍表达的必需蛋白，并在 RNA 加工中发挥作用。 SMA 患者的 SMN1 基因纯合缺失，但保留了至少一份几乎相同的 SMN2 基因。 SMN2 基因产生低水平的 SMN 蛋白，导致运动神经元变性和骨骼肌萎缩。由于亚效型 SMN2 基因的较高拷贝数可降低疾病的严重程度，因此迄今为止大多数 SMA 治疗发现工作都集中在增加 SMN 的表达上。增强 SMN 功能或纠正 SMN 依赖性下游事件可能代表额外的治疗途径。然而，目前对 SMA 治疗方法开发的限制是对合适治疗靶点的了解有限。该研究项目旨在识别和表征控制表达和功能的细胞因子 SMN 蛋白的研究，最终目标是确定 SMA 治疗干预的新途径。为了实现这一目标，我们将采用一个新开发的发现平台，该平台利用 SMN 缺陷引发的细胞增殖缺陷，作为 NIH3T3 成纤维细胞中人类 SMN2 基因产生的功能性 SMN 水平的稳健表型读数，并调节内源性小鼠 Smn 的敲低。我们的初步研究表明，该系统至少部分地再现了疾病中起作用的机制。在目标 1 中，我们将进行全基因组 cDNA 筛选，以确定其过度表达可改善 Smn 缺陷的 NIH3T3 成纤维细胞的细胞增殖表型的基因。我们提出的表型筛选策略的一个独特优势是有可能识别通过多种作用机制作用于 SMN 生物学的修饰基因。在目标 2 中，我们将使用一组正交测定来确定遗传筛选揭示的候选修饰剂以及已知模拟其效果的任何化合物是否 i) 增加 SMN2 基因表达，ii) 增强 SMN 功能，或 iii) 影响SMN 相关的下游事件。重要的是，我们还将测试这些修饰剂提高从小鼠胚胎干细胞分化而来的 SMA 运动神经元存活率的能力，以确定它们的疾病相关性和治疗潜力。总的来说，我们的研究旨在确定 SMN 表达和功能的遗传修饰因子，作为开发 SMA 新型治疗方法的候选靶点。它们还可能导致基因和小分子的发现，作为研究 SMN 生物学和 SMA 疾病机制基本方面的新研究工具。