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）-婴儿期死亡的最常见遗传原因-是一种运动神经元疾病，由运动神经元存活（SMN）蛋白水平降低引起，目前尚无有效治疗方法。因此，迫切需要确定可以恢复SMN水平或可以纠正SMN消耗下游的缺陷的治疗。SMN是一种在RNA加工中广泛表达和发挥功能的必需蛋白质。SMA患者具有SMN 1基因的纯合缺失，并保留至少一个几乎相同的SMN 2基因拷贝。SMN 2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。由于亚型SMN 2基因的较高拷贝数降低了疾病的严重程度，因此迄今为止，SMA的大多数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其他治疗途径。然而，目前SMA治疗开发的限制是对合适治疗靶点的了解有限。该研究项目旨在鉴定和表征控制表达和功能的细胞因子， SMN蛋白的最终目标是确定SMA治疗干预的新途径。为了实现这一目标，我们将采用一种新开发的发现平台，该平台使用SMN缺陷引发的细胞增殖缺陷作为NIH 3 T3成纤维细胞中人SMN 2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，这个系统至少部分地概括了疾病中起作用的机制。在目的1中，我们将进行全基因组cDNA筛选，以确定其过表达改善Smn缺陷的NIH 3 T3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通过多种作用机制鉴定作用于SMN生物学的修饰基因的潜力。在目标2中，我们将使用一组正交试验来确定遗传筛选揭示的候选修饰剂以及已知模拟其作用的任何化合物是否i）增加SMN 2基因表达，ii）增强SMN功能，或iii）影响SMN依赖性下游事件。重要的是，我们还将测试这些修饰剂改善从小鼠胚胎干细胞分化的SMA运动神经元存活的能力，以确定其疾病相关性和治疗潜力。总的来说，我们的研究旨在鉴定SMN表达的遗传修饰剂，并将其作为开发SMA新型治疗方法的候选靶点。它们还可能导致发现基因和小分子作为新的研究工具，以研究SMN生物学和SMA疾病机制的基本方面。