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

描述(申请人提供)：脊髓性肌萎缩症(SMA)是婴儿最常见的遗传死亡原因，是一种运动神经元疾病，由存活运动神经元(SMN)蛋白水平降低引起，目前尚无有效的治疗方法。因此，迫切需要确定能够恢复SMN水平或纠正SMN耗竭下游赤字的治疗方法。SMN是一种在RNA加工过程中普遍表达并发挥功能的必需蛋白质。SMA患者有SMN1基因的纯合缺失，并保留了至少一个几乎相同的SMN1基因的副本。SMN2基因产生低水平的SMN蛋白，导致运动神经元退化和骨骼肌萎缩。由于较高拷贝数的低构型SMN2基因降低了疾病的严重性，到目前为止，大多数SMA的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依赖的下游事件可能是另一种治疗途径。然而，目前对SMA治疗方法开发的一个限制是对合适的治疗靶点的有限了解。这项研究项目旨在识别和表征控制表达和功能的细胞因子 对SMN蛋白的研究，最终目标是确定治疗SMA的新途径。为了实现这一目标，我们将使用一个新开发的发现平台，该平台使用SMN缺乏引发的细胞增殖缺陷作为NIH3T3成纤维细胞中人类SMN基因产生的功能性SMN水平的稳健表型读数，并调控内源性小鼠SMN的下调。我们的初步研究表明，这个系统至少部分概括了在疾病中发挥作用的机制。在目标1中，我们将进行全基因组的cDNA筛选，以确定其过表达改善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通过多种作用机制识别作用于SMN生物学的修饰基因。在目标2中，我们将使用一组正交分析来确定通过遗传筛选揭示的候选修饰物以及任何已知模拟其作用的化合物是否：i)增加SMN基因的表达，ii)增强SMN的功能，或iii)影响SMN依赖的下游事件。重要的是，我们还将测试这些修饰物提高从小鼠胚胎干细胞分化的SMA运动神经元存活的能力，以确定它们与疾病的相关性和治疗潜力。总之，我们的研究旨在确定SMN表达的遗传修饰物，并作为开发SMA新的治疗方法的候选靶点。它们还可能导致发现基因和小分子，作为研究SMN生物学和SMA疾病机制的基本方面的新研究工具。

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.