A Functional Cell-Based Screen for Potential SMA Therapeutic Compounds

描述（由申请人提供）：脊髓性肌萎缩症（SMA）是婴儿期死亡的最常见遗传原因，但没有有效的治疗方法。SMA是由运动神经元存活（SMN）蛋白水平降低引起的-反映了SMN 1基因的纯合丢失，但保留了几乎相同的SMN 2基因。低SMN水平导致运动神经元变性和肌肉力量丧失。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。由于SMA患者和SMN 2基因拷贝数较高的小鼠模型显示疾病严重程度降低，因此大多数药物筛选工作都集中在增强SMN 2基因的全长SMN表达。这可能是通过增加SMN 2转录或通过纠正剪接缺陷而发生的，这导致它是亚型的。然而，大多数现有的筛选试验并不直接评估SMN功能，而是测量报告基因活性和/或SMN蛋白积累。因此，对总体水平影响较小但对SMN功能有显著影响的化合物可能未被检测到。此外，没有筛选作用于疾病途径而不是疾病触发因素的化合物。目前的建议旨在解决这些缺点。我们已经产生了小鼠成纤维细胞系与调节敲低内源性SMN。将mSmn表达降低到与I型患者组织中发现的水平相似的水平会触发这些细胞的生长停滞-这是一种可以通过RNAi抗性人SMN的异位表达来纠正的表型。为了支持该测定反映了在疾病中起作用的机制的观点，SMN的不同点突变形式纠正生长缺陷的能力与其在小鼠模型中挽救运动表型的效力成比例。我们将开发该细胞系的改良版本，其中细胞增殖依赖于人SMN 2基因产生的SMN水平。未处理的细胞系将显示生长减少，而增强SMN 2转录或剪接、稳定SMN蛋白或纠正下游缺陷的试剂将促进增殖增加。因此，这种新模型将为SMA中的多个潜在治疗靶点提供功能读数。在对已知基准化合物的重现性和响应性进行初步测试后，我们将对已知显示生物活性的4，000种化合物进行中试筛选。设计了二次筛选，以消除通过非疾病相关机制起作用的化合物，并区分在SMN 2水平起作用的化合物和在下游起作用的化合物。该筛选旨在为后续研究提供对化合物作用机制和优先级排序的初步了解。这些将分为三种类型：i）与学术和药物筛选中心合作，对经验证的检测方法进行大规模高通量筛选，ii）分析命中化合物指示的潜在疾病机制，iii）研究命中化合物在SMA小鼠模型中的治疗潜力。 公共卫生相关性：脊髓性肌萎缩症（SMA）是婴儿死亡的最常见遗传原因，但没有有效的治疗方法。我们正在开发新的细胞模型，使我们能够模拟培养皿中的缺陷。作为新的治疗策略的第一步，我们将测试数千种化合物，以确定它们纠正这些细胞中SMA相关缺陷的能力。

DESCRIPTION (provided by applicant): Spinal muscular atrophy (SMA) is the most common genetic cause of death in infancy, but no effective treatment is available. SMA is caused by reduced levels of the survival motor neuron (SMN) protein - reflecting homozygous loss of the SMN1 gene but preservation of the nearly-identical SMN2 gene. Low SMN levels lead to motor neuron degeneration and loss of muscle strength. There is therefore an urgent need to identify treatments that can restore SMN levels or can correct the deficits downstream of SMN depletion. Since SMA patients and mouse models with higher copy numbers of the SMN2 gene show reduced disease severity, most drug screening efforts have focused on enhancing expression of full-length SMN from the SMN2 gene. This may occur either by increasing SMN2 transcription or by correcting the splicing defect, which leads it to be hypomorphic. However, most of the existing screening assays do not directly assess SMN function but instead measure reporter gene activity and/or SMN protein accumulation. Therefore, compounds that have a small effect on overall levels but a significant effect on SMN function may go undetected. Moreover, compounds acting on the disease pathway rather than on the disease trigger are not screened for. The current proposal aims to address each of these shortcomings. We have generated mouse fibroblast lines with regulated knockdown of endogenous SMN. Reducing mSmn expression to a level similar to that found in tissues of type I patients triggers growth arrest in these cells - a phenotype that can be corrected by ectopic expression of RNAi-resistant human SMN. In support of the idea that this assay mirrors the mechanisms at play in the disease, the ability of different point mutant forms of SMN to correct the growth defect is proportional to their potency in rescuing the motor phenotype in mouse models. We will develop a modified version of this cell line in which cell proliferation is dependent on SMN levels produced by the human SMN2 gene. The untreated cell line will show reduced growth, while agents that enhance SMN2 transcription or splicing, stabilize SMN protein, or correct downstream defects will promote increased proliferation. This new model will therefore provide a functional readout for multiple potential therapeutic targets in SMA. Following initial testing of the assay for reproducibility and responsiveness to known benchmarking compounds, we will perform a pilot screen of 4,000 chemical compounds known to show biological activity. Secondary screens have been devised to eliminate compounds acting through mechanisms that are not disease-related and to distinguish between those acting at the level of SMN2 and others acting downstream. The screen is designed to provide a first insight into mechanism(s) of action and priority ranking of compounds for follow-up studies. These will be of three types: i) partnerships with academic and pharmaceutical screening centers to pursue a large-scale high-throughput screen on the validated assay, ii) analysis of potential disease mechanisms indicated by the hit compounds, and iii) investigation of therapeutic potential of the hit compounds in mouse models of SMA. PUBLIC HEALTH RELEVANCE: Spinal muscular atrophy (SMA) is the most common genetic cause of death in infancy, but no effective treatment is available. We are developing new cell models which allow us to mimic the defect in the culture dish. As a first step toward new therapeutic strategies, we will test thousands of chemical compounds for their ability to correct the SMA-related defect in these cells.