Small molecule screen to suppress expression of mutant huntington

描述(申请人提供)：亨廷顿病(HD)是一种常染色体显性遗传性神经退行性疾病，以运动、认知和情绪异常为特征，持续发展，直到发病约20年后死亡。HD是由Huntingtin(HTT)基因外显子1的CAG重复序列扩大引起的。疾病的发病机制在很大程度上是突变的转录本和蛋白表达的结果，它们具有神经毒性特性。因此，抑制突变等位基因的表达是一种很有前途的治疗方法，到目前为止，通过抗体、寡核苷酸和siRNA策略在细胞和动物模型中进行了探索。与任何击倒方法一样，特别是在中枢神经系统，使用这些方法的交付、可逆性和偏离目标的效果的问题仍然没有解决。令人惊讶的是，HTT表达的调控并没有引起人们的关注：HTT的确切启动子区域是Wang等人今年才发现的，而且只有几个转录因子被证明调控HTT的表达。另一方面，我们最近发现了一个转录本，Huntingtin反义(HTTAS_V1)，它是从HD基因的HTT反义链中产生的。HTTAS_v1下调HTT的表达，而其自身的表达受重复长度的调节。因此，我们推测，小分子诱导的HTT表达下调或HTTAS_v1表达上调可能为HD治疗提供直接而有效的方法，有可能避免需要外源核酸注射的HTT抑制方法固有的一些困难。在这里，我们提出了一个小分子高通量筛选(HTS)来抑制HTT的表达，通过测量小分子对HTT和HTTAS_v1启动子的影响来进行分析。这项研究将与国家高级翻译科学中心(NCATS，团队负责人Marc Ferrer博士)合作进行。在具体目标1中，我们将设计用于高温超导的细胞系。首先，我们首先将HTT和HTTAS_v1的最佳启动子区域分别克隆到一个能同时用Renilla和萤火虫荧光素酶检测启动子活性的重合报告载体中。这种结构是由美国国立卫生研究院专门为HTS检测而开发的。然后，我们将获得稳定的细胞系(使用Invitgen的FLP-inTM T-RexTM系统)，其中包含HTTAS_v1启动子或HTT启动子与报告构建体融合，以及控制系。在具体目标2中，NCATS将筛选约3800种先前由FDA批准用于人类的化合物。我们将测试筛选阳性的化合物，以确定它们对细胞系和HD患者成纤维细胞内源性HTT水平的影响。我们的直接目标是验证这些方法，然后这些方法可以应用于40万个小分子的大规模高温超导。我们的长期目标是使用这种方法生产出可用于HD治疗的小分子。

DESCRIPTION (provided by applicant): Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder, characterized by abnormalities of movement, cognition and emotion, with relentless progression until death ~20 years after disease onset. HD is caused by an expanded CAG repeat in exon 1 of the huntingtin (HTT) gene. Disease pathogenesis is largely a result of expression of the mutant transcript and protein, which have neurotoxic properties. Suppressing the expression of the mutant allele is therefore a promising therapeutic approach, thus far pursued in cell and animal models with antibody, oligonucleotide and siRNA strategies. As with any knockdown approach, especially in the CNS, problems of delivery, reversibility, and off-target effects using these methods remain unsolved. Surprisingly, the regulation of HTT expression has received little attention: the exact promoter region of HTT was just identified by Wang et al this year, and only a few transcription factors had been shown to regulate HTT expression. On the other hand, we have recently discovered a transcript, huntingtin antisense (HTTAS_v1), generated from the strand antisense to HTT at the HD locus. HTTAS_v1 down-regulates HTT expression, while its own expression is regulated by repeat length. We therefore hypothesize that small molecule-induced down regulation of HTT expression or upregulation of HTTAS_v1 expression may provide direct and powerful approaches to HD therapy, with the potential of avoiding some of the difficulties inherent in approaches to HTT suppression that require administration of exogenous nucleic acids. Here, we propose a pilot high throughput screen (HTS) of small compounds to suppress HTT expression, assayed by measuring the effect of the small molecules on the HTT and HTTAS_v1 promoters. This study will be performed in collaboration with the National Center for Advancing Translational Sciences (NCATS, Dr. Marc Ferrer, Team Leader). In specific aim 1, we will engineer cell lines for use in HTS. First, we will first separately clone the optimal promoter regions of HTT and HTTAS_v1 into a coincidence reporter vector in which promoter activity can be measured by both Renilla and firefly luciferase simultaneously. The construct was developed at the NIH specifically for HTS assays. We will then generate stable cell lines (using the Flp-inTM T-RexTM system from Invitrogen) containing the HTTAS_v1 promoter or the HTT promoter fused to the reporter construct, as well as control lines. In Specific Aim 2, NCATS will screen ~3800 compounds previously approved by the FDA for human use. We will test compounds that screen positive to determine their effect on endogenous levels of HTT in cell lines and in fibroblasts from HD patients. Our immediate goal is to validate methods that can then be applied to a large scale HTS of > 400,000 small molecules. Our long term goal is to use this method to yield small molecules that can be used as therapy in HD.