Small molecule screen to suppress expression of mutant huntington

描述（由申请人提供）：亨廷顿病（HD）是一种常染色体显性遗传神经退行性疾病，其特征为运动、认知和情感异常，在发病后约20年内持续进展直至死亡。HD是由亨廷顿蛋白（HTT）基因外显子1中的CAG重复扩增引起的。疾病的发病机制主要是突变体转录物和蛋白质表达的结果，其具有神经毒性特性。因此，抑制突变等位基因的表达是一种有前途的治疗方法，迄今为止，在细胞和动物模型中采用抗体、寡核苷酸和siRNA策略。与任何敲除方法一样，特别是在CNS中，使用这些方法的递送、可逆性和脱靶效应的问题仍未解决。令人惊讶的是，HTT表达的调控很少受到关注：今年Wang等人刚刚确定了HTT的确切启动子区域，并且只有少数转录因子被证明可以调控HTT表达。另一方面，我们最近发现了一个转录本，亨廷顿蛋白反义（HTTAS_v1），产生自链反义HTT在HD基因座。HTTAS_v1下调HTT表达，而其自身的表达受重复序列长度的调节。因此，我们假设小分子诱导的HTT表达下调或HTTAS_v1表达上调可能为HD治疗提供直接而有力的方法，并有可能避免需要给予外源性核酸的HTT抑制方法中固有的一些困难。在这里，我们提出了一种抑制HTT表达的小分子化合物的试点高通量筛选（HTS），通过测量小分子对HTT和HTTAS_v1启动子的影响来测定。本研究将与国家转化科学推进中心（NCATS，Marc Ferrer博士，团队负责人）合作进行。在具体目标1中，我们将工程化用于HTS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重合报告载体中，其中启动子活性可以同时通过海肾和萤火虫荧光素酶测量。该构建体是在NIH专门为HTS测定开发的。然后，我们将产生含有与报告构建体融合的HTTAS_v1启动子或HTT启动子的稳定细胞系（使用来自Invitrogen的Flp-inTM T-RexTM系统）以及对照系。在具体目标2中，NCATS将筛选约3800种先前经FDA批准用于人类的化合物。我们将测试筛选阳性的化合物，以确定其对HD患者细胞系和成纤维细胞中HTT内源性水平的影响。我们的近期目标是验证可以应用于> 400，000个小分子的大规模HTS的方法。我们的长期目标是使用这种方法产生可用于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.