Small molecule screen to suppress expression of mutant huntington

抑制突变亨廷顿表达的小分子筛选

基本信息

批准号：
8621121
负责人：
RUSSELL L MARGOLIS
金额：
$ 24.3万
依托单位：
JOHNS HOPKINS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-12-15 至 2015-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8621121
关键词：
Adult Alleles Animal Model Antibodies Attention Automobile Driving Biological Assay Brain region CAG repeat Cell Line Cell model Cells Cessation of life Clinical Trials Cognition Collaborations Development Disease Disease Progression Down-Regulation Drug Targeting Emotions Engineering Exons FDA approved Face Fibroblasts Firefly Luciferases Funding Genes Genetic Goals Human Huntington Disease Immunization Individual Inherited Length Luc Gene Measures Methods Movement Mutation Neurodegenerative Disorders Nucleic Acids Oligonucleotides Onset of illness Pathogenesis Pathway interactions Patients Promoter Regions Property Proteins Regulation Renilla Reporter Research Site Small Interfering RNA Specificity System Testing Therapeutic Therapeutic Agents Toxic effect Transcript Translational Research United States National Institutes of Health Up-Regulation Validation Work design disorder control high throughput screening homologous recombination human Huntingtin protein interest knock-down mouse model mutant neurotoxic novel strategies polyglutamine prevent promoter public health relevance research study slow potential small molecule stable cell line tool transcription factor vector working group

项目摘要

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.

描述（由申请人提供）：亨廷顿病（HD）是一种常染色体显性神经退行性疾病，其特征是运动、认知和情感异常，并在发病约 20 年后不断进展直至死亡。 HD 是由亨廷顿蛋白 (HTT) 基因外显子 1 中的 CAG 重复序列扩展引起的。疾病发病机制很大程度上是突变转录本和蛋白质表达的结果，它们具有神经毒性。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 策略进行研究。与任何击倒方法一样，尤其是在中枢神经系统中，使用这些方法的递送、可逆性和脱靶效应问题仍未得到解决。令人惊讶的是，HTT表达的调控很少受到关注：Wang等人今年才确定了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），是由 HD 基因座上的 HTT 反义链生成的。 HTTAS_v1 下调 HTT 表达，而其自身表达受重复长度调节。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 治疗提供直接而有效的方法，并有可能避免需要施用外源核酸的 HTT 抑制方法中固有的一些困难。在这里，我们提出了一种小化合物的试点高通量筛选（HTS），以抑制 HTT 表达，通过测量小分子对 HTT 和 HTTAS_v1 启动子的影响进行测定。这项研究将与国家转化科学促进中心（NCATS，Marc Ferrer 博士，团队负责人）合作进行。在具体目标 1 中，我们将设计用于 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，其中海肾和萤火虫荧光素酶可以同时测量启动子活性。该构建体是在 NIH 专门用于 HTS 测定而开发的。然后，我们将生成包含 HTTAS_v1 启动子或与报告构建体融合的 HTT 启动子的稳定细胞系（使用 Invitrogen 的 Flp-inTM T-RexTM 系统）以及对照系。在具体目标 2 中，NCATS 将筛选 FDA 先前批准用于人类用途的约 3800 种化合物。我们将测试筛选出阳性的化合物，以确定它们对 HD 患者细胞系和成纤维细胞中 HTT 内源水平的影响。我们的近期目标是验证可应用于超过 400,000 个小分子的大规模 HTS 的方法。我们的长期目标是利用这种方法生产可用于 HD 治疗的小分子。