Small molecule screen to suppress expression of mutant huntington

抑制突变亨廷顿表达的小分子筛选

• 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
• 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

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（申请人提供）：亨廷顿舞蹈病（HD）是一种常染色体显性的神经退行性疾病，以运动、认知和情绪异常为特征，发病后20年左右持续发展直至死亡。亨廷顿舞蹈症是由亨廷顿蛋白（HTT）基因外显子1中CAG重复扩增引起的。疾病的发病机制主要是由于具有神经毒性的突变转录物和蛋白质的表达。因此，抑制突变等位基因的表达是一种很有前景的治疗方法，迄今为止，在细胞和动物模型中采用抗体、寡核苷酸和siRNA策略进行了研究。与任何击倒方法一样，特别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解决。令人惊讶的是，HTT的表达调控很少受到关注：HTT的确切启动子区域今年才被Wang等人确定，只有少数转录因子被证明可以调节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），由HD位点的HTT链反义产生。HTTAS_v1下调HTT表达，自身表达受重复长度调控。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，并有可能避免一些需要外源性核酸抑制HTT方法固有的困难。在这里，我们提出了一个小分子化合物的先导高通量筛选（HTS）来抑制HTT的表达，通过测量小分子对HTT和HTTAS_v1启动子的影响来检测。本研究将与国家推进转化科学中心（NCATS， Marc Ferrer博士，组长）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，我们将HTT和HTTAS_v1的最佳启动子区域分别克隆到一个巧合报告载体中，在该载体中启动子活性可以同时被Renilla和萤火虫荧光素酶测量。该构建体是在NIH专门为HTS检测开发的。然后，我们将生成稳定的细胞系（使用来自Invitrogen的flip - intm T-RexTM系统），其中包含HTTAS_v1启动子或与报告基因构建融合的HTT启动子，以及控制系。在Specific Aim 2中，NCATS将筛选约3800种先前被FDA批准用于人用的化合物。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，然后将其应用于含有100万小分子的大规模高温超导。我们的长期目标是利用这种方法产生可用于治疗HD的小分子。