Targeting trinucleotide repeats-induced transcriptional silencing in Friedreich's

靶向弗里德赖希氏病中三核苷酸重复诱导的转录沉默

• 批准号: 8191283
• 负责人: Marek Napierala
• 金额: $ 3.95万
• 依托单位: UNIVERSITY OF TX MD ANDERSON CAN CTR
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-09-30 至 2012-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8191283
• 关键词: Ataxia; Biochemical; Biological Assay; Caucasians; Caucasoid Race; Cell Line; Cells; Chromosomes; Clinical; Code; Collection; Cytomegalovirus; Development; Disease; Eligibility Determination; Ensure; Epigenetic Process; Firefly Luciferases; Fragile X Syndrome; Friedreich Ataxia; Gene Expression; Genes; Genetic; Genetic Transcription; Goals; Green Fluorescent Proteins; Health; Human Cell Line; In Vitro; Inherited; Introns; Lead; Libraries; Luciferases; Malignant Neoplasms; Measures; Messenger RNA; Methods; Molecular; Molecular Bank; Monitor; Mutate; Mutation; Neurodegenerative Disorders; Patients; Production; Proteins; Protocols documentation; Reporter; Reporter Genes; Research; Research Design; Screening procedure; Signal Transduction; Specificity; System; Systems Analysis; Testing; Therapeutic; Therapeutic Effect; Trinucleotide Repeats; United States National Institutes of Health; Validation; base; design; design and construction; effective therapy; follow-up; frataxin; gene induction; high throughput screening; improved; innovation; lymphoblastoid cell line; nervous system disorder; novel therapeutics; programs; promoter; red fluorescent protein; research study; response

DESCRIPTION (provided by applicant): Friedreich's ataxia is a severe autosomal recessive neurodegenerative disease, the most frequent inherited ataxia in Caucasians. It is caused by transcriptional silencing of the FXN gene induced by expansion mutation of the GAA repeats located in the first intron of this gene. Importantly, coding sequence of the FXN gene is intact and fully capable of expressing functional frataxin. Friedreich's ataxia patients homozygous for GAA expansion have very low frataxin mRNA and protein levels when compared with heterozygous carriers and healthy controls. Currently there is no effective treatment for Friedreich's ataxia. Thus far, a very limited number of compounds have been shown to alleviate GAA-induced transcriptional silencing to a small extent in vitro. The objective of this project is to design, construct and characterize a high-throughput screening strategy aimed towards discovering new pharmacological probes capable to stimulate gene expression blocked by pathologically expanded GAA repeats. To achieve these goals we propose the following specific aims: (i) Development of a cell-based reporter assay for identification of compounds capable to alleviate GAA repeats-induced transcriptional silencing. We will construct and test the reporter green fluorescent protein (GFP) minigene containing long intronic GAA repeats. Our preliminary experiments showed a significant silencing of this reporter gene by a tract of 560 GAA repeats. In order to improve the signal-to-background ratio of the screen, a longer GAA repeat tract will be introduced to the reporter. Additionally, the endogenous frataxin promoter will be used to express the reporter minigene. Experiments will be conducted to ensure that the silencing of the GFP reporter gene is mimicking transcriptional inhibition of the FXN gene. We will also develop selectivity assay using an independent reporter red fluorescent protein (RFP) minigene without GAA repeats. (ii) Configuration of the assay for a high-throughput screen. A counter-screen strategy based on firefly luciferase reporter containing long intronic GAA repeats will be developed. Additionally, follow-up protocol involving lymphoblastoid cell lines from Friedreich's ataxia patients will be developed to verify specificity of hits towards FXN gene induction. We will also conduct a pilot screen using NIH Clinical Collection library of compounds. Collectively, these studies will result in the development of the HTS assay ready for implementation into the screening program at the Molecular Libraries Probe Production Centers Network. Furthermore, we will propose a strategy of lead development in the follow-up research program designed to discover new therapeutic probes for Friedreich's ataxia and perhaps other diseases caused by transcriptional silencing. PUBLIC HEALTH RELEVANCE: This project can lead to the discovery of new compounds with a therapeutic activity towards Friedreich's ataxia, the most common inherited ataxia. Results of this study may also be applicable in the development of innovative therapeutic approaches for Fragile X syndrome and other neurological diseases as well as cancer.

描述（由申请人提供）：弗里德赖希共济失调是一种严重的常染色体隐性神经退行性疾病，是白种人中最常见的遗传性共济失调。它是由 FXN 基因的转录沉默引起的，该沉默是由位于该基因第一个内含子中的 GAA 重复序列的扩展突变引起的。重要的是，FXN 基因的编码序列是完整的并且完全能够表达功能性frataxin。与杂合子携带者和健康对照相比，GAA 扩增纯合子的 Friedreich 共济失调患者的 frataxin mRNA 和蛋白质水平非常低。目前尚无针对弗里德赖希共济失调的有效治疗方法。迄今为止，只有非常有限的化合物已被证明可以在体外小程度地减轻 GAA 诱导的转录沉默。该项目的目标是设计、构建和表征高通量筛选策略，旨在发现能够刺激被病理性扩展的 GAA 重复阻断的基因表达的新药理学探针。为了实现这些目标，我们提出以下具体目标：（i）开发基于细胞的报告测定法，用于鉴定能够减轻 GAA 重复诱导的转录沉默的化合物。我们将构建并测试含有长内含子 GAA 重复序列的报告绿色荧光蛋白 (GFP) 小基因。我们的初步实验表明该报告基因被 560 个 GAA 重复序列显着沉默。为了提高屏幕的信背景比，将向记者引入更长的GAA重复序列。此外，内源性 frataxin 启动子将用于表达报告小基因。将进行实验以确保 GFP 报告基因的沉默模拟 FXN 基因的转录抑制。我们还将使用不含 GAA 重复的独立报告红色荧光蛋白 (RFP) 小基因开发选择性测定。 (ii) 高通量筛选测定的配置。将开发基于包含长内含子 GAA 重复的萤火虫荧光素酶报告基因的反筛选策略。此外，还将制定涉及弗里德赖希共济失调患者的淋巴母细胞系的后续方案，以验证 FXN 基因诱导命中的特异性。我们还将使用 NIH 临床保藏化合物库进行试点筛选。总的来说，这些研究将导致 HTS 测定的开发，准备好实施到分子库探针生产中心网络的筛选计划中。此外，我们将在后续研究计划中提出先导开发策略，旨在发现弗里德赖希共济失调以及可能由转录沉默引起的其他疾病的新治疗探针。 公共健康相关性：该项目可以发现对弗里德赖希共济失调（最常见的遗传性共济失调）具有治疗活性的新化合物。这项研究的结果也可能适用于开发脆性 X 综合征和其他神经系统疾病以及癌症的创新治疗方法。