Targeting trinucleotide repeats-induced transcriptional silencing in Friedreich's

描述（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。它是由位于FXN基因第一个内含子的GAA重复序列扩增突变引起的FXN基因转录沉默引起的。重要的是，FXN基因的编码序列是完整的，完全能够表达功能性的fraataxin。与杂合子携带者和健康对照相比，GAA扩增纯合子的弗里德赖希共济失调患者的frataxin mRNA和蛋白水平非常低。目前还没有有效的治疗弗里德赖希共济失调的方法。到目前为止，非常有限的化合物在体外被证明可以在很小程度上缓解gaa诱导的转录沉默。该项目的目标是设计、构建和表征一种高通量筛选策略，旨在发现能够刺激被病理扩展的GAA重复序列阻断的基因表达的新药理学探针。为了实现这些目标，我们提出了以下具体目标：(i)开发一种基于细胞的报告基因试验，用于鉴定能够减轻GAA重复诱导的转录沉默的化合物。我们将构建并检测含有长内含子GAA重复序列的绿色荧光蛋白（GFP）报告基因。我们的初步实验表明，这一报告基因被560个GAA重复序列显著沉默。为了提高屏幕的信本比，将向报告者引入更长的GAA重复通道。此外，内源性frataxin启动子将用于表达报告基因。将进行实验以确保GFP报告基因的沉默模仿FXN基因的转录抑制。我们还将开发使用不含GAA重复序列的独立报告红色荧光蛋白（RFP）小基因的选择性试验。（ii）高通量筛选的分析配置。将开发一种基于含有长内含子GAA重复序列的萤火虫荧光素酶报告基因的反筛选策略。此外，将开发涉及弗里德赖希共济失调患者淋巴母细胞样细胞系的后续方案，以验证命中对FXN基因诱导的特异性。我们还将使用NIH临床收集化合物库进行试点筛选。总的来说，这些研究将导致HTS分析的发展，准备在分子文库探针生产中心网络的筛选程序中实施。此外，我们将在后续研究计划中提出先导开发策略，旨在发现新的治疗探针，用于治疗弗里德赖希共济失调和其他由转录沉默引起的疾病。

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.