Identification of compounds stabilizing the GAA repeats in Friedreich’s ataxia cells

鉴定稳定 Friedreich 共济失调细胞中 GAA 重复序列的化合物

• 批准号: 9507188
• 负责人: Jeannine Gerhardt
• 金额: $ 8.48万
• 依托单位: WEILL MEDICAL COLL OF CORNELL UNIV
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-04-01 至 2020-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9507188
• 关键词: Address; Ataxia; Biological Models; Cardiac Myocytes; Caucasians; Cell Differentiation process; Cells; Childhood; Color; DNA; DNA Structure; DNA biosynthesis; DNA replication fork; DNA-Directed DNA Polymerase; Development; Disease; Disease Progression; Fibroblasts; Friedreich Ataxia; Genes; Genetic Transcription; Genome; Genomics; Goals; Heart Abnormalities; Human; Immunofluorescence Microscopy; Impairment; Inherited; Introns; Lead; Length; Longevity; Metabolic; Molecular; Monitor; Nerve Degeneration; Neurons; Parents; Pathology; Patients; Play; Polymerase; Polyploidy; Population; Proliferating; Reactive Oxygen Species; Replication-Associated Process; Role; Stem cells; Stress; Structure; Symptoms; Testing; Therapeutic; Treatment Efficacy; Trinucleotide Repeat Expansion; Trinucleotide Repeats; Undifferentiated; Work; early childhood; experimental study; frataxin; improved; in vivo; induced pluripotent stem cell; nerve stem cell; nervous system disorder; oxidation; prevent; programs; small molecule; stem cell differentiation; tool; young adult

PROJECT SUMMARY/ABSTRACT Friedreich's ataxia (FRDA) is caused by the expansion of a trinucleotide repeat located in the first intron of the Frataxin (FXN) gene, which leads to reduced FXN gene transcription. The extent of Frataxin reduction depends on the GAA repeat length. The GAA repeats continue to expand in FRDA patients, aggravating symptoms and contributing to disease progression. The mechanism for repeat expansion is not clearly understood. It is proposed that an altered DNA replication program and replication fork stalling at secondary repeat structures could cause DNA polymerase slippage and repeat expansion. However, it remains unclear whether secondary repeat structures are formed in vivo at the endogenous FXN gene locus in patients. Furthermore it is unknown whether an altered replication program plays a role in repeat expansion in FRDA cells. Using a unique approach, which monitors the DNA replication in single DNA molecules by multi-color immunofluorescence microscopy (SMARD), our preliminary results show inaccuracies in the DNA replication process in FRDA cells. The goals of this proposal are to determine the cause of replication fork stalling at the expanded GAA repeats and to release the replication fork block to stabilize the repeats in FRDA cells. These aims will address several fundamental aspects of the molecular mechanisms causing repeat expansion in FRDA patients. Can replication be detected in differentiate cells such as cardiomyocytes and if yes, is the replication program altered and can the replication program be corrected at the endogenous FXN locus in FRDA cells? Does the replication fork stall at the proposed secondary repeat structures at the endogenous disease locus in FRDA cells in vivo? Can small molecules release the stalled replication forks? In order to answer these questions we will use the newly established FRDA induced pluripotent stem cells (iPSCs) that contain expanding GAA repeats. FRDA iPSCs can be differentiated to neuronal cell and cardiomyocytes; which makes these cells a perfect tool to study the reason for the repeat instability and decrease FXN transcription in undifferentiated and differentiated FRDA cells. We will employ SMARD to determine the DNA replication at the endogenous disease locus in FRDA cells. In addition, we will test whether small molecules can release the replication fork stall and stabilize the GAA repeats in FRDA cells. Understanding how repeat expansion occurs in the native chromosomal context in FRDA cells is an important prerequisite for the development of effective therapeutic treatments for FRDA.

项目概要/摘要 弗里德赖希共济失调 (FRDA) 是由位于第一个内含子的三核苷酸重复序列的扩增引起的 Frataxin (FXN) 基因，导致 FXN 基因转录减少。 Frataxin 减少的程度取决于 关于 GAA 重复长度。 GAA 重复在 FRDA 患者中继续扩大，加重症状并导致 有助于疾病进展。重复扩展的机制尚不清楚。这是 提出改变 DNA 复制程序和复制叉在二级重复结构处停滞 可能导致 DNA 聚合酶滑动和重复扩增。但目前尚不清楚是否属于二次 重复结构是在患者的内源性 FXN 基因位点体内形成的。此外还未知 改变的复制程序是否在 FRDA 细胞的重复扩增中发挥作用。使用独特的 方法，通过多色免疫荧光监测单个 DNA 分子中的 DNA 复制 显微镜（SMARD），我们的初步结果显示 FRDA 细胞中 DNA 复制过程不准确。 该提案的目标是确定扩展 GAA 处复制叉停滞的原因 重复序列并释放复制叉块以稳定 FRDA 细胞中的重复序列。这些目标将 解决导致 FRDA 重复扩增的分子机制的几个基本方面 患者。能否在心肌细胞等分化细胞中检测到复制，如果是，是否是复制 程序发生改变，FRDA 细胞内源 FXN 位点的复制程序能否得到纠正？ 复制叉是否在内源性疾病基因座的拟议二级重复结构处停滞？ 体内FRDA细胞？小分子可以释放停滞的复制叉吗？为了回答这些 为了解决这些问题，我们将使用新建立的 FRDA 诱导多能干细胞 (iPSC)，其中包含 扩大 GAA 重复序列。 FRDA iPSC可分化为神经元细胞和心肌细胞；这使得 这些细胞是研究 FXN 重复不稳定和减少 FXN 转录的原因的完美工具 未分化和分化的 FRDA 细胞。我们将使用 SMARD 来确定 DNA 复制情况 FRDA 细胞中的内源性疾病位点。另外，我们会测试小分子是否可以释放 复制叉停滞并稳定 FRDA 细胞中的 GAA 重复序列。了解重复扩展是如何发生的 在 FRDA 细胞的天然染色体环境中，是开发有效的 FRDA 的治疗方法。