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

项目总结/摘要 弗里德赖希共济失调（FRDA）是由位于第一内含子的三核苷酸重复序列的扩增引起的。 Frataxin（FXN）基因，其导致FXN基因转录减少。Frataxin减少的程度取决于 GAA重复序列长度GAA重复序列在FRDA患者中继续扩大，加重症状， 促进疾病进展。重复扩增的机制尚不清楚。是 提出改变的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。然而，目前尚不清楚， 在患者体内内源性FXN基因座处形成重复结构。此外，未知 改变的复制程序是否在FRDA细胞中的重复扩增中起作用。使用唯一 一种通过多色免疫荧光监测单个DNA分子中DNA复制的方法 通过使用显微镜（SMARD），我们的初步结果显示FRDA细胞中DNA复制过程的不准确性。 本建议书的目标是确定复制叉在扩展GAA上停止的原因 重复并释放复制叉阻断以稳定FRDA细胞中的重复。这些目标将 解决几个基本方面的分子机制，造成重复扩增FRDA 患者在分化的细胞如心肌细胞中是否可以检测到复制，如果可以， 在FRDA细胞中，复制程序是否可以在内源性FXN基因座处得到纠正？ 复制叉是否在内源性疾病位点的二级重复结构处停滞， 体内FRDA细胞？小分子能释放停滞的复制叉吗？为了回答这些 我们将使用新建立的FRDA诱导多能干细胞（iPSC）， 扩增GAA重复序列。FRDA iPSC可以分化为神经元细胞和心肌细胞;这使得 这些细胞是研究重复不稳定性和减少FXN转录的原因的完美工具， 未分化和分化的FRDA细胞。我们将采用SMARD来确定DNA复制在 FRDA细胞中的内源性疾病基因座。此外，我们还将测试小分子是否能释放出 复制叉停止并稳定FRDA细胞中的GAA重复序列。了解重复扩展是如何发生的 在FRDA细胞中的天然染色体背景下是开发有效的 FRDA的治疗方法。

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.