Small Molecular Therapeutics for Friedreich's Ataxia

描述（由申请人提供）：弗里德赖希共济失调（FRDA）是一种遗传性神经退行性疾病，由核编码的线粒体蛋白共济失调蛋白缺乏引起。目前还没有有效的治愈或治疗FRDA的方法。98%的FRDA患者中发现的DMA异常是共济失调蛋白基因第一个内含子中GAA三联体的不稳定过度扩张，该三联体采用三重DMA结构，干扰基因转录。在先前的研究中，我们已经成功地开发了合成的吡咯-咪唑聚酰胺靶向GAA重复DMA。这些分子以高亲和力结合双链体GAA DNA，并缓解源自FRDA患者的淋巴细胞中共济失调蛋白基因的抑制。我们现在希望探索这些分子是否会激活神经元细胞中的frataxin基因，以及FRDA的小鼠敲入模型，并确定这些分子的药理学特性。我们将探索聚酰胺组成和功能之间的关系与一系列新的分子。去卷积显微镜将用于监测FRDA患者培养细胞系和淋巴细胞中荧光染料-聚酰胺缀合物的亚细胞定位和摄取动力学。将使用实时PCR来确定聚酰胺对人FRDA细胞系、从FRDA供体血液分离的淋巴样细胞和从扩增的共济失调蛋白基因敲入小鼠建立的神经元细胞系中共济失调蛋白mRNA表达的影响。聚酰胺对细胞共济失调蛋白的影响将通过蛋白质印迹法测定，聚酰胺处理的全基因组影响将通过DNA微阵列分析评估。将在正常小鼠中进行动物研究，以确定生物利用度、组织分布、药代动力学、化合物在血清中的半衰期、毒性和最大耐受剂量。扩增的GAA等位基因敲入小鼠将用于确定GAA特异性化合物是否在体内激活共济失调蛋白基因表达。如果聚酰胺不能穿过血脑屏障，将研究替代化学品和输送系统。 该提案旨在开发治疗遗传性神经系统疾病弗里德赖希共济失调（FRDA）的新药。FRDA是一种遗传性疾病，其中受影响基因的一个区域，称为frataxin，通过添加简单序列GAA的重复来扩大大小。这些重复序列使基因失活，我们已经开发出小分子，通过靶向GAA重复序列来逆转这种失活。

DESCRIPTION (provided by applicant): Friedreich's ataxia (FRDA) is an inherited neurodegenerative disease caused by a deficiency in the nuclear-encoded mitochondrial protein frataxin. At present there is no effective cure or treatment for FRDA. The DMA abnormality found in 98% of FRDA patients is the unstable hyper-expansion of a GAA triplet in the first intron of the frataxin gene, which adopts a triplex DMA structure that interferes with gene transcription. In prior studies, we have successfully developed synthetic pyrrole-imidizole polyamides to target GAA repeat DMA. These molecules bind duplex GAA DNA with high affinity and relieve repression of the frataxin gene in lymphoid cells derived from FRDA patients. We now wish to explore whether these molecules will activate the frataxin gene in neuronal cells, and in a mouse knock-in model for FRDA, and to determine the pharmacological properties of these molecules. We will explore the relationship between polyamide composition and function with a new series of molecules. Deconvolution microscopy will be used to monitor the subcellular localization and kinetics of uptake of fluorescent dye-polyamide conjugates in cultured cell lines and in lymphoid cells from FRDA patients. Real-time PCR will be used to determine the effects of polyamides on frataxin mRNA expression in human FRDA cell lines, lymphoid cells isolated from FRDA donor blood, and in neuronal cell lines established from expanded frataxin knock-in mice. The effects of polyamides on cellular frataxin protein will be determined by western blotting, and the genome-wide effects of polyamide treatment will be assessed by DNA microarray analysis. Animal studies will be performed in normal mice to determine the bioavailability, tissue distribution, pharmacokinetics, half-lives of the compounds in serum, toxicity, and maximum tolerated dosage. Expanded GAA allele knock-in mice will be used to determine whether the GAA-specific compounds activate frataxin gene expression in vivo. If polyamides fail to cross the blood-brain barrier, alternative chemistries and delivery systems will be investigated. This proposal is aimed at the development of new drugs to treat the inherited neurological disease Friedreich's ataxia (FRDA). FRDA is a genetic disease, in which a region of the affected gene, called frataxin, is expanded in size by the addition of repeats of the simple sequence GAA. These repeats inactivate the gene, and we have developed small molecules that reverse this inactivation by targeting the GAA repeats.