Small Molecular Therapeutics for Friedreich's Ataxia

描述(申请人提供)：Friedreich‘s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Frataxin缺乏引起。目前，FRDA尚无有效的治愈或治疗方法。在98%的FRDA患者中发现的DMA异常是Frataxin基因第一内含子中的GAA三联体不稳定的过度扩张，采用了干扰基因转录的三重DMA结构。在以前的研究中，我们已经成功地合成了针对GAA重复DMA的吡咯-咪唑聚酰胺。这些分子以高亲和力结合双链GAA DNA，并解除FRDA患者来源的淋巴细胞中Frataxin基因的抑制。我们现在希望探索这些分子是否会激活神经细胞中的Frataxin基因，以及在FRDA的小鼠敲入模型中，并确定这些分子的药理学特性。我们将用一系列新的分子来探索聚酰胺组成和功能之间的关系。去卷积显微镜将被用来监测荧光染料-聚酰胺结合物在培养细胞系和FRDA患者的淋巴细胞中的亚细胞定位和摄取动力学。实时荧光定量聚合酶链式反应将被用来确定聚酰胺对人FRDA细胞系、从FRDA供者血中分离的淋巴细胞以及从扩增的Frataxin基因敲除小鼠建立的神经细胞系中Frataxin mRNA表达的影响。多胺对细胞Frataxin蛋白的影响将通过蛋白质印迹来确定，而聚酰胺处理的全基因组影响将通过DNA微阵列分析来评估。将在正常小鼠身上进行动物研究，以确定这些化合物的生物利用度、组织分布、药代动力学、血清半衰期、毒性和最大耐受量。扩大的GAA等位基因敲入小鼠将被用来确定GAA特异性化合物是否在体内激活Frataxin基因的表达。如果聚酰胺不能通过血脑屏障，将研究替代的化学物质和输送系统。 这项建议旨在开发治疗遗传性神经系统疾病弗里德里希共济失调(FRDA)的新药。FRDA是一种遗传性疾病，在这种疾病中，受影响的基因的一个区域，称为Frataxin，通过增加简单序列GAA的重复而扩大大小。这些重复使基因失活，我们已经开发出通过靶向GAA重复来逆转这种失活的小分子。

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.