Small Molecular Therapeutics for Friedreich's Ataxia

弗里德里希共济失调(FRDA)是一种遗传性神经退行性疾病，由核编码缺陷引起 线粒体蛋白Frataxin。目前，FRDA尚无有效的治愈或治疗方法。发现的DMA值异常 98%的FRDA患者是Frataxin基因第一内含子中GAA三联体的不稳定超扩增，这 采用干扰基因转录的三链DNA结构。在之前的研究中，我们成功地开发出 合成的吡咯-咪唑聚酰胺靶向GAA重复序列DNA，这些分子与双链GAA DNA结合 FRDA患者来源的淋巴细胞中Frataxin基因的亲和力和解除抑制。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，以及在小鼠的敲入模型中 FRDA，并确定这些分子的药理性质。我们将探索两者之间的关系 聚酰胺的组成和功能具有一系列新的分子。反卷积显微镜将用于监测 荧光染料-聚酰胺偶联物在培养细胞中的亚细胞定位和摄取动力学 FRDA患者的淋巴样细胞。实时定量聚合酶链式反应将被用来确定聚酰胺类药物对Frataxin的影响 人FRDA细胞系、FRDA供体血分离的淋巴样细胞和神经细胞系的mRNA表达 由扩大的Frataxin基因敲除小鼠建立。聚酰胺对细胞Frataxin蛋白的影响将是 通过Western blotting确定，聚酰胺处理的全基因组效应将通过DNA进行评估 微阵列分析。动物研究将在正常小鼠身上进行，以确定生物利用度、组织分布、 药物动力学、药物在血清中的半衰期、毒性和最大耐受量。扩展的GAA 等位基因敲入小鼠将被用来确定GAA特异性化合物是否激活Frataxin基因表达 在活体内。如果聚酰胺不能通过血脑屏障，替代的化学物质和输送系统将 调查过了。 这项提议旨在开发治疗遗传性神经性疾病弗里德里希共济失调的新药 (FRDA)。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 添加简单序列GAA的重复。这些重复序列使基因失活，我们开发出了小分子 通过靶向GAA来逆转这种失活的分子会重复。

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 DMAabnormality 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 DMAstructure that interferes with gene transcription. In prior studies, we have successfullydeveloped 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 geneexpression 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.