Small Molecular Therapeutics for Friedreich's Ataxia

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 线粒体蛋白共济蛋白。目前还没有有效的治愈或治疗FRDA的方法。发现DMA异常 在98%的FRDA患者中，是共济失调蛋白基因第一内含子中GAA三联体的不稳定过度扩增， 采用干扰基因转录的三链DMA结构。在先前的研究中，我们成功地开发了 合成吡咯-咪唑聚酰胺靶向GAA重复DNA。这些分子结合双链体GAA DNA， 在源自FRDA患者的淋巴样细胞中，FRATAXIN具有亲和力并解除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元细胞中的frataxin基因，并在小鼠基因敲入模型中， FRDA，并确定这些分子的药理学特性。我们将探讨 聚酰胺的组成和功能与一系列新的分子。反卷积显微镜将用于监测 荧光染料-聚酰胺缀合物在培养的细胞系中的亚细胞定位和摄取动力学， FRDA患者的淋巴细胞。实时PCR将用于确定聚酰胺对共济失调蛋白的影响。 人FRDA细胞系、从FRDA供体血液分离的淋巴样细胞和神经元细胞系中的mRNA表达 从扩增的共济失调蛋白基因敲入小鼠建立。聚酰胺对细胞共济失调蛋白的影响将是 通过蛋白质印迹法确定，聚酰胺处理的全基因组效应将通过DNA印迹法评估。 微阵列分析。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， 药物动力学、化合物在血清中的半衰期、毒性和最大耐受剂量。扩展GAA 将使用等位基因敲入小鼠来确定GAA特异性化合物是否激活共济失调蛋白基因表达 in vivo.如果聚酰胺不能穿过血脑屏障，替代化学品和递送系统将被使用。 研究了 这一提议旨在开发治疗遗传性神经系统疾病弗里德赖希共济失调的新药 （FRDA）。FRDA是一种遗传性疾病，其中受影响的基因区域称为frataxin，其大小被 添加简单序列GAA的重复。这些重复序列使基因重复， 通过靶向GAA重复序列逆转这种失活的分子。

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.