Small Molecular Therapeutics for Friedreich's Ataxia

弗里德赖希共济失调的小分子疗法

基本信息

批准号：
7560344
负责人：
JOEL M. GOTTESFELD
金额：
$ 41.4万
依托单位：
SCRIPPS RESEARCH INSTITUTE, THE
依托单位国家：
美国
项目类别：
财政年份：
2006
资助国家：
美国
起止时间：
2006-04-01 至 2011-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7560344
关键词：
Adopted Affect Affinity Alanine Alleles Animal Model Animals Antibodies Binding Biological Availability Blood - brain barrier anatomy Blood donor Blood specimen Cell Line Cell Nucleus Cells Chemistry Cultured Cells DNA DNA Binding DNA Microarray Chip DNA Sequence DNA Structure DNase-I Footprinting Development Drug Kinetics Drug or chemical Tissue Distribution Fluorescent Dyes Friedreich Ataxia Funding Gene Expression Gene Targeting Generations Genes Genetic Transcription Grant Half-Life Hereditary Disease Housekeeping Gene Human Inherited Introns Kinetics Knock-in Mouse Ligands Link Lymphoid Cell Measures Messenger RNA Microarray Analysis Microscopy Mitochondrial Proteins Modeling Molecular Monitor Mus Neurodegenerative Disorders Neurons Nuclear Nylons Patients Pharmaceutical Preparations Property Proteins Pyrroles RNA Repression Research Series Serum Short Tandem Repeat Site Specificity System Therapeutic Time Tissues Toxic effect Transcription Regulatory Protein Triplet Multiple Birth United States National Institutes of Health Western Blotting base cell type dosage established cell line frataxin genome-wide in vivo mRNA Expression nervous system disorder research study small molecule synthetic construct uptake

项目摘要

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.

弗里德里希（Friedreich）的共济失调（FRDA）是一种遗传性神经退行性疾病，原因是核编码不足线粒体蛋白frataxin。目前，FRDA尚无有效的治疗或治疗方法。发现了dmaabnormality 在98％的FRDA患者中，在Frataxin基因的第一个内含子中，GAA三重态的不稳定过度膨胀，该基因是该基因的第一例。采用了干扰基因转录的三重三体结构。在先前的研究中，我们已经成功地开发了合成吡咯 - 硅质聚酰胺以靶向GAA重复DMA。这些分子与高高结合双链GAA DNA 来自FRDA患者的淋巴样细胞中Frataxin基因的亲和力和缓解。我们现在想探索这些分子是否会激活神经元细胞中的Frataxin基因，并在小鼠敲入模型中激活 FRDA，并确定这些分子的药理特性。我们将探索多酰胺组成和功能与一系列新分子。反卷积显微镜将用于监测在培养的细胞系中的荧光染料 - 聚丙烯酰胺偶联物的摄取的亚细胞定位和动力学来自FRDA患者的淋巴细胞。实时PCR将用于确定聚酰胺对Frataxin的影响在人FRDA细胞系，从FRDA供体血液中分离的淋巴样细胞和神经元细胞系中的mRNA表达由扩展的法付蛋白敲入小鼠建立。聚酰胺对细胞Frataxin蛋白的影响将是通过蛋白质印迹确定，多酰胺处理的全基因组效应将通过DNA评估微阵列分析。动物研究将在正常小鼠中进行，以确定生物利用度，组织分布，药代动力学，血清中的化合物的半衰期，毒性和最大耐受剂量。扩展的GAA 等位基因敲入小鼠将用于确定GAA特异性化合物是否激活Frataxin Geneexpression 体内。如果聚酰胺无法越过血脑屏障，则替代化学和输送系统将是调查。该建议旨在开发新药来治疗遗传性神经疾病弗里德里希共济失调（FRDA）。 FRDA是一种遗传疾病，其中受影响的基因（称为Frataxin）的大小扩大了添加简单序列GAA的重复序列。这些重复使基因失活，我们已经发展出很小的通过靶向GAA重复的分子，可以逆转这种灭活。