TAT-Mediated Delivery of Frataxin for Friedreichs Ataxia

TAT 介导的 Frataxin 递送治疗弗里德赖希共济失调

• 批准号: 7093253
• 负责人: Ronald Mark Payne
• 金额: $ 17.52万
• 依托单位: INDIANA UNIV-PURDUE UNIV AT INDIANAPOLIS
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-05-02 至 2008-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7093253
• 关键词: ataxia; birth; genes; mitochondria; proteins

DESCRIPTION (provided by applicant): The goal of this translational research project is the preclinical development of a novel therapy for Friedreich's Ataxia (FA). FA is the most common human ataxia and results from inadequate production of the Frataxin protein most often due to a triplet expansion in the nuclear FRDA gene. The gene cannot be translated. Frataxin is an iron-binding protein targeted to the mitochondrial matrix. In its absence, iron accumulation in mitochondria causes oxidant stress destroying mitochondrial and nuclear function. Progressive ataxia and cardiomyopathy are prominent clinical findings with early death from hypertrophic cardiomyopathy. Experiments in cell culture have shown that replacement of the FRDA gene can restore mitochondrial function indicating that if Frataxin can be produced, the cell will be rescued. To overcome the limitations of viral vectors for delivering gene products to mitochondria inside of cells, we will use protein transduction domains (PTD) that cross cell membranes. We have recently shown that the Transactivator of Transcription (TAT) peptide from the human immunodeficiency virus can deliver proteins to mitochondria. We have further developed methods to localize these proteins to mitochondria by including a mitochondrial targeting sequence (MTS) in the fusion protein construct. Our preliminary data shows that a TAT-Frataxin fusion protein crosses both cell and mitochondrial membranes and is localized because the MTS is recognized and cleaved leaving the fusion protein trapped in the mitochondria. This 2 year project will: 1) Develop and test TAT-Frataxin fusion proteins using both cells in culture, and normal mice, for correct localization, processing, and assembly. The most efficient fusion construct for the Frataxin protein will be determined (year 1). 2) Show that TAT-Frataxin can rescue the phenotype of fibroblast cells in culture from FRDA patients (year 1). 3) Show that TAT-Frataxin can rescue the phenotype of an animal model transgenic for conditional loss of the FRDA gene in heart and brain (year 2). These mice are now in hand. The long term goal of this project is the development of a novel technology supporting therapy development for FA. The identification of this candidate therapeutic will allow preclinical evaluation of the strategy in an animal model of FA and will be used to establish a larger multidisciplinary team approach for treatment of this disease through use of the U01, or other, funding mechanism (PAR-02-139).

描述（由申请人提供）：该翻译研究项目的目标是弗里德里希共济失调（FA）的新疗法的临床前开发。 FA是最常见的人类共济失调，是由于核FRDA基因的三胞胎扩张而导致的Frataxin蛋白产生不足所致。该基因无法翻译。 Frataxin是一种针对线粒体基质的铁结合蛋白。在缺乏线粒体中的铁积累会导致氧化应激破坏线粒体和核功能。进行性共济失调和心肌病是显着的临床发现，肥厚性心肌病早已死亡。细胞培养的实验表明，FRDA基因的替换可以恢复线粒体功能，表明如果可以产生frataxin，则将拯救细胞。为了克服将基因产物传递到细胞内部线粒体的病毒载体的局限性，我们将使用跨细胞膜的蛋白质转导域（PTD）。我们最近表明，来自人类免疫缺陷病毒的转录（TAT）肽的反式激活剂可以将蛋白质传递给线粒体。我们进一步开发了通过在融合蛋白构建体中包括线粒体靶向序列（MTS）来将这些蛋白质定位到线粒体的方法。我们的初步数据表明，TAT-弗拉托蛋白融合蛋白越过细胞和线粒体膜，并且由于MTS被识别并裂解，因此将融合蛋白捕获在线粒体中。这个2年的项目将：1）使用培养物和正常小鼠中的两个细胞来开发和测试TAT-弗拉托蛋白融合蛋白，以正确定位，加工和组装。将确定Frataxin蛋白的最有效融合构建体（第1年）。 2）表明TAT-弗拉托蛋白可以从FRDA患者的培养中挽救成纤维细胞的表型（1年级）。 3）表明TAT-氟二辛可以挽救动物模型转基因的表型，以使心脏和大脑中FRDA基因的有条件丧失（2年级）。这些老鼠现在在手里。该项目的长期目标是开发FA的新技术支持治疗开发。该候选治疗方法的鉴定将允许在FA动物模型中对该策略进行临床前评估，并将通过使用U01或其他资金机制（PAR-02-139）来建立更大的多学科团队方法来治疗该疾病。