TAT-Mediated Delivery of Frataxin for Friedreichs Ataxia

描述(由申请者提供)：该转译研究项目的目标是开发一种治疗Friedreich共济失调(FA)的新疗法。FA是人类最常见的共济失调，是由于Frataxin蛋白生产不足造成的，最常见的原因是核FRDA基因的三联体扩张。这种基因不能被翻译。Frataxin是一种针对线粒体基质的铁结合蛋白。在缺乏铁的情况下，铁在线粒体中的积累会导致氧化应激破坏线粒体和核的功能。进行性共济失调和心肌病是肥厚型心肌病早期死亡的显著临床表现。细胞培养实验表明，FRDA基因的替换可以恢复线粒体功能，这表明如果能够产生Frataxin，细胞将被拯救。为了克服病毒载体向细胞内线粒体运送基因产品的局限性，我们将使用跨越细胞膜的蛋白质转导结构域(PTD)。我们最近已经证明，人类免疫缺陷病毒的转录反式激活因子(TAT)肽可以将蛋白质运送到线粒体。我们进一步发展了通过在融合蛋白构建中包括线粒体靶向序列(MTS)来定位这些蛋白质的方法。我们的初步数据显示，TAT-Frataxin融合蛋白跨越细胞膜和线粒体膜并被定位，因为MTS被识别和切割，使融合蛋白被困在线粒体中。这个为期两年的项目将：1)开发和测试TAT-Frataxin融合蛋白，使用培养细胞和正常小鼠，以进行正确的定位、加工和组装。Frataxin蛋白最有效的融合结构将被确定(1年)。2)表明TAT-Frataxin能挽救FRDA患者(1岁)培养的成纤维细胞表型。3)表明TAT-Frataxin可以挽救FRDA基因在心脏和脑中条件性丢失的转基因动物模型的表型(第2年)。这些老鼠现在已经控制住了。该项目的长期目标是开发一种支持FA治疗发展的新技术。这种候选疗法的确定将允许在FA的动物模型中对该策略进行临床前评估，并将被用于通过U01或其他资助机制建立一个更大的多学科团队方法来治疗这种疾病(PAR-02-139)。

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).