TAT-Mediated Delivery of Frataxin for Friedreichs Ataxia

描述（由申请人提供）：该转化研究项目的目标是临床前开发弗里德赖希共济失调（FA）的新疗法。FA是最常见的人类共济失调，并且是由Frataxin蛋白的不充分产生引起的，最常见的是由于核FRDA基因中的三联体扩增。基因不能翻译。Frataxin是一种靶向线粒体基质的铁结合蛋白。在缺乏铁的情况下，线粒体中的铁积累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肥厚型心肌病早期死亡的主要临床表现。细胞培养中的实验表明，FRDA基因的替换可以恢复线粒体功能，这表明如果可以产生Frataxin，细胞将被拯救。为了克服病毒载体将基因产物递送到细胞内线粒体的局限性，我们将使用跨细胞膜的蛋白转导结构域（PTD）。我们最近发现，人类免疫缺陷病毒的转录反式激活因子（达特）肽可以将蛋白质传递到线粒体。我们已经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）来将这些蛋白定位于线粒体的方法。我们的初步数据显示，TAT-Frataxin融合蛋白穿过细胞和线粒体膜，并且由于MTS被识别和切割而被定位，从而使融合蛋白被捕获在线粒体中。这个为期两年的项目将：1）使用培养物中的细胞和正常小鼠开发和测试TAT-Frataxin融合蛋白，用于正确定位、加工和组装。将确定Frataxin蛋白的最有效融合构建体（第1年）。2)显示TAT-Frataxin可以拯救来自FRDA患者（第1年）的培养物中的成纤维细胞的表型。3)显示TAT-Frataxin可以挽救心脏和大脑中FRDA基因条件性丢失的转基因动物模型的表型（第2年）。这些老鼠现在在手中。该项目的长期目标是开发一种支持FA治疗开发的新技术。这种候选治疗药物的确定将允许在FA动物模型中对该策略进行临床前评价，并将用于通过使用U 01或其他资助机制（PAR-02-139）建立更大的多学科团队方法来治疗这种疾病。

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).