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）：该转化研究项目的目标是针对弗里德赖希共济失调（FA）的新型疗法的临床前开发。 FA 是最常见的人类共济失调，通常是由于核 FRDA 基因的三联体扩增导致 Frataxin 蛋白产生不足所致。该基因无法翻译。 Frataxin 是一种靶向线粒体基质的铁结合蛋白。如果缺乏铁，线粒体中铁的积累会导致氧化应激，从而破坏线粒体和核功能。进行性共济失调和心肌病是肥厚性心肌病早期死亡的突出临床表现。细胞培养实验表明，替换 FRDA 基因可以恢复线粒体功能，这表明如果可以产生 Frataxin，细胞将得到拯救。为了克服病毒载体将基因产物传递到细胞内线粒体的局限性，我们将使用跨细胞膜的蛋白质转导域（PTD）。我们最近证明，来自人类免疫缺陷病毒的转录反式激活因子（TAT）肽可以将蛋白质递送至线粒体。我们进一步开发了通过在融合蛋白构建体中包含线粒体靶向序列（MTS）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-Frataxin 融合蛋白穿过细胞膜和线粒体膜并被定位，因为 MTS 被识别并裂解，使融合蛋白被困在线粒体中。这个为期 2 年的项目将： 1) 使用培养细胞和正常小鼠开发和测试 TAT-Frataxin 融合蛋白，以进行正确的定位、处理和组装。 Frataxin 蛋白最有效的融合构建体将被确定（第一年）。 2) 表明 TAT-Frataxin 可以挽救 FRDA 患者培养物中成纤维细胞的表型（第 1 年）。 3) 表明 TAT-Frataxin 可以挽救心脏和大脑中 FRDA 基因条件性缺失的转基因动物模型的表型（第 2 年）。这些老鼠现在已经到手了。该项目的长期目标是开发支持 FA 治疗开发的新技术。这种候选疗法的确定将允许在 FA 动物模型中对该策略进行临床前评估，并将用于建立一个更大的多学科团队方法，通过使用 U01 或其他资助机制 (PAR-02) 来治疗这种疾病。 -139）。