Non-viral Delivery of Proteins to Mitochondria

该项目的目标是开发将蛋白质定向输送到线粒体的新技术。 线粒体功能缺陷在人类疾病中很常见，并与显著的 死亡率和/或出生缺陷。为了克服病毒载体在将基因产品运送到 对于细胞内的线粒体，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，来自人类的转录反式激活因子(TAT)肽 免疫缺陷病毒可以将蛋白质运送到线粒体。我们已经进一步开发了方法来 通过在线粒体中包括线粒体靶向序列(MTS)来定位这些蛋白质 融合蛋白构建。TAT-融合蛋白跨越细胞膜和线粒体膜，是 定位是因为MTS被识别和切割，而融合蛋白被困在 线粒体。该项目将使用一个多学科团队来检验TAT-融合蛋白的假设 可以将生物活性蛋白靶向完整动物的线粒体。我们将：1)检验假设 TAT多肽可以在体外传递一种活性线粒体蛋白。一种融合蛋白，由以下成分组成 TAT和小鼠线粒体三功能蛋白(TFP)将被构建并在细胞内进行测试 文化。2)挽救TFP缺失的转基因动物的表型。这将检验这一假设 TFP可以在体内以足够的量被输送到组织中，以恢复生物功能。3)测试 ！假设TAT多肽可以将蛋白质运送到线粒体的不同区段。Tat Will ！与膜间空间蛋白融合，以表明转导的靶序列 蛋白质在TAT融合蛋白中仍然起作用。4)确定新型PTD的有效性 将蛋白质输送到线粒体。将对其他PTD进行测试，以确定其在过路时的有效性 线粒体膜。这项工作的意义在于一部小说的发现和应用 线粒体功能缺陷患者的治疗。这项工作的科学重要性在于 开发一种可用于将基因产品运送到线粒体的非病毒平台技术 在多种细胞类型和组织中，这将对多个学科的科学家有价值。

The goal of this project is to develop novel technology for targeted delivery of proteins to mitochondria. Defects in mitochondrial function are common in human diseases and are associated with significant mortality and/or birth defects. 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. The TAT-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 project will use a multidisciplinary team to test the hypothesis that TAT-fusion proteins can target biologically active proteins to mitochondria in the intact animal. We will: 1) Test the hypothesis that the TAT peptide can deliver an active mitochondrial protein in vitro. A fusion protein consisting of TAT and the mouse mitochondrial Trifunctional Protein (TFP) will be constructed and tested in cell culture. 2) Rescue the phenotype of an animal transgenic for loss of TFP. This will test the hypothesis that TFP can be delivered to tissues in vivo in adequate amounts to restore biological function. 3) Test !the hypothesis that TAT peptide can deliver proteins to different compartments in mitochondria. TAT will !be fused to an intermembranous space protein to show that the targeting sequence of the transduced protein remains operative in TAT fusion proteins. 4) Determine the effectiveness of novel PTDs for delivering proteins to mitochondria. Other PTDs will be tested to determine their effectiveness at crossing mitochondrial membranes. The significance of this work is the discovery and application of a novel therapy for patients with defects in mitochondrial function. The scientific importance of this work is the development of a non-viral platform technology that can be used to deliver gene products to mitochondria in multiple cell types and tissues that will be of value to scientists in multiple disciplines.