Non-viral Delivery of Proteins to Mitochondria

超出提供的空间。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/或出生缺陷有关。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。TAT-融合蛋白跨越细胞膜和线粒体膜，由于MTS被识别和切割，融合蛋白被困在线粒体中，因此被定位。该项目将使用一个多学科团队来测试TAT融合蛋白可以将生物活性蛋白靶向完整动物的线粒体的假设。我们将：1)验证TAT多肽可以在体外运送活性线粒体蛋白的假设。将构建由TAT和小鼠线粒体三功能蛋白(TFP)组成的融合蛋白，并在细胞培养中进行测试。2)挽救TFP缺失的转基因动物的表型。这将检验这样一种假设，即TFP可以在体内被适量地输送到组织中，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域。TAT将被融合到膜间空间蛋白上，以表明转导蛋白的靶向序列在TAT融合蛋白中仍然有效。4)确定新型PTD向线粒体运送蛋白质的有效性。将对其他PTD进行测试，以确定它们穿越线粒体膜的有效性。这项工作的意义在于发现并应用了一种新的治疗线粒体功能缺陷的方法。这项工作的科学重要性是开发一种非病毒平台技术，该技术可用于将基因产品输送到多种细胞类型和组织中的线粒体，这将对多学科的科学家具有价值。表演网站========================================Section End===========================================

EXCEED THE SPACE PROVIDED. 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. PERFORMANCE SITE ========================================Section End===========================================