Non-viral Delivery of Proteins to Mitochondria

该项目的目标是开发新技术，将蛋白质靶向递送到线粒体。 线粒体功能缺陷在人类疾病中很常见，并且与显著的 死亡率和/或出生缺陷。为了克服病毒载体用于将基因产物递送至 在细胞内的线粒体中，我们将使用跨细胞膜的蛋白转导结构域（PTD）。 我们最近已经表明，来自人类的转录反式激活因子（达特）肽， 免疫缺陷病毒可以将蛋白质递送到线粒体。我们进一步开发了方法， 将这些蛋白定位于线粒体，通过在线粒体中包括线粒体靶向序列（MTS）， 融合蛋白构建体。TAT融合蛋白穿过细胞膜和线粒体膜， 因为MTS被识别并切割，留下融合蛋白被捕获在 线粒体该项目将使用一个多学科的团队来测试TAT融合蛋白的假设， 可以将生物活性蛋白质靶向完整动物体内的线粒体。我们将：1）测试假设 达特肽可以在体外递送活性线粒体蛋白。一种融合蛋白，其由以下组成： 达特和小鼠线粒体三功能蛋白（TFP）将在细胞中构建和测试。 文化2)挽救转基因动物TFP缺失的表型。这将检验假设 TFP可以以足够的量递送到体内组织以恢复生物功能。3)测试 !达特肽可以将蛋白质递送到线粒体中的不同隔室的假设。达特将 !与膜间空间蛋白融合，以显示转导的靶向序列 蛋白在达特融合蛋白中保持有效。4)确定新型PTD的有效性， 将蛋白质输送到线粒体。将测试其他PTD，以确定其在过境时的有效性 线粒体膜这项工作的意义在于发现和应用了一种新颖的 治疗线粒体功能缺陷的患者。这项工作的科学重要性在于 开发可用于将基因产物递送至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.