Non-viral Delivery of Proteins to Mitochondria

蛋白质非病毒递送至线粒体

基本信息

批准号：
6829679
负责人：
Ronald Mark Payne
金额：
$ 13.1万
依托单位：
WAKE FOREST UNIVERSITY HEALTH SCIENCES
依托单位国家：
美国
项目类别：
财政年份：
2003
资助国家：
美国
起止时间：
2003-12-01 至 2005-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/6829679
关键词：
Non viral Delivery Proteins Mitochondria

项目摘要

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===========================================

超出所提供的空间。该项目的目标是开发将蛋白质靶向递送至线粒体的新技术。线粒体功能缺陷在人类疾病中很常见，并且与显着的死亡率和/或出生缺陷相关。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。 TAT 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。该项目将利用多学科团队来检验 TAT 融合蛋白可以将生物活性蛋白靶向完整动物线粒体的假设。我们将： 1) 测试 TAT 肽可以在体外传递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，并在细胞培养物中进行测试。 2) 拯救转基因动物的 TFP 损失表型。这将检验以下假设：TFP 可以以足够的量递送至体内组织以恢复生物功能。 3) 测试 TAT 肽可以将蛋白质递送至线粒体不同区室的假设。 TAT将与膜间间隙蛋白融合以显示转导蛋白的靶向序列在TAT融合蛋白中保持有效。 4) 确定新型 PTD 将蛋白质递送至线粒体的有效性。其他 PTD 将进行测试以确定其穿过线粒体膜的有效性。这项工作的意义在于发现并应用了一种治疗线粒体功能缺陷患者的新疗法。这项工作的科学重要性在于开发一种非病毒平台技术，该技术可用于将基因产物传递到多种细胞类型和组织中的线粒体，这对多个学科的科学家都具有价值。表演网站==========================================部分结束====== =======================================