Non-viral Delivery of Proteins to Mitochondria

蛋白质非病毒递送至线粒体

• 批准号: 6734503
• 负责人: Ronald Mark Payne
• 金额: $ 26.16万
• 依托单位: WAKE FOREST UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-12-01 至 2007-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6734503
• 关键词: SDS polyacrylamide gel electrophoresis; electron microscopy; genetically modified animals; laboratory mouse; mitochondria; mitochondrial disease /disorder; mitochondrial membrane; peptides; positron emission tomography; protein protein interaction; protein transport; technology /technique development; tissue /cell culture; virus protein; western blottings

DESCRIPTION (provided by applicant): 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.

描述（由申请人提供）：该项目的目的是开发新技术，以靶向蛋白质向线粒体递送。线粒体功能的缺陷在人类疾病中很常见，并且与死亡率和/或出生缺陷有关。为了克服将基因产物传递到细胞内部线粒体的病毒载体的局限性，我们将使用跨细胞膜的蛋白质转导域（PTD）。我们最近表明，来自人类免疫缺陷病毒的转录（TAT）肽的反式激活剂可以将蛋白质传递给线粒体。我们进一步开发了通过在融合蛋白构建体中包括线粒体靶向序列（MTS）来将这些蛋白质定位到线粒体的方法。 TAT融合蛋白跨越细胞和线粒体膜，并且由于MTS被识别和裂解而被定位，使融合蛋白被困在线粒体中。该项目将使用一个多学科团队来检验以下假设：TAT融合蛋白可以将生物活性蛋白靶向完整动物的线粒体。我们将：1）检验以下假设：TAT肽可以在体外递送活性线粒体蛋白。将在细胞培养物中构建和测试，该融合蛋白由TAT和小鼠线粒体三功能蛋白（TFP）组成。 2）营救动物转基因的表型，以损失TFP。这将检验以下假设：TFP可以足够恢复生物学功能的体内递送到组织中。 3）检验了TAT肽可以将蛋白质输送到线粒体中不同隔室的假设。 TAT将融合到膜间空间蛋白上，以表明转导蛋白的靶向序列在TAT融合蛋白中仍然可操作。 4）确定新型PTD对将蛋白质输送到线粒体的有效性。将测试其他PTD，以确定其在穿越线粒体膜时的有效性。这项工作的重要性是针对线粒体功能缺陷的患者发现和应用新的疗法。这项工作的科学重要性是开发了一种非病毒平台技术，该技术可用于在多种细胞类型和组织中向线粒体传递基因产品，这对多个学科的科学家具有价值。