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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：
7169906
负责人：
Ronald Mark Payne
金额：
$ 21.99万
依托单位：
INDIANA UNIV-PURDUE UNIV AT INDIANAPOLIS
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 2007-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7169906
关键词：
Ablation Adverse effects Animals Biochemical Biological Biological Assay Biological Process Brain CKB gene Cell membrane Cells Cessation of life Characteristics Child Chimeric Proteins Cleaved cell Complementary DNA Complex Congenital Abnormality Cultured Cells Data Defect Development Discipline Disease Effectiveness Gene Proteins Gene Transfer Genes Genetic Transcription Genome Goals Green Fluorescent Proteins Growth HIV Heart Hepatocyte In Vitro Laboratories Left Liposome-Mediated Gene Transfer Liver Localized Malate Dehydrogenase Mediating Membrane Methods Mitochondria Mitochondrial DNA Mitochondrial Diseases Mitochondrial Matrix Mitochondrial Proteins Modeling Mus NIH 3T3 Cells Newborn Infant Nuclear Patient currently pregnant Patients Peptide T Peptides Phenotype Process Proteins Rate Reporting Research Personnel Research Proposals Risk Role Scientist Side Skeletal Muscle Sudden infant death syndrome System Techniques Technology Tertiary Protein Structure Testing Tissues Trans-Activators Transfection Transgenic Animals Transgenic Mice Viral Viral Vector Work base cell type cellular transduction dosage fetal gene therapy human disease improved in vivo mitochondrial creatine kinase mitochondrial membrane mortality multidisciplinary new technology novel protein function pup repaired research study targeted delivery

项目摘要

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.

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，并且与重大疾病相关。死亡率和/或出生缺陷。克服病毒载体将基因产物传递给人类的局限性在细胞内的线粒体中，我们将使用跨细胞膜的蛋白质转导域（PTD）。我们最近发现，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输送到线粒体。我们进一步开发了方法通过在线粒体中包含线粒体靶向序列（MTS）将这些蛋白质定位到线粒体融合蛋白构建体。 TAT 融合蛋白穿过细胞膜和线粒体膜，局部化，因为 MTS 被识别并裂解，使融合蛋白被困在线粒体。该项目将利用多学科团队来检验 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 肽可以在体外传递活性线粒体蛋白。融合蛋白由以下组成 TAT 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) 拯救转基因动物的TFP 损失表型。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 !假设 TAT 肽可以将蛋白质递送到线粒体的不同区室。 TAT 将 !与膜间空间蛋白融合以显示转导的靶向序列 TAT 融合蛋白中的蛋白质仍然有效。 4) 确定新型 PTD 的有效性将蛋白质输送到线粒体。其他 PTD 将进行测试以确定其穿越时的有效性线粒体膜。这项工作的意义在于发现并应用了一种新颖的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，这对于多个学科的科学家来说都是有价值的。