Investigation of Endocytosis Involved in Electrotransfection

电转染中涉及的内吞作用的研究

基本信息

批准号：
9066493
负责人：
FAN YUAN
金额：
$ 29.83万
依托单位：
DUKE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-09-03 至 2018-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9066493
关键词：
Binding Cell membrane Cells Chemicals Complex Cultured Cells Cytoplasm Cytoskeleton DNA DNA Binding Data Development Disease Dominant-Negative Mutation Electrophoresis Electroporation Endocytosis Enzymes Gene Delivery Gene Expression Gene Transfer Genes Goals Health Histocompatibility Testing Human In Vitro Intracellular Transport Investigation Label Lead Mediating Membrane Methodology Methods Muscle Outcome Pathway interactions Patients Pharmacologic Substance Physiologic pulse Research Shapes Small Interfering RNA Solid Neoplasm Subcutaneous Tissue System Technology Testing Time Tissues Transmembrane Transport Tumor Tissue Vaccination Vesicle Work base clinical application electric field gene therapy immunogenic improved in vivo inhibitor/antagonist innovation knock-down novel novel strategies plasmid DNA subcutaneous temporal measurement theories treatment effect tumor uptake viral gene delivery

项目摘要

DESCRIPTION (provided by applicant): The long-term goal of this project is to develop novel systems/methodologies for improving electric field- mediated gene delivery in vivo. This delivery technology, also known as electrotransfection, has been used in gene therapy and DNA vaccination. A challenge for the technology is low efficiency in gene transfer, especially in vivo applications. To improve electrotransfection efficiency (eTE), many studies have been devoted to optimization of electric field parameters (e.g., pulse shape and field strength) for improving transient pore creation in cell membrane (i.e., electroporation) or plasmid DNA (pDNA) transport through the pores via electrophoresis. However, the improvement in eTE has now reached a plateau. One of the key barriers to further improvement is the lack of understanding of pDNA transport pathways in cells. Recent studies have shown that electrotransfection depends on formation of stable complexes between pDNA and plasma membrane induced by applied electric field, and that eTE can be significantly reduced by treating cells with inhibitors of endocytosis. Furthermore, the preliminary data in this proposal showed that knocking down expression of genes involved in endocytosis could reduce eTE in both tumor and normal human primary cells. These observations suggest that completely new strategies need to be developed for further improving eTE. The objective of the proposed study is to determine pathways for intracellular transport of pDNA in electrotransfection. The overall hypothesis is that adsorptive endocytosis is a key pathway for transport of membrane-bound pDNA in electrotransfection. The rationale for the study is that manipulation of new pathways for pDNA transport can lead to development of completely new strategies for improving eTE. The hypothesis will be tested through a systematic investigation of mechanisms that can influence pDNA transport via endocytic pathways in cultured cells (Specific Aim 1). By understanding the mechanisms, the project will develop novel strategies for improving eTE in cultured cells (Specific Aim 2) and three tissues in vivo: normal subcutaneous, normal muscle, and solid tumor (Specific Aim 3). The investigation will use fluorescent markers to label pDNA, cell membrane, and intracellular vesicles for co-localization analysis. The endocytic pathways will be selectively blocked by pharmaceutical inhibitors, dominant-negative mutants, or small interfering RNA (siRNA) that can knock down expression of specific genes in endocytic pathways. The study will quantify dynamic interactions between pDNA and membrane as well as pDNA distributions in the cytoplasm at different time points after electric field application. To facilitate in vivo studies f mechanisms, a unique enabling platform will be developed, which allows non-invasive observation of pDNA and cells in subcutaneous and tumor tissues at high spatial and temporal resolutions. This integrated research is significant and innovative because it will lead to better understanding of pDNA transport mechanisms and development of completely new strategies for improving eTE in vivo, which are critical for clinical applications of electrotransfection.

描述（由申请人提供）：该项目的长期目标是开发用于改善体内电场介导的基因输送的新型系统/方法。该递送技术，也称为电转染，已用于基因治疗和DNA疫苗接种。该技术的挑战是基因转移的效率低，尤其是体内应用。为了提高电转染效率（ETE），许多研究致力于优化电场参数（例如脉冲形状和野外强度），以改善细胞膜中的短暂孔隙孔（即电穿孔）或质粒DNA（PDNA）（PDNA）通过孔通过电气传输。但是，ETE的改善现已达到平稳状态。进一步改善的关键障碍之一是缺乏对细胞中pDNA传输途径的了解。最近的研究表明，电转染取决于通过施加的电场诱导的pDNA和质膜之间稳定复合物的形成，并且通过用内吞作用抑制剂治疗细胞可以显着降低ETE。此外，该提案中的初步数据表明，击倒参与内吞作用的基因表达可以减少肿瘤和正常人原代细胞的ETE。这些观察结果表明，需要制定全新的策略以进一步改善ETE。拟议的研究的目的是确定pDNA在电转染中细胞内转运的途径。总体假设是，吸附性内吞作用是膜结合pDNA在电转染中运输的关键途径。该研究的理由是，操纵pDNA运输的新途径可以导致发展全新的ETE策略。该假设将通过系统地研究可以影响培养细胞中内吞途径的机制进行系统研究（特定的目标1）。通过了解机制，该项目将开发用于改善培养细胞中ETE的新型策略（特定目标2）和三个组织：正常皮下，正常肌肉和实体肿瘤（特定的目标3）。该研究将使用荧光标记来标记pDNA，细胞膜和细胞内囊泡进行共定位分析。内吞途径将被药物抑制剂，显性阴性突变体或小型干扰RNA（siRNA）选择性阻止，这些RNA可以击倒内吞途径中特定基因的表达。该研究将在电场应用后的不同时间点量化pDNA和膜之间的动态相互作用以及细胞质中的pDNA分布。为了促进体内研究F机制，将开发一个独特的启示式平台，从而可以在高空间和时间分辨率下对皮下和肿瘤组织中的pDNA和细胞进行非侵入性观察。这项综合研究具有重要意义和创新性，因为它将更好地理解pDNA传输机制，并开发了改善体内ETE的全新策略，这对于电子转染的临床应用至关重要。