Investigation of Endocytosis Involved in Electrotransfection

描述（由申请人提供）：本项目的长期目标是开发用于改善电场介导的体内基因递送的新系统/方法。这种传递技术，也称为电转染，已用于基因治疗和DNA疫苗接种。该技术面临的一个挑战是基因转移效率低，特别是体内应用。为了提高电转染效率（埃特），许多研究致力于优化电场参数（例如，脉冲形状和场强）用于改善细胞膜中的瞬时孔产生（即，电穿孔）或质粒DNA（pDNA）通过电泳运输通过孔。然而，埃特的改善现在已经达到了平台期。进一步改进的关键障碍之一是缺乏对细胞中pDNA转运途径的理解。最近的研究表明，电转染依赖于由外加电场诱导的pDNA和质膜之间的稳定复合物的形成，并且可以通过用内吞作用抑制剂处理细胞来显著降低埃特。此外，该提案中的初步数据表明，敲低参与内吞作用的基因的表达可以降低肿瘤和正常人原代细胞中的埃特。这些观察结果表明，需要开发全新的策略来进一步改善埃特。所提出的研究的目的是确定pDNA在电转染中的细胞内转运途径。总的假设是吸附性内吞作用是电转染中膜结合pDNA转运的关键途径。该研究的基本原理是操纵pDNA转运的新途径可以导致开发用于改善埃特的全新策略。将通过系统研究可影响培养细胞中pDNA通过内吞途径转运的机制来检验该假设（特定目的1）。通过了解这些机制，该项目将开发新的策略，以改善培养细胞（特定目标2）和三种体内组织（正常皮下组织、正常肌肉和实体瘤）中的埃特（特定目标3）。该研究将使用荧光标记物标记pDNA、细胞膜和胞内囊泡用于共定位分析。内吞途径将被药物抑制剂、显性失活突变体或小干扰RNA（siRNA）选择性阻断，所述药物抑制剂、显性失活突变体或小干扰RNA（siRNA）可以敲低内吞途径中特定基因的表达。该研究将量化pDNA和膜之间的动态相互作用以及在电场施加后的不同时间点在细胞质中的pDNA分布。为了促进机制的体内研究，将开发一个独特的使能平台，该平台允许以高空间和时间分辨率非侵入性地观察皮下和肿瘤组织中的pDNA和细胞。这项综合研究是重要的和创新的，因为它将导致更好地理解pDNA转运机制和开发全新的策略，以改善体内埃特，这是电转染的临床应用的关键。

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.