Investigation of Endocytosis Involved in Electrotransfection

描述（由申请人提供）：该项目的长期目标是开发新的系统/方法来改善电场介导的基因在体内的传递。这种传递技术，也被称为电转染，已用于基因治疗和DNA疫苗接种。该技术面临的一个挑战是低效率的基因转移，特别是在体内应用。为了提高电转染效率（eTE），许多研究致力于优化电场参数（如脉冲形状和场强），以改善细胞膜上的瞬态孔形成（即电穿孔）或质粒DNA （pDNA）通过电泳通过孔的运输。然而，电子商务的改善现在已经达到了一个平台期。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，电转染依赖于外加电场诱导pDNA与质膜之间形成稳定的复合物，并且用内吞作用抑制剂处理细胞可以显著降低eTE。此外，本提案的初步数据表明，敲低参与内吞作用的基因表达可以降低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来进一步改善te。该研究的目的是确定电转染中pDNA细胞内转运的途径。总的假设是，吸附内吞作用是电转染中膜结合pDNA运输的关键途径。这项研究的基本原理是，操纵pDNA运输的新途径可以导致改善eTE的全新策略的发展。这一假设将通过对培养细胞中通过内吞途径影响pDNA运输的机制的系统研究来验证（Specific Aim 1）。通过了解机制，该项目将开发新的策略来改善培养细胞（特异性目标2）和三种体内组织：正常皮下组织，正常肌肉组织和实体肿瘤（特异性目标3）中的eTE。该研究将使用荧光标记标记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干扰RNA （siRNA）选择性阻断，siRNA可以敲低内吞途径中特定基因的表达。本研究将量化电场作用后不同时间点pDNA与膜之间的动态相互作用以及细胞质中pDNA的分布。为了促进体内机制的研究，将开发一个独特的支持平台，允许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大意义和创新性，因为它将有助于更好地了解pDNA转运机制，并开发出改善体内eTE的全新策略，这对电转染的临床应用至关重要。

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.