Investigation of Endocytosis Involved in Electrotransfection

描述(申请人提供)：该项目的长期目标是开发新的系统/方法来改进电场介导的体内基因传递。这种递送技术，也被称为电转染，已被用于基因治疗和DNA疫苗接种。这项技术面临的一个挑战是基因转移效率低，特别是在体内应用。为了提高电转染效率，许多研究致力于优化电场参数(如脉冲形状和电场强度)，以改善细胞膜上的瞬时孔形成(即电穿孔)或通过电泳法将质粒DNA(PDNA)运输到孔中。然而，ETE的改善现在已经进入平台期。进一步改进的关键障碍之一是缺乏对细胞内PDNA运输途径的了解。最近的研究表明，电转染依赖于外加电场诱导的pDNA与质膜之间形成稳定的络合物，而用内吞抑制剂处理细胞可以显著减少ETE。此外，这项提案中的初步数据表明，下调参与内吞作用的基因的表达可以减少肿瘤和正常人类原代细胞中的ETE。这些观察结果表明，需要开发全新的策略来进一步改进ETE。这项研究的目的是确定电转染中pDNA在细胞内的转运途径。总的假设是，吸附内吞作用是电转染中膜结合的pDNA转运的关键途径。这项研究的基本原理是，操纵PDNA运输的新途径可以导致开发出改善ETE的全新策略。这一假说将通过对可通过培养细胞的内吞途径影响PDNA运输的机制的系统研究来检验(具体目标1)。通过对机制的了解，该项目将开发新的策略来改善培养细胞(特定目标2)和体内三种组织：正常皮下组织、正常肌肉和实体肿瘤(特定目标3)的ETE。这项研究将使用荧光标记来标记PDNA、细胞膜和细胞内小泡，以进行共定位分析。内吞途径将被药物抑制剂、显性负性突变体或小干扰RNA(SiRNA)选择性地阻断，这些小干扰RNA可以抑制内吞途径中特定基因的表达。本研究将对电场作用后不同时间点PDNA与细胞膜的动态相互作用以及PDNA在细胞质中的分布进行定量研究。为了促进体内机制的研究，将开发一个独特的使能平台，允许以高空间和时间分辨率对皮下和肿瘤组织中的PDNA和细胞进行非侵入性观察。这一综合研究具有重要的意义和创新性，因为它将有助于更好地了解pDNA的转运机制，并开发出全新的策略来改善体内的ETE，这对电转基因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.