Non-Canonical Pathways for Electrogene Transfer

电基因转移的非规范途径

• 批准号: 10246268
• 负责人: FAN YUAN
• 金额: $ 31.23万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-20 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10246268
• 关键词: 3-Dimensional; Autophagocytosis; Biological; Buffers; Cell Nucleus; Cell Survival; Cell Therapy; Cells; Chemicals; Clinical Trials; Cytoplasm; DNA; Data; Development; Diffusion; Disease; Electrophoresis; Endocytosis; Extracellular Space; Gel; Gene Delivery; Gene Expression; Gene Transfer; Genes; Goals; Hydrogels; Immune response; In Vitro; Infection; Innate Immune Response; Intracellular Transport; Investigation; Literature; Mediating; Molecular; Nuclear; Optics; Outcome; Pathway interactions; Pharmaceutical Preparations; Pharmacology; Physiologic pulse; Protocols documentation; Research; Resolution; Solid Neoplasm; Techniques; Technology; Testing; Vaccination; Vesicle; Viral Vector; Virus Diseases; base; cell type; clinical application; cytotoxicity; disorder prevention; epigenome editing; experimental study; extracellular; gene therapy; genome editing; improved; in vivo; non-viral gene delivery; novel; novel strategies; plasmid DNA; three-dimensional modeling; trafficking; transgene expression; two-dimensional; uptake; viral gene delivery

Project Summary The long-term goal of the project is to develop a general strategy for improving clinical applications of electrotransfection (ET). The technology has been widely used for gene delivery in different applications, such as genome and epigenome editing, cell and gene therapies, and vaccination for prevention of diseases. However, the technique is currently limited by its low efficiency. Only a tiny fraction of plasmid DNA (pDNA) molecules in extracellular space can be delivered into the nucleus of cells for target gene expression. As a result, ET requires to use buffers with high pDNA concentration and electric pulses with high energy, which can cause cytotoxicity and induce undesired immune responses in cells. To improve the efficiency, the overall objective of the proposed study is to understand molecular mechanisms of pDNA transport in cells. Understanding the mechanisms is critical for development of a general strategy for improving the efficiency of ET, in which intracellular pathways will be manipulated to enhance pDNA transport to the nucleus, and reduce its degradation in the cytoplasm. The central hypothesis in the study is that intracellular transport of electrotransfected pDNA is mediated by vesicles in noncanonical pathways that overlap with those for endocytosis and autophagy. To test the hypothesis, the study will investigate specific pathways involved in cellular uptake and intracellular transport of electrotransfected pDNA (Aim 1). The investigation will be based on quantitative analysis of spatial and temporal distributions of pDNA in cells and its associations with endocytic and autophagic markers. Meanwhile, components in intracellular pathways will be manipulated to identify those that can be used to enhance ET efficiency and cell viability (Aim 2). The manipulation will include treatment of cells with different electric pulses and pharmacological agents, or changing expression levels of specific genes in cells prior to or post ET. The investigations in Aims 1 and 2 will use cells from two dimensional (2D) culture. To understand how ET in 2D differ from that in 3D microenvironment, the proposed study will investigate mechanisms of ET in 3D cell constructs (Aim 3). Results from the mechanistic study will be used, as a proof of principle, to enhance electrogene transfer in solid tumors in vivo (Aim 3). Taken together, the proposed study will reveal new mechanisms of ET in both 2D and 3D models, and develop a more general strategy for improving ET efficiency and cell viability for all cell types. The increase in efficiency will also decrease the amount of pDNA required for ET, thereby reducing undesired innate immune responses to ET. Compared to empirical, trial-and-error approaches used currently in the literature, the new strategy will be more efficient, versatile, and practical, which is critical for improving ET in clinical applications.

项目摘要 该项目的长期目标是制定一种一般策略，以改善电转染的临床应用 （ET）。该技术已被广泛用于基因递送的不同应用，例如基因组和表观基因组 用于预防疾病的编辑，细胞和基因疗法以及疫苗接种。但是，该技术目前受到 其低效率。只能将细胞外空间中的一小部分质粒DNA（PDNA）分子传递到 细胞核用于靶基因表达。结果，ET需要使用具有高pDNA浓度和电的缓冲液 具有高能量的脉冲，可能导致细胞毒性并诱导细胞中不希望的免疫反应。改善 效率是拟议研究的总体目标是了解细胞中pDNA转运的分子机制。 了解机制对于制定提高ET效率的一般策略至关重要，其中 细胞内途径将被操纵以增强pDNA转运到细胞核，并减少其降解 细胞质。研究中的中心假设是，电转录pDNA的细胞内转运是由 非规范途径中的囊泡与内吞和自噬的囊泡重叠。为了检验假设，该研究 将研究参与电转染pDNA细胞摄取和细胞内转运的特定途径（AIM 1）。该研究将基于对细胞中pDNA的空间和时间分布的定量分析 与内吞和自噬标记的关联。同时，将操纵细胞内途径中的组件 确定可用于提高ET效率和细胞活力的人（AIM 2）。操纵将包括 用不同的电脉冲和药理学剂处理细胞，或变化特定基因的表达水平 在ET之前或发布之前的细胞中。目标1和2中的研究将使用来自二维（2D）培养的细胞。到 了解2D中的ET与3D微环境中的ET不同，拟议的研究将研究ET的机制 在3D细胞构建体中（AIM 3）。机械研究的结果将作为原理证明，以增强电气 体内实体瘤中的转移（AIM 3）。综上所述，拟议的研究将揭示两个2D的ET的新机制 和3D模型，并制定了提高所有细胞类型的ET效率和细胞活力的更一般策略。这 效率的提高还将减少ET所需的pDNA量，从而减少不希望的先天免疫 对ET的响应。与经验，文献中目前使用的经验试验方法相比，新策略将是 更有效，多功能和实用，这对于改善临床应用中的ET至关重要。