Non-Canonical Pathways for Electrogene Transfer

项目摘要 该项目的长期目标是开发一种通用策略，以改进电转基因的临床应用。 (ET)。这项技术已被广泛用于基因传递的不同应用，如基因组和表观基因组 编辑、细胞和基因治疗，以及预防疾病的疫苗接种。然而，这项技术目前受到以下限制 其效率较低。细胞外空间中只有一小部分质粒DNA(PDNA)分子可以被输送到 细胞核用于靶基因的表达。因此，ET需要使用高pDNA浓度和带电的缓冲液 高能量的脉冲，可引起细胞毒性并在细胞中诱导不希望看到的免疫反应。为了改善 为了提高效率，这项拟议研究的总体目标是了解PDNA在细胞中运输的分子机制。 了解这些机制对于制定提高ET效率的总体战略至关重要，在该战略中 细胞内途径将被操纵，以加强pDNA到细胞核的运输，并减少其在 细胞质。这项研究的中心假设是电转染的pDNA的细胞内转运是由 与内吞和自噬途径重叠的非规范途径中的小泡。为了检验这一假设，这项研究 将研究参与细胞摄取和细胞内转运的电转染pDNA(AIM 1)。这项研究将基于对PDNA在细胞和其上的时空分布的定量分析。 与内吞和自噬标记物的关系。同时，细胞内通路中的成分将被操纵 确定可用于提高ET效率和细胞存活率的药物(目标2)。该操作将包括 用不同的电脉冲和药物处理细胞，或改变特定基因的表达水平 在ET之前或之后的单元格中。AIMS 1和AIMS 2的研究将使用来自二维(2D)培养的细胞。至 了解2D环境中的ET与3D微环境中的ET有何不同，拟议的研究将探讨ET的机制 在3D单元格构造中(目标3)。机理研究的结果将被用来作为加强电基因的原理的证明 实体瘤体内转移(目标3)。综上所述，这项拟议的研究将揭示两种二维ET的新机制 和3D模型，并开发更通用的策略来提高ET效率和所有细胞类型的细胞存活率。这个 效率的提高也将减少ET所需的pDNA数量，从而减少不受欢迎的先天免疫 对ET的响应。与目前文献中使用的经验的、试错的方法相比，新的策略将是 更高效、更通用、更实用，这对提高ET在临床应用中的应用至关重要。

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.