Non-Canonical Pathways for Electrogene Transfer

项目摘要 该项目的长期目标是制定一个改善电转染临床应用的总体策略 （ET）。该技术已被广泛应用于基因组和表观基因组等不同应用中的基因递送 编辑、细胞和基因治疗以及预防疾病的疫苗接种。然而，该技术目前受到以下限制： 其低效率。细胞外空间中只有一小部分质粒DNA（pDNA）分子可以被递送到细胞内。 用于靶基因表达的细胞核。因此，ET需要使用具有高pDNA浓度和电活性的缓冲液， 具有高能量的脉冲，其可引起细胞毒性并在细胞中诱导不期望的免疫应答。提高 为了提高效率，所提出的研究的总体目标是了解细胞中pDNA转运的分子机制。 了解这些机制对于制定提高ET效率的总体战略至关重要，其中 将操纵细胞内途径以增强pDNA向细胞核的转运，并减少其在细胞内的降解。 细胞质该研究的中心假设是电转染的pDNA的细胞内转运是由 囊泡在非经典途径，与那些重叠的内吞和自噬。为了验证这一假设， 将研究涉及电转染pDNA的细胞摄取和细胞内转运的特定途径（Aim 1）。该研究将基于定量分析pDNA在细胞中的空间和时间分布及其 与内吞和自噬标记物的关联。与此同时，细胞内通路中的成分将被操纵， 以鉴定可用于增强ET效率和细胞活力的那些（目的2）。操纵将包括 用不同的电脉冲和药物治疗细胞，或改变特定基因的表达水平 在ET之前或之后的细胞中。目的1和2中的研究将使用来自二维（2D）培养的细胞。到 了解ET在2D与3D微环境中的不同，拟议的研究将探讨ET的机制 在3D细胞构建中（目标3）。从机制研究的结果将被用来，作为一个原则的证明，以提高电基因 体内实体瘤转移（目的3）。综上所述，拟议的研究将揭示ET在2D和2D中的新机制。 和3D模型，并开发一种更通用的策略，用于提高所有细胞类型的ET效率和细胞活力。的 效率的提高也将减少ET所需的pDNA的量，从而减少不希望的先天免疫 对ET的回应与目前文献中使用的经验性试错方法相比，新策略将 更高效、通用和实用，这对于在临床应用中改进ET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.