Electric Field - Forced Gene Transfer In Solid Tumors

描述(由申请人提供)：本申请的总体目标 就是提高实体瘤中的电场强制基因转移。基因治疗 在改善不同疾病的治疗方面具有巨大的潜力，包括 实体瘤，但它受到了基因传递困难的阻碍 特定的细胞。其他调查人员和我们最近的报告显示 证明了通过使用 脉冲电场。然而，现有的优化电力系统的方法 场强迫基因转移，在电脉冲特性方面(例如， 场强和脉冲持续时间)，仍然是经验的和不完整的。这个 这个提议中的基本假设是电场强迫的DNA 细胞附近的运输决定了基因转移量 通过细胞质膜上的暂时性气孔 活体电穿孔。为了验证这一假设，我们将系统地量化 DNA间质转运机制的研究进展 体外(目标1)和体内(目标2)的电脉冲。定量化 将基于我们实验室开发的独特实验方法，它可能 导致识别用于改进的有效脉冲序列 DNA的间隙运输。改善了已确定的交通状况 通过对肿瘤组织进行修饰，电场将进一步增强 结构，使用基质酶、高渗溶液或凋亡剂 (目标3)。这些化学处理瞬间增加了间质间隙。 从而降低对DNA运输的抵抗力。量化结果来自 具体目标1至3中的运输研究将最终用于 提高白细胞介素12的转染率和治疗效果 小鼠移植实体瘤中的基因(目标4)。建议的目标是 研究是为了建立有效的实验方案来改进 质粒DNA的间隙运输，这反过来可能导致增加 在不增加毒性的情况下提高实体瘤的转染率。

Description (provided by applicant): The overall objective of this application is to improve electric field-forced gene transfer in solid tumors. Gene therapy has a great potential to improve treatment of different diseases, including solid tumors, but it has been impeded by the difficulties of gene delivery to specific cells. Recent reports by other investigators and ourselves have demonstrated that plasmid DNA delivery can be enhanced through the use of pulsed electric fields. However, present methods for optimizing electric field-forced gene transfer, in terms of electric pulse characteristics (e.g., field strength and pulse duration), are still empirical and incomplete. The fundamental hypothesis in this proposal is that electric field-forced DNA transport in the vicinity of cells determines the amount of gene transfer through the transient pores in the plasma membrane of cells created during in vivo electroporation. To test this hypothesis, we will systematically quantify the mechanisms of interstitial transport of DNA in response to different electric pulses both ex vivo (Aim 1) and in vivo (Aim 2). The quantification will be based on unique experimental methods developed in our labs, and it may result in an identification of effective pulse sequences for improving interstitial transport of DNA. The improved transport in the identified electric fields will be further increased through modifications in tumor tissue structures, using matrix enzymes, hypertonic solutions, or apoptotic agents (Aim 3). These chemical treatments transiently increase the interstitial space and thus decrease the resistance to DNA transport. Quantitative results from the transport studies in Specific Aims 1 through 3 will finally be used to improve transfection efficiency and therapeutic efficacy of interleukin- 12 gene in solid tumors transplanted in mice (Aim 4). The goal of the proposed study is to establish effective experimental protocols for improving interstitial transport of plasmid DNA which in turn may result in an increase in transfection efficiency in solid tumors without increasing toxicity.