Novel gene delivery and expression system

描述（由申请人提供）：要实现非病毒基因治疗作为癌症治疗方式的前景，必须在几个关键步骤中取得突破性技术，其中包括：1)开发一种简化但有效的基因传递系统，以提高基因传递效率；2)有效的基因表达系统，延长治疗性基因表达时间；3)开发肿瘤靶向基因，降低全身毒性；4)开发一种新的策略来克服对所提出的治疗方法的抵抗。本应用程序的目标是通过探索两种创新方法来解决前两个关键问题。为了解决传递问题，申请人将开发对流增强传递(CED)-结合电穿孔传递（EPD）系统进行基因传递。其基本原理是，CED增加了注入的DNA在肿瘤中的分布，而EPD会在细胞膜上产生孔以允许DNA摄取。这种新策略将增加吸收DNA的细胞总数，提高基因表达水平和治疗效果。为了确保拟议的CED适用于大分子质粒DNA输注，申请人通过提出两种改进的策略对传统的CED进行了创新：流体动力学CED和针阵列CED。然后，申请人将开发IFN增强表达系统，其中IFN基因由CMV/IRF7融合启动子驱动。IFN产物通过反馈调控激活IRF7启动子，增强IFN基因表达，延缓IFN基因表达终止。因此，基因表达的持续时间将会延长。申请人将结合新的CED-EPD递送系统和ifn增强表达系统，以提高对肿瘤的治疗效果。我们的假设是肿瘤中IFN的高水平积累会导致有效的肿瘤消退。我们的目标是开发一种简单有效的非病毒基因疗法来治疗肿瘤。我们提出的实验有很高的成功概率，因为我们有一个优秀的模型系统，在非病毒基因治疗领域的经验，以及完成任务所需的所有资源。用于发展这种治疗方法的原发肿瘤模型是经常发生在口腔的鳞状细胞癌。

DESCRIPTION (provided by applicant): To realize the promise of non-viral gene therapy as a treatment modality for cancer, breakthrough technologies in several key steps have to be made, which includes 1) to develop a simplified but effective gene delivery system to increase the gene delivery efficiency; 2) an effective gene expression system to extend the duration of therapeutic gene expression; 3) develop a tumor-targeted gene to minimize the systemic toxicity; and 4) develop a novel strategy to overcome the resistance to the proposed therapeutic approach. The goal of this application aims to addresses the first two critical issues by exploring two innovative approaches. To address the delivery issues, the applicant will develop convection-enhanced delivery (CED)-combined with the electroporation delivery (EPD) system for gene delivery. The rationale is that CED increases the distribution of infused DNA in tumors and EPD will generate pores in cell membrane to allow DNA uptake. This novel strategy will increase the total number of cells to uptake DNA and increase the level of gene expression and therapeutic efficacy. To ensure that the proposed CED works on macromolecule plasmid DNA infusion, the applicant has innovated the conventional CED by proposing two modified strategies: a hydrodynamic CED and a needle array CED. Then, the applicant will develop an IFN-enhanced expression system, in which the IFN gene is driven by a CMV/IRF7 fusion promoter. The IFN product will activate the IRF7 promoter by feedback regulation to enhance the IFN gene expression and delay the termination of IFN gene expression. As a result, the gene expression duration will be extended. The applicant will combine the novel CED-EPD delivery system and the IFN-enhanced expression system to increase the therapeutic efficacy against tumors. Our hypothesis is that a high level accumulation of IFN in tumors will cause effective tumor regression. Our goal is to develop a simplified and effective non-viral gene therapy for treating tumors. The experiments that we propose have a high probability of success because we have an excellent model system in place, experience in the non-viral gene therapy field, and all the resources necessary to complete the task. The primary tumor model used for developing this therapeutic approach is squamous cell carcinoma that often occurs in oral cavity.