HYPERTHERMIA MEDIATED GENE THERAPY APPROACH FOR CANCER

该项目的长期目标是开发一种可诱导的癌症基因治疗方法，其中使用热疗作为触发，同时控制针对肿瘤细胞和肿瘤血管的有效治疗基因的空间和时间表达。如果成功，热诱导基因治疗方法将对癌症基因治疗产生重大影响。原因是目前癌症基因治疗面临两个障碍：有效地将基因治疗载体输送到肿瘤组织和在肿瘤细胞中选择性表达治疗基因。拟议的项目为第二个障碍提供了一个新颖的解决方案。还将试图通过使用编码分泌产物的基因来绕过第一个障碍。这样的系统可以潜在地最大限度地杀灭肿瘤细胞，同时将对正常组织的细胞毒性降至最低。这一提议最重要的新方面有两个：一个强大的可诱导基因表达系统，以及对肿瘤细胞和肿瘤血管的双管齐下的联合攻击。在特定目标1中，利用报告基因绿色荧光蛋白(GFP)和腺病毒和腺相关病毒载体，在体外和体内表征选定启动子的热诱导能力。体外实验将在组织培养的肿瘤细胞中进行，体内实验将在啮齿动物背部皮肤褶皱窗肿瘤模型中进行。这些模型允许在活体内，实时。绿色荧光蛋白表达的非侵入性检测。将检测报告基因在临床可达到的范围内(39-43摄氏度)的依赖于温度的基因表达和报告基因的热诱导动力学(时间过程)。在特定目的2中，将建立新的小鼠和大鼠背部皮肤褶皱窗腔肿瘤模型。将稳定表达GFP基因的大鼠乳腺癌细胞系R3230Ac用于同基因Fischer 344大鼠或免疫缺陷裸鼠的肿瘤形成。希望在绿色荧光蛋白和罗丹明标记的脂质体作为荧光血流示踪剂的帮助下，肿瘤细胞和肿瘤相关血管都可以清晰地显示出来。这样的模型将是描述治疗性基因有效性的强大工具。在具体目标3中，将构建同时针对肿瘤细胞和肿瘤血管的热诱导治疗基因的腺病毒和腺相关病毒载体，并将在组织培养细胞中表征其基因表达效率。在特定目的4中，将在特定目的2中建立的窗口室模型中评价在特定目的3中构建的载体的抗肿瘤效果。将检测抗血管、抗血管生成和抗肿瘤作用。预计通过这一项目，将检验所提出的高温调控基因治疗方法的可行性，并从机制水平上了解其潜在的抗肿瘤作用。

The long term objective of this project is to develop an inducible cancer gene therapy approach in which hyperthermia is used as a trigger to control both spatial and temporal expression of potent therapeutic genes that target tumor cells and tumor vasculature simultaneously. If successful, the heat-induced gene therapy approach will have a significant impact on cancer gene therapy. The reason is that there are currently two hurdles facing cancer gene therapy: efficient delivery of gene therapy vectors to the tumor mass and selective expression of therapeutic genes in tumor cells. The proposed project offers a novel solution to the second hurdle. Attempts will also be made to circumvent the first hurdle by using genes that encode secreted products. Such a system can potentially maximize tumor cell killing while minimizing cytotoxicity to normal tissues. The most important novel aspects of this proposal are twofold: a powerful inducible gene expression system and a combined two-pronged attack on both the tumor cells and tumor vasculature. In Specific Aim 1, the heat inducibility of a selected promoter will be characterized in vitro and in vivo using a reporter gene green fluorescence protein (GFP) with adenovirus and adeno- associated virus vectors. In vitro experiments will be conducted in tissue cultured tumor cells while in vivo experiments will be conducted in rodent dorsal skin fold window chamber tumor models. These models allow in vivo, real time. and non-invasive examination of GFP expression. Temperature-dependent gene expression in the range that is clinically achievable (39-43 degrees C) and heat induction kinetics (time course) of the reporter gene will be examined. In Specific Aim 2, novel mouse and rat dorsal skin fold window chamber tumor models will be established. A rat mammary adenocarcinoma cell line R3230Ac will be stably transduced with a constitutively expressed GFP gene and used to form tumors in syngeneic Fischer 344 rats or immunodeficient nude mice. It is hoped with the help of GFP and rhodamine-Iabeled liposomes as fluorescent blood flow tracers, both the tumor cells and tumor- associated vasculature can be visualized without ambiguity. Such models will be powerful tools to characterize the efficacy of therapeutic genes. In Specific aim 3, adenoviral and adeno- associated viral vectors with heat-inducible therapeutic genes that target both the tumor cells and tumor vasculature simultaneously will be built and their gene expression efficiency will be characterized in tissue cultured cells. In Specific aim 4, the anti-tumor efficacy of the vectors built in Specific Aim 3 will be evaluated in the window chamber models established in Specific Aim 2. Anti-vascular, anti-angiogenic, and anti-tumor effects will all be examined. It is anticipated that through this project, the feasibility of the proposed hyperthermia-regulated gene therapy approach will be examined and its potential anti-tumor effects will be understood at the mechanistic level.