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，在该方法中，热疗被用作触发器来控制同时靶向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：一是基因治疗载体有效地递送到肿瘤肿块，二是治疗基因在肿瘤细胞中的选择性表达。拟议的项目为第二个障碍提供了一种新颖的解决方案。人们还将尝试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死肿瘤细胞，同时最大限度地减少对正常组织的细胞毒性。这一建议最重要的新颖之处在于两个方面：一个强大的诱导基因表达系统和对肿瘤细胞和肿瘤血管的双管齐下的联合攻击。在特异性目标1中，将使用报告基因绿色荧光蛋白（GFP）与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性。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皮肤褶窗室肿瘤模型中进行。这些模型允许在体内，实时。无创检测GFP表达。在临床可达到的温度范围内（39-43℃）的温度依赖性基因表达和报告基因的热诱导动力学（时间过程）将被检查。在Specific Aim 2中，我们将建立新的小鼠和大鼠背皮肤褶皱窗室肿瘤模型。一种大鼠乳腺腺癌细胞系R3230Ac将稳定地用组成性表达的GFP基因转导，并用于在同基因Fischer 344大鼠或免疫缺陷裸鼠中形成肿瘤。希望在GFP和罗丹明标记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的血管系统都能清晰可见。这些模型将成为表征治疗性基因功效的有力工具。在特异性目标3中，将构建具有热诱导治疗基因的腺病毒和腺相关病毒载体，同时靶向肿瘤细胞和肿瘤血管，并在组织培养细胞中表征其基因表达效率。在Specific aim 4中，将在Specific aim 2中建立的窗室模型中评估在Specific aim 3中构建的载体的抗肿瘤效果。抗血管，抗血管生成和抗肿瘤作用都将被检查。预计通过本项目，将检验所提出的高温调节基因治疗方法的可行性，并在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.