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.
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