Despite the use of multimodality therapy, malignant gliomas are uniformly fatal underscoring the need for a superior therapeutic modality. Suicide gene therapy represents an emerging experimental therapeutic approach. The basic principle being that using an enzyme/prodrug combination one could deliver much higher doses of a therapeutic agent to tumor tissue while avoiding systemic toxicity. We have identified a novel form of the cytosine deaminase (CD) gene that is 100 fold more active (yeast CD) than the commonly used bacterial CD. Preliminary results demonstrate that expression of CD in 9L tumors followed by systemic administration of the prodrug 5FC results in tumor specific production of 5FU, an anti-cancer agent, and tumor cytotoxicity. A major problem with most in vivo gene therapy studies is that it is difficult to monitor efficiency of gene delivery as well as gene function. Since therapeutic outcome is very dependent on the level of gene expression and activity, in this proposal we will develop methodologies that take advantage of the fact that the fluorine group in 5FU can be detected by 19F MRS.
In Specific Aim 1 we will utilize MR to quantitatively measure 5FU production after administration of various doses of prodrug (1A) or enzyme (1C). In addition, changes in diffusion and perfusion will be evaluated as measures of 5FU mediated tumor cytotoxicity (1B). Since 5FU rapidly diffuses out of cells and does not accumulate over time within a CD transduced cell we will construct in Specific Aim 2 a CD-Uracil Phospho Ribosyl Transferase (UPRT) fusion. We hypothesize that in cells expressing this fusion the 5FU produced by CD will be phosphorylated thus trapping it within the cell. This will result in accumulation of a fluorine signal within the cell thus facilitating 19F imaging and therefore imaging of areas of the tumor wherein successful gene delivery has occurred. This construct will then be tested in a tumor therapy experiment and either using stable cell lines (Specific Aim 2) or an adenoviral vector (Specific Aim 3), MRI/S tumor data will be compared with histopathology, animal survival, changes in tumor volumes, tumor growth rates, and cell kill to determine which specific or combined MR-observable parameter(s) can be utilized as surrogate markers for predicting therapeutic outcome using this gene therapy paradigm. Overall, these studies will provide the foundation for evaluating the effectiveness of MRI/S for assessment of brain tumor CD gene therapy. In addition, these results will provide the foundation for using these MR methods for studying CD gene therapy in tumor in the clinic when Phase I/II clinical trials are initiated.
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