The overall goal of this research proposal is to develop a widely applicable method for noninvasive imaging of gene expression in target tissue transduced by a multiple gene delivery vector. The applicants have previously demonstrated that HSV1-tk gene expression can be measured and imaged with currently available clinical equipment using radiolabeled substrates that are selectively metabolized by HSV1-tk and accumulate in transduced tissue. The current proposal seeks to demonstrate that imaging gene expression can be extended to a broad range of genes that are of therapeutic interest. The approach will use multiple gene delivery vectors that include HSV1-tk as a """"""""marker gene"""""""" and a different """"""""therapeutic gene"""""""" in the same expression cassette. The focus of this proposal is to demonstrate that proportional and consistent co-expression of both """"""""therapeutic"""""""" and """"""""marker"""""""" genes can be achieved in target tissue (over a wide range of gene expression) following transduction with a multiple gene retroviral vector. The applicants propose to test several existing multiple gene delivery vector constructs (multiple gene expression cassettes) for their ability to provide sufficient and proportional levels of co-expression of two or more genes in transduced tissue. The research will be directed at evaluating different multiple gene delivery vector constructs that include HSV1-tk (as a marker gene) and three genes of therapeutic interest [double mutant dihydrofolate reductase (DHFR), mutant low affinity nerve growth factor receptor (NTP), and granulocyte-macrophage colony stimulating factor (GM-CSF)] that have different sites of gene product activity (intracellular, cell membrane, and extracellular). By including activator-repressor elements in the bicistronic vectors, the applicants will also test the applicability of noninvasive imaging of HSV1-tk expression to monitor changes in gene expression induced by exogenous modulatory stimuli. These studies will be performed first in tissue culture and then in animals using clinical imaging techniques.
