SYSTEMATIC EVALUATION OF CHIMERIC MARKER GENES ON DICISTRONIC TRANSCRIPTION UNITS FOR REGULATED EXPRESSION OF TRANSGENES IN-VITRO AND IN-VIVO

Plasmid expression vectors combining human cytokine cDNAs and selectab le marker genes on dicistronic transcription units were functionally c haracterized in vitro and in vivo. The internal ribosome entry sequenc e (IRES) of encephalomyocarditis virus mediated cap-independent transl ation of the downstream cistron. After cationic lipofection of cells w ith a dicistronic construct containing the Neo(r) gene downstream of a human interleukin-2 (IL-2) cDNA, all G418-resistant clones secreted h igh amounts of IL-2. Reversal of the order of the cDNAs was associated with less efficient transgene expression and represented no advantage in comparison to separate expression cassettes. To combine direct in vitro selection of expression with in vivo elimination of cytokine-sec reting cells, an improved chimeric cDNA of the Neo(r) and herpes simpl ex virus (HSV) thymidine kinase (TK) genes was constructed and shown t o confer sensitivity to ganciclovir concentrations that can be achieve d in human patients. This chimeric marker was coupled on dicistronic c onstructs with a granulocyte colony-stimulating factor (G-CSF) cDNA as a molecule with easily detectable bioactivity in vivo. Subcutaneous i mplantation of pCMV.GCSF.ires TK/NEO-transfected CMS-5 cells into syng eneic BALB/c mice resulted in excessive leukocytosis and progressively growing tumors. Treatment with ganciclovir led to normalization of le ukocyte counts in all animals, whereas complete regression of tumors w as observed in only 3/5 mice. Hypermethylation of the transfected prom oter was demonstrated in both ganciclovir-resistant tumors. Thus, tran scription units combining selectable markers and genes of interest all ow selection of high producer cells in vitro and efficient elimination of transgene-expressing cells in vivo. However, cells that hypermethy late transfected genes to terminate gene expression in vivo may escape conditional ablation.