Systematic Evaluation of Chimeric Marker Genes on Dicistronic Transcription Units for Regulated Expression of Transgenes In Vitro and In Vivo

Abstract

Plasmid expression vectors combining human cytokine cDNAs and selectable marker genes on dicistronic transcription units were functionally characterized in vitro and in vivo. The internal ribosome entry sequence (IRES) of encephalomyocarditis virus mediated cap-independent translation of the downstream cistron. After cationic lipofection of cells with a dicistronic construct containing the Neo^r gene downstream of a human interleukin-2 (IL-2) cDNA, all G418-resistant clones secreted high 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-secreting cells, an improved chimeric cDNA of the Neo^r and herpes simplex virus (HSV) thymidine kinase (TK) genes was constructed and shown to confer sensitivity to ganciclovir concentrations that can be achieved in human patients. This chimeric marker was coupled on dicistronic constructs with a granulocyte colony-stimulating factor (G-CSF) cDNA as a molecule with easily detectable bioactivity in vivo. Subcutaneous implantation of pCMV.GCSF.ires TK/NEO-transfected CMS-5 cells into syngeneic BALB/c mice resulted in excessive leukocytosis and progressively growing tumors. Treatment with ganciclovir led to normalization of leukocyte counts in all animals, whereas complete regression of tumors was observed in only 3/5 mice. Hypermethylation of the transfected promoter was demonstrated in both ganciclovir-resistant tumors. Thus, transcription units combining selectable markers and genes of interest allow selection of high producer cells in vitro and efficient elimination of transgene-expressing cells in vivo. However, cells that hypermethylate transfected genes to terminate gene expression in vivo may escape conditional ablation.

Overview summary

Expression vectors with dicistronic transcription units composed of cDNAs of a selectable marker gene and of a transgene of interest under control of a single promoter were constructed systematically and tested with regard to mandatory coupling of the expression of the transgene to a selectable phenotype of transfected cells. Insertion of a neomycin phosphotransferase gene whose translation is mediated by a picornaviral internal ribosome entry sequence downstream of an interleukin-2 (IL-2) or granulocyte colony-stimulating factor (G-CSF) cDNA resulted in high-level cytokine secretion in virtually every stably transfected clone under selection with neomycin. An optimized chimeric selectable marker with neomycin phosphotransferase and thymidine kinase activity in analogous constructs permitted additional negative selection of cytokine-expressing murine fibrosarcoma cells in vitro and in vivo. Animal experiments demonstrated that this arrangement leads to elimination of all cells expressing the transfected constructs whereas cells that silence transcription through hypermethylation may escape negative selection in vivo.