803. Functional Analysis of Various Gene Linkers Serving Proportional Coexpression of PET Marker and Therapeutic Genes

Top of pageAbstract Objective: To improve the overall level of gene expression of the second gene of interest in our universal HSV-1 amplicon vectors (Jacobs et al. 2003) using alternative gene linkers (eukaryotic IRES, 2A) and/or mutant HSV-1 thymidine kinase (tk39; Black et al. 1996). Background: The gene expression cassette cdIREStkgfp (Jacobs et al. 2003) was originally engineered to assess for the proportionality of co-expression of the therapeutic E. coli cytosine deaminase gene, cd, and the HSV-1-tkgfp fusion gene (fusion of the herpes simplex virus type 1 thymidine kinase gene serving as PET-marker gene and the gene coding for green fluorescent protein, gfp, as a reporter gene for cell culture; Jacobs et al. 1999). Functional evaluation had shown an attenuation of the IRES-mediated expression of the second fusion gene compared to the expression of the gene at the 5'-primed end of the IRES-element. Methods: First, the ECMV-derived IRES-element was replaced by an eucaryotic IRES-element (Ie; Chappell et al. 2000) as well as by the 2A-element of the food and mouth disease (FMD) virus (deFelipe et al. 1999). Thereafter, human Gli36dEGFR glioma cells (as well as BHK, Vero2-2, U87 cells) were infected with an MOI=1 of HSV-CITG, HSV-CIeTG or HSV-C2ATG, and relative GFP-expression was compared. 24 h after infection, a single cell analysis of relative GFP-expression was performed by means of the MPI-Tool imaging software (Jacobs et al. 2003). Second, the HSV-1-tk gene was replaced by the gene mutant HSV-1-tk39 showing a higher enzymatic efficiency than the wild type HSV-1-tk (Black et al. 1996). Gli36dEGFR cells were infected with an MOI=3 of HSV-CITG or HSV-CITk39G, and an MTT-assay was performed employing different concentrations of ganciclovir (0.01-10 g/ml). Results: In cell culture, the relative level of TKGFP-expression mediated by HSV-CIeTG-and HSV-C2ATG-infection was 65.2+21.9% and 86.6+12.4%, respectively, compared to HSV-CITG infected cells. These data indicate a further attenuation- instead of the expected improvement- of expression of the second gene in all tested cell lines. In contrast, the exchange of wild type HSV-1-tk to its mutant tk39 showed a significant increase in protein activity (LD50 HSV-CITG: 2.7 g/ml GCV; LD50 HSV-CITk39G: 0.023 g/ml GCV). Conclusion: No significant improvement of the expression of the second gene was achieved by replacing the ECMV-derived IRES-element with the alternative linkers, eukaryotic IRES and 2A-FMD, in all cell lines tested. However, an increasing activity of the second gene product with respect to its TK-activity could be achieved by replacing the wild type tk-gene with the tk39 mutant possessing a higher enzymatic efficiency.