Improved co-expression of multiple genes in vectors containing internal ribosome entry sites (IRESes) from human genes.

Incorporation of an internal ribosome entry site (IRES) into the gene therapy vector represents a promising strategy to efficiently co-express several gene products from the same promoter. However, vector systems that utilize the encephalomyocarditis virus IRES express the downstream gene much less efficiently than the upstream gene. In this study, we compared four IRESes isolated from human genes against the EMCV IRES, using beta-galactosidase and chloramphenicol acetyl transferase genes as reporters, to evaluate their potential for providing better expression of the downstream gene. We found that an IRES from the eukaryotic initiation factor 4G gene mediates greater than 100-fold higher expression of the downstream gene compared with the EMCV IRES in four different cell lines tested. Other mammalian IRESes displayed more variable results and smaller enhancement of downstream gene expression in three different cell lines tested. Furthermore, while the efficiency of the IRES from the vascular endothelium growth factor gene was not significantly higher than the EMCV IRES under normoxic conditions, expression was significantly increased under hypoglycemic conditions, suggesting that the VEGF IRES could be exploited in cancer gene therapy to preferentially target expression of therapeutic genes at the relatively hypoglycemic cores of tumors.