A novel synthetic mammalian promoter derived from an internal ribosome entry site

Introduction of specific mutations into a synthetic internal ribosome entry site (IRES(GTX)) derived from the GTX homeodomain protein revealed additional transcriptional activity. This novel synthetic P(GTX) promoter exhibited consensus core promoter modules such as the initiator (Inr) and the partial downstream promoter elements (DPE) and mediated high-level expression of a variety of transgenes including the human vascular endothelial growth factor 121 (VEGF(121)), the human placental secreted alkaline phosphatase (SEAP), and the Bacillus stearothermophilus-derived secreted alpha-amylase (SAMY) in Chinese hamster ovary cells (CHO-K1) and a variety of other mammalian and human cell lines. The spacing between Inr and DPE modules was found to be critical for promoter performance since introduction of a single nucleotide (resulting in P(GTX2)) doubled the SEAP expression levels in CHO-K1. P(GTX2) reached near 70% of P(SV40)-driven expression levels and outperformed constitutive phosphoglycerate kinase (P(PGK)) and human ubiquitin C (P(hUBC)) promoters in CHO-K1. Also, P(GTX2) was successfully engineered for macrolide-inducible transgene expression. Owing to its size of only 182 bp, P(GTX2) is one of the smallest eukaryotic promoters. Although P(GTX2) was found to be a potent promoter, it retained its IRES(GTX)-specific translation-initiation capacity. Synthetic DNAs, which combine multiple activities in a most compact sequence format may foster advances in therapeutic engineering of mammalian cells.