Plasmid based gene therapy approaches often lack long term transgene expression in vivo due to silencing or loss of the vector. One way to overcome these limitations is to combine non-silenced promoters with strong viral enhancers. Here we combine cytomegalovirus (CMV) derived enhancer elements with the strong, human elongation factor 1 alpha (EF1a) promoter in a plasmid backbone devoid of potentially immunostimulating CpG sequences. The transgene expression of plasmids containing either the murine or human immediate early enhancer were monitored in vivo. The human CMV enhancer led to an enhanced and prolonged transgene signal compared to the murine enhancer. The elevated expression in the case of the human enhancer correlated with a higher plasmid copy number found in the liver two months after gene delivery. The transgene expression could be even further increased by using a new synthetic promoter, SCEP (shuffled CMV EF1 promoter) instead of the EF1 promoter, in combination with the human CMV enhancer.

Secondly, to reach a tissue specific and high expression in liver carcinoma a plasmid with the AFP promoter was combined with the human CMV enhancer.