Sustained, high transgene expression in liver with plasmid vectors using optimized promoter-enhancer combinations.

BACKGROUND: Plasmid-based gene therapy approaches often lack long-term transgene expression in vivo as a result of silencing or loss of the vector. One way to overcome these limitations is to combine nonsilenced promoters with strong enhancers.
METHODS: In the present study, we combine murine or human cytomegalovirus (CMV)-derived enhancer elements with the human elongation factor 1α (EF1α) promoter in a plasmid backbone devoid of potentially immunostimulating cytosine-guanine repeat sequences. Luciferase transgene activity was monitored in mouse liver after hydrodynamic plasmid delivery.
RESULTS: Luciferase activity of a CMV-promoter driven plasmid rapidly declined within days, whereas the activity of the EF1α driven plasmid remained high for 2 weeks (murine enhancer) and detectable for > 80 days (human enhancer). Expression levels clearly correlated with higher plasmid copy number found in the liver at 2 months after gene delivery. Furthermore, we developed a novel synthetic CMV-EF1α hybrid promoter (SCEP) combining the high activity of CMV and sustained activity of EF1α promoter. The SCEP led to a constitutive three-fold increase in expression levels compared to the EF1α promoter in vivo.
CONCLUSIONS: This novel combination of enhancer and promoter element with optimized plasmid backbones will pave the way for more efficient nonviral approaches in gene therapy.