title: Sustained, high transgene expression in liver with plasmid vectors using optimized promoter-enhancer combinations.
abstract: 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.
authors: Department of Pharmacy, University of Munich, Germany.
authors: Terese
authors: T
authors: Magnusson
authors: Rudolf
authors: R
authors: Haase
authors: Martin
authors: M
authors: Schleef
authors: Ernst
authors: E
authors: Wagner
authors: Manfred
authors: M
authors: Ogris
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D018899:CpG Islands / Q000235:genetics; D004742:Enhancer Elements, Genetic; D005260:Female; D015870:Gene Expression / Q000235:genetics*; D005786:Gene Expression Regulation; D018014:Gene Transfer Techniques; D015316:Genetic Therapy; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D008099:Liver / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D016513:Mice, SCID; D009928:Organ Specificity / Q000235:genetics; D010957:Plasmids / Q000235:genetics*; D011401:Promoter Regions, Genetic; D019076:Transgenes / Q000235:genetics*