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J Gene Med. 2011 Jul;13(7-8):382-91. doi: 10.1002/jgm.1585.

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

Author information

1
Department of Pharmacy, University of Munich, Germany.

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.

PMID:
21721074
DOI:
10.1002/jgm.1585
[Indexed for MEDLINE]

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