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Increased in vitro and in vivo transgene expression levels mediated through cis ‐acting elements
Author(s) -
Johansen Jens,
Tornøe Jens,
Møller Arne,
Johansen Teit E.
Publication year - 2003
Publication title -
the journal of gene medicine
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.689
H-Index - 91
eISSN - 1521-2254
pISSN - 1099-498X
DOI - 10.1002/jgm.444
Subject(s) - transgene , biology , reporter gene , enhancer , microbiology and biotechnology , woodchuck hepatitis virus , gene expression , transfection , in vivo , gene , genetics , hepatitis b virus , virus , hepadnaviridae
Background Gene therapy for neurodegenerative diseases depends critically on the vector system to direct sustained and stable expression of the transgene. It is, however, a commonly observed phenomenon that transgene expression from currently available vectors is down‐regulated following ex vivo gene transfer to the central nervous system (CNS). In an attempt to circumvent this problem, we have systematically evaluated the potential of different cis ‐acting elements to increase and stabilize transgene expression in vitro and after grafting of engineered cell lines to the CNS. Methods Plasmid vector constructs incorporating Woodchuck hepatitis post‐transcriptional regulatory element (WPRE), cHS4 insulator elements and/or the translational enhancer element SP163 were produced. Stable, polyclonal cultures of HiB5 cells were generated by transfection with reporter constructs, and in vitro transgene mRNA and protein levels were determined. Finally, HiB5 clones engineered to express the enhanced green fluorescent protein (EGFP) were grafted to the rat striatum and expression levels were evaluated. Results Inserting the WPRE element downstream of the open reading frame (ORF) of a reporter gene and flanking the transcriptional unit with cHS4 insulator elements significantly increased protein and mRNA expression levels. Surprisingly, the SP163 element, previously reported to be a translational enhancer, apparently did not promote any translational enhancing activity. Furthermore, the SP163 element exerted a negative effect on transcription. The ability of cHS4 and WPRE elements to stabilize in vivo transgene expression was demonstrated by transplantation of HiB5 clones containing expression constructs into the rat striatum. Conclusion The data suggest that incorporating cis ‐acting elements in gene therapy vectors may result in improvements to currently available therapeutic vectors. Copyright © 2003 John Wiley & Sons, Ltd.