Comparison of promoter region constructs for in vivo intramuscular expression.
BACKGROUND
High transgene expression is generally expected after gene transfer. However, different level, kinetics and localization of expression might be needed for relevant therapeutic applications. Former studies have compared various promoter regions driving gene expression leading to conflicting results. In the present work, two promoter families have been compared using the efficient in vivo intramuscular electrotransfer technique.


METHODS
Three promoter regions were constructed by associating the strong ubiquitous cytomegalovirus (CMV) enhancer-promoter to its homologous intron A or to a heterologous intron, or to a hybrid intron. Promoter regions derived from the muscle creatine kinase (MCK) promoter were also studied. The expression of the same transgene (SeAP or neurotrophin-3) under control of these different promoters was compared after plasmid electrotransfer in mouse tibialis-cranialis skeletal muscle.


RESULTS
Heterologous intron association to the CMV promoter did not modify gene expression kinetics nor increase gene expression level. Usefulness of intron A or hybrid intron association to the CMV promoter depended on the gene. The various MCK promoters drove efficient gene expression but lower than that obtained with the CMV promoter. Furthermore, peak value was reached earlier with MCK promoter regions (14 days).


CONCLUSIONS
For applications of gene transfer restricted to skeletal muscle, the MCK promoter or a MCK promoter variant would be a promising alternative to the CMV promoter. Indeed, it has been demonstrated that the use of MCK promoter limits humoral and cell-mediated immune responses. Furthermore, the MCK promoter decreases the initial expression peak that may be detrimental, drives a sustained gene expression, and improves gene transfer safety.
INSERM U640, F-75006 Paris, France.
Emmanuelle E
EE
Fabre
Pascal
P
Bigey
Cécile
C
Orsini
Daniel
D
Scherman
D000469:Alkaline Phosphatase; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D052283:Creatine Kinase, MM Form / Q000235:genetics; D003587:Cytomegalovirus / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D005260:Female; D058851:GPI-Linked Proteins; D015870:Gene Expression; D018014:Gene Transfer Techniques; D017930:Genes, Reporter; D006801:Humans; D007527:Isoenzymes / Q000235:genetics; D051379:Mice; D016513:Mice, SCID; D018482:Muscle, Skeletal / Q000378:metabolism*; D041681:NIH 3T3 Cells; D020933:Neurotrophin 3 / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic
D017931:DNA Primers; D058851:GPI-Linked Proteins; D007527:Isoenzymes; D020933:Neurotrophin 3; D052283:Creatine Kinase, MM Form; D000469:Alkaline Phosphatase; C019839:alkaline phosphatase, placental