Enhancement of a constitutively active promoter for gene therapy by a positive feed-back transcriptional activator mechanism.
Success of gene replacement therapy depends on long-term, high level expression of the transgene. Gene therapy vectors incorporating a promoter of a constitutively active eukaryotic gene may allow long-term expression in vivo, but the expression level may be insufficient for therapeutic effects. To enhance transcription from eukaryotic promoters, a strategy with dicistronic vectors encoding the therapeutic gene of interest together with a transcription factor that binds and activates the promoter was tested. Expression vectors for the chimeric tet repressor/VP16 transcription factor (tTA) driven by the human beta-actin promoter were constructed, and tandem tet operators were inserted within the promoter. This arrangement significantly enhanced expression of G-CSF in fibroblasts to higher levels than the immediate/early CMV promoter. Stably transfected fibroblast clones produced up to 2.4 microg G-CSF per 10(6) cells x 24 h. After injection of genetically engineered cells into SCID mice, the enhanced beta-actin promoter construct resulted in marked leukocytosis, whereas the unmodified promoter had only a marginal therapeutic effect. Transcription factor-enhanced, feed-back-activated human promoters may thus achieve higher expression levels than viral control elements, and may be advantageous for gene therapy due to high constitutive activity in vivo.
Veelken
H
H
Department of Internal Medicine I (Hematology/Oncology), Freiburg University Medical Center, Hugstetter Strasse 55, D-79106 Freiburg, Germany.
Leutgeb
B
B
Kulmburg
P
P
Fiebig
H H
HH
Mackensen
A
A
Lindemann
A
A
D000199:Actins / Q000235:genetics*; D000818:Animals; D002460:Cell Line; D004251:DNA Transposable Elements; D004926:Escherichia coli / Q000235:genetics; D025461:Feedback, Physiological; D005347:Fibroblasts / Q000378:metabolism; D015316:Genetic Therapy; D005822:Genetic Vectors; D016179:Granulocyte Colony-Stimulating Factor / Q000096:biosynthesis* / Q000235:genetics; D017405:Herpes Simplex Virus Protein Vmw65 / Q000235:genetics*; D006801:Humans; D051379:Mice; D009875:Operator Regions, Genetic; D011401:Promoter Regions, Genetic; D012097:Repressor Proteins / Q000235:genetics; D015533:Transcriptional Activation; D014162:Transfection
D000199:Actins; D004251:DNA Transposable Elements; D017405:Herpes Simplex Virus Protein Vmw65; D012097:Repressor Proteins; C034811:tetracycline resistance-encoding transposon repressor protein; D016179:Granulocyte Colony-Stimulating Factor