Intermittent, repetitive corticosteroid-induced upregulation of platelet levels after adenovirus-mediated transfer to the liver of a chimeric glucocorticoid-responsive promoter controlling the thrombopoietin cDNA.
For many in vivo gene therapy clinical applications, it is desirable to control the expression of the transferred transgene using pharmacologic agents. To evaluate the feasibility of accomplishing this using corticosteroids, pharmacologic agents widely used in clinical medicine, we constructed replication deficient adenoviral (Ad) vectors containing an expression cassette with a chimeric promoter comprised of five glucocorticoid response elements (GRE) and the chloramphenicol acetyltransferase reporter gene (AdGRE.CAT) or the murine thrombopoietin cDNA (AdGRE.mTPO). In vitro studies showed the vectors functioned as expected, with marked glucocorticoid-induced upregulation of the CAT or mTPO transgenes. To evaluate the inducibility of the GRE promoter in vivo, the AdGRE. CAT vector was administered intravenously to C57B1/6 mice, and CAT activity was quantified in liver before and after intraperitoneal administration of dexamethasone. The GRE promoter activity was dependent on the dexamethasone dose, with a 100-fold increase in CAT expression with 50 microg dexamethasone, similar to the levels observed in vivo with the Rous sarcoma virus long terminal repeat constitutive promoter. After dexamethasone administration, maximum CAT activity was observed at day 2, with a slow decline to baseline levels by 2 weeks. Based on these observations, we hypothesized that a single administration of an Ad vector-mediated transfer of the chimeric GRE inducible promoter driving the mTPO cDNA would enable repetitive administration of corticosteroids to repetitively upregulate platelet levels for 1 to 2 weeks. The data show that this occurs, with dexamethasone administration every 3 weeks associated with 1-week elevations (at each 3-week interval) of serum mTPO levels, megakaryocyte numbers in bone marrow, and platelet levels fourfold to sixfold over baseline. Thus, with the appropriate promoter, it is possible to use a commonly used pharmacologic agent to upregulate the expression of a newly transferred gene on demand.
Division of Pulmonary and Critical Care Medicine, The New York Hospital-Cornell Medical Center, New York, NY, USA.
K
K
Narumi
M
M
Suzuki
W
W
Song
M A
MA
Moore
R G
RG
Crystal
D000260:Adenoviruses, Human / Q000235:genetics*; D000818:Animals; D001792:Blood Platelets / Q000166:cytology*; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D004274:DNA, Recombinant / Q000235:genetics; D003907:Dexamethasone / Q000494:pharmacology; D005260:Female; D005786:Gene Expression Regulation / Q000187:drug effects*; D017930:Genes, Reporter; D005813:Genes, Synthetic; D005822:Genetic Vectors / Q000008:administration & dosage / Q000235:genetics*; D006367:HeLa Cells / Q000378:metabolism; D006410:Hematopoiesis / Q000502:physiology*; D006801:Humans; D007275:Injections, Intravenous; D008099:Liver / Q000378:metabolism*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D010976:Platelet Count; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011965:Receptors, Glucocorticoid / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000502:physiology; D013926:Thrombopoietin / Q000096:biosynthesis / Q000235:genetics* / Q000502:physiology
D018076:DNA, Complementary; D004274:DNA, Recombinant; D011965:Receptors, Glucocorticoid; D011993:Recombinant Fusion Proteins; D003907:Dexamethasone; D013926:Thrombopoietin; D015500:Chloramphenicol O-Acetyltransferase