title: In vivo ligand-inducible regulation of gene expression in a gutless adenoviral vector system.
abstract: Transcriptional regulation that is rapid, reversible, and repeatedly inducible would greatly enhance the safety and efficacy of many gene therapy strategies. We developed a chimeric ligand-inducible regulation system based on the human estrogen receptor. This system has two components, the responsive promoter driving expression of the transgene of interest, and the ligand-inducible chimeric transcription factor. The transcription factor is composed of a novel DNA binding domain and a modified estrogen receptor ligand-binding domain. A point mutation in the ligand-binding domain significantly reduces estrogen binding while allowing binding of the estrogen antagonist, tamoxifen. We used a gutless adenoviral vector system and incorporated both components into two separate vectors. A single gutless vector encoding both system components was also generated. The tamoxifen-mediated induciblity of transgene expression of the gutless vector system was compared in vitro and in vivo with the analogous components incorporated into early generation, E1/E2a/E3-deficient adenoviral vectors. In normal mice, both the gutless vector and early generation systems displayed inducibility in the presence of tamoxifen. Importantly, the gutless vector system was inducible to extremely high levels, at least four times over a 2-month period. In contrast, the early generation vector system was inducible only once. Furthermore, the early generation system displayed significant toxicity, as evidenced by extremely high liver enzyme levels, abnormal liver pathology, and rapid loss of vector DNA from the liver, while the gutless vector system displayed minimal toxicity. These data directly demonstrate the improved in vivo function of the tamoxifen-inducible transcriptional regulation system in the context of the gutless adenoviral vectors.
authors.affiliation: Genetic Therapy, Inc. (A Novartis Company), Gaithersburg, MD 20878, USA.
authors.forename: Dennis
authors.initials: D
authors.lastname: Zerby
authors.forename: Kiran
authors.initials: K
authors.lastname: Sakhuja
authors.forename: P Seshidhar
authors.initials: PS
authors.lastname: Reddy
authors.forename: Heather
authors.initials: H
authors.lastname: Zimmerman
authors.forename: Dawn
authors.initials: D
authors.lastname: Kayda
authors.forename: Shanthi
authors.initials: S
authors.lastname: Ganesh
authors.forename: Scott
authors.initials: S
authors.lastname: Pattison
authors.forename: Terrence
authors.initials: T
authors.lastname: Brann
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Kadan
authors.forename: Michael
authors.initials: M
authors.lastname: Kaleko
authors.forename: Sheila
authors.initials: S
authors.lastname: Connelly
mesh_terms: D000256:Adenoviridae / Q000235:genetics*; D000818:Animals; D056486:Chemical and Drug Induced Liver Injury; D003673:Defective Viruses / Q000235:genetics; D043169:Endostatins / Q000096:biosynthesis / Q000235:genetics; D005786:Gene Expression Regulation; D005822:Genetic Vectors / Q000633:toxicity; D006367:HeLa Cells; D006801:Humans; D008024:Ligands; D008099:Liver / Q000378:metabolism / Q000473:pathology; D008107:Liver Diseases / Q000378:metabolism / Q000473:pathology; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D011960:Receptors, Estrogen / Q000235:genetics / Q000378:metabolism; D020845:Selective Estrogen Receptor Modulators / Q000494:pharmacology; D013629:Tamoxifen / Q000494:pharmacology; D014158:Transcription, Genetic
chemical_list: D043169:Endostatins; D008024:Ligands; D011960:Receptors, Estrogen; D020845:Selective Estrogen Receptor Modulators; D013629:Tamoxifen; D008156:Luciferases