title: Long-term and tight control of gene expression in mouse skeletal muscle by a new hybrid human transcription factor.
abstract: Diseases requiring frequent and lifelong injections of recombinant proteins would be more efficaciously treated by intramuscular delivery of genes encoding secretable proteins. However, the success of this approach largely depends on our capability to temporally regulate transcription of delivered genes. Therefore, we sought to generate a humanized transcription factor to regulate transgene expression in muscle. A novel 4-hydroxytamoxifen (4-OHT)-dependent transcriptional regulator (called HEA-3) was constructed by fusing in-frame the DNA binding domain of the human hepatocyte nuclear factor-1alpha (HNF1alpha), which is not expressed in muscle cells, a G(521)R mutant of the ligand binding domain of human estrogen receptor-alpha (ERalpha), and the activation domain derived from human nuclear factor-kappaB p65 subunit (NF-kappaB p65). We demonstrate that an artificial promoter containing multimeric HNF1alpha binding sites is silent in muscles and in cell lines that lack endogenous HNF1alpha. HEA-3 stimulated transcription from this target promoter in a stringent 4-OHT-dependent manner. The dynamic range of transgene regulation was high, because of the low basal activity and high inducibility of the system. Ex vivo, HEA-3 increased expression of the transfected reporter gene by more than 1000-fold in a ligand-dependent manner. In vivo, HEA-3 stimulated by more than 100-fold, the expression of secreted alkaline phosphatase after delivery as plasmid DNA into mouse muscles. Moreover, long-term modulation of the expression of intramuscularly delivered mouse erythropoietin was achieved in immunocompetent mice.
authors.affiliation: Department of Molecular and Cellular Biology, Istituto di Ricerche di Biologia Molecolare, I.R.B.M.-P. Angeletti, Pomezia, Roma, Italy.
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Roscilli
authors.forename: Cira Daniela
authors.initials: CD
authors.lastname: Rinaudo
authors.forename: Monica
authors.initials: M
authors.lastname: Cimino
authors.forename: Elisabetta
authors.initials: E
authors.lastname: Sporeno
authors.forename: Stefania
authors.initials: S
authors.lastname: Lamartina
authors.forename: Gennaro
authors.initials: G
authors.lastname: Ciliberto
authors.forename: Carlo
authors.initials: C
authors.lastname: Toniatti
mesh_terms: D000818:Animals; D015153:Blotting, Western; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins; D004305:Dose-Response Relationship, Drug; D018014:Gene Transfer Techniques; D015316:Genetic Therapy / Q000379:methods; D005822:Genetic Vectors; D006367:HeLa Cells; D051537:Hepatocyte Nuclear Factor 1; D051538:Hepatocyte Nuclear Factor 1-alpha; D051539:Hepatocyte Nuclear Factor 1-beta; D006801:Humans; D008024:Ligands; D051379:Mice; D008807:Mice, Inbred BALB C; D008856:Microscopy, Fluorescence; D018482:Muscle, Skeletal / Q000378:metabolism; D016328:NF-kappa B / Q000378:metabolism; D009687:Nuclear Proteins; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013629:Tamoxifen / Q000031:analogs & derivatives* / Q000494:pharmacology; D013997:Time Factors; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D015533:Transcriptional Activation; D014162:Transfection; D019076:Transgenes
chemical_list: D004268:DNA-Binding Proteins; C496487:HNF1A protein, human; C496495:HNF1B protein, human; D051538:Hepatocyte Nuclear Factor 1-alpha; C496488:Hnf1a protein, mouse; C496496:Hnf1b protein, mouse; D008024:Ligands; D016328:NF-kappa B; D009687:Nuclear Proteins; D014157:Transcription Factors; D013629:Tamoxifen; D051537:Hepatocyte Nuclear Factor 1; D051539:Hepatocyte Nuclear Factor 1-beta; C016601:afimoxifene; D004247:DNA