title: 5-Azacytidine prevents transgene methylation in vivo.
abstract: Retroviral sequence can silence transgene expression in vitro and in vivo. We report that this effect can be efficiently prevented by in vivo administration of the demethylating agent 5-azacytidine (aza-C). We engineered the U937 human cell line with a retroviral vector consisting of the thymidine kinase suicide gene (tk), which induces sensitivity to ganciclovir (gcv) and through an IRES sequence, the bacterial beta-galactosidase gene (lacZ) as a marker gene. About 90% of the U937 cells expressed the transgene. By injecting the transduced U937 cells in severe combined immunodeficient disease (SCID) mice, we generated a tumor which, during in vivo treatment with aza-C, maintained the high expression of lacZ and tk genes at the baseline values. LacZ-positive cells in the tumour masses after death was weak (1-2%) in the control group, while in mice treated with aza-C it was maintained at 90%. The delay in tumour onset was significantly longer when animals were treated with both aza-C and gcv (P < 0.0001) compared with animals treated with gcv or with aza-C alone. The prevention of silencing phenomena has important implications for gene therapy, because an efficient transduction associated with appropriate drug therapy, might be a powerful strategy for successful application of gene therapy protocols.
authors.affiliation: Department of Clinical and Experimental Medicine, University of Perugia, Italy.
authors.forename: M
authors.initials: M
authors.lastname: Di Ianni
authors.forename: A
authors.initials: A
authors.lastname: Terenzi
authors.forename: K
authors.initials: K
authors.lastname: Perruccio
authors.forename: R
authors.initials: R
authors.lastname: Ciurnelli
authors.forename: F
authors.initials: F
authors.lastname: Lucheroni
authors.forename: R
authors.initials: R
authors.lastname: Benedetti
authors.forename: M F
authors.initials: MF
authors.lastname: Martelli
authors.forename: A
authors.initials: A
authors.lastname: Tabilio
mesh_terms: D000818:Animals; D000998:Antiviral Agents / Q000008:administration & dosage; D001374:Azacitidine / Q000627:therapeutic use*; D019175:DNA Methylation / Q000187:drug effects*; D015774:Ganciclovir / Q000008:administration & dosage; D015870:Gene Expression / Q000187:drug effects; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000008:administration & dosage* / Q000235:genetics; D006801:Humans; D007763:Lac Operon / Q000235:genetics; D051379:Mice; D016513:Mice, SCID; D012190:Retroviridae / Q000235:genetics*; D013937:Thymidine Kinase / Q000235:genetics; D020298:U937 Cells
chemical_list: D000998:Antiviral Agents; D013937:Thymidine Kinase; D001374:Azacitidine; D015774:Ganciclovir