title: Detection of plasmid DNA vectors following gene transfer to the murine airways.
abstract: Non-viral gene therapy is being considered as a treatment for cystic fibrosis. In clinical studies and in studies using the mouse airways as a model, current formulations result in only transient transgene expression. A number of reasons for this have been proposed including the loss of plasmid DNA from cells. The aim of these studies was to investigate why transgene expression from non-viral vectors is transient in the mouse lung. Plasmid DNA encoding the luciferase reporter gene was complexed with the cationic lipid GL67 and delivered to the mouse airways. The persistence of plasmid DNA in the mouse lungs was investigated using quantitative PCR and Southern hybridization. Results showed that intact plasmid DNA persisted in the mouse lung in the absence of any detectable luciferase activity. The de novo methylation of plasmid DNA in vivo was investigated as a potential cause of this transient gene expression but results suggested that plasmid DNA does not become de novo methylated in the mouse lung. Therefore processes other than the loss of plasmid DNA from the lung or the de novo methylation of plasmid DNA vectors must be responsible for the transient transgene expression.
authors.affiliation: GeneMedicine Research Group, Nuffield Department of Clinical Laboratory Sciences, University of Oxford, John Radcliffe Hospital, Oxford, UK.
authors.forename: I A
authors.initials: IA
authors.lastname: Pringle
authors.forename: S
authors.initials: S
authors.lastname: Raman
authors.forename: W W
authors.initials: WW
authors.lastname: Sharp
authors.forename: S H
authors.initials: SH
authors.lastname: Cheng
authors.forename: S C
authors.initials: SC
authors.lastname: Hyde
authors.forename: D R
authors.initials: DR
authors.lastname: Gill
mesh_terms: D000256:Adenoviridae / Q000235:genetics; D000280:Administration, Inhalation; D000818:Animals; D001483:Base Sequence; D015139:Blotting, Southern / Q000379:methods; D001992:Bronchoalveolar Lavage Fluid / Q000737:chemistry; D003550:Cystic Fibrosis / Q000628:therapy; D004247:DNA / Q000008:administration & dosage / Q000032:analysis*; D019175:DNA Methylation; D015870:Gene Expression; D017930:Genes, Reporter; D015316:Genetic Therapy / Q000379:methods*; D008081:Liposomes; D008156:Luciferases / Q000235:genetics; D008168:Lung / Q000737:chemistry* / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D010957:Plasmids; D016133:Polymerase Chain Reaction; D013997:Time Factors; D014162:Transfection / Q000379:methods*; D019076:Transgenes
chemical_list: D008081:Liposomes; D004247:DNA; D008156:Luciferases