title: Optimisation of real-time quantitative RT-PCR for the evaluation of non-viral mediated gene transfer to the airways.
abstract: Naked plasmid DNA and DNA/liposome complexes are currently being considered as gene therapy treatments for cystic fibrosis (CF) pulmonary disease. Current methods of gene delivery to the airways result only in transient correction of the CF ion transport defect, and disease treatment is likely to require repeated administrations of vector. However, it is unclear if repeat administration will be tolerated by CF individuals. Technologies including TaqMan (Applied Biosystems) real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) can be used to determine the efficacy of gene transfer formulations. TaqMan RT-PCR assays were designed and optimised to detect plasmid vector-derived and endogenous gene expression. Subsequently, these assays were used to quantify vector-derived mRNA after delivery of naked DNA and DNA/liposome formulations expressing human and murine cystic fibrosis transmembrane conductance regulator (CFTR) to the mouse airways. Vector-derived mRNA was detected in samples following the delivery of naked DNA or DNA/liposomes to the mouse airways, and no reduction in vector-derived mRNA was observed upon repeat administration, a finding that is consistent with the murine and human CFTR being tolerated by the mouse. Although it remains to be seen if CF patients can tolerate long-term expression of wild-type CFTR, these data demonstrate that TaqMan RT-PCR is an effective tool to accurately quantify transgene expression in the airways.
authors.affiliation: Nuffield Department of Clinical Laboratory Sciences, University of Oxford, John Radcliffe Hospital, Oxford OX3 9DU, UK.
authors.forename: A C
authors.initials: AC
authors.lastname: Rose
authors.forename: C A
authors.initials: CA
authors.lastname: Goddard
authors.forename: W H
authors.initials: WH
authors.lastname: Colledge
authors.forename: S H
authors.initials: SH
authors.lastname: Cheng
authors.forename: D R
authors.initials: DR
authors.lastname: Gill
authors.forename: S C
authors.initials: SC
authors.lastname: Hyde
mesh_terms: D000818:Animals; D003550:Cystic Fibrosis / Q000628:therapy*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics / Q000378:metabolism*; D005260:Female; D015870:Gene Expression; D018014:Gene Transfer Techniques; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors; D006801:Humans; D008081:Liposomes; D051379:Mice; D008807:Mice, Inbred BALB C; D010957:Plasmids; D012333:RNA, Messenger / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019076:Transgenes
chemical_list: C505032:CFTR protein, human; D008081:Liposomes; D012333:RNA, Messenger; D019005:Cystic Fibrosis Transmembrane Conductance Regulator