An immunocytochemical assay to detect human CFTR expression following gene transfer.
BACKGROUND
To assess gene therapy treatment for cystic fibrosis (CF) in clinical trials it is essential to develop robust assays that can accurately detect transgene expression in human airway epithelial cells. Our aim was to develop a reproducible immunocytochemical assay for human CFTR protein which can measure both endogenous CFTR levels and augmented CFTR expression after gene delivery.


METHODS
We characterised an antibody (G449) which satisfied the criteria for use in clinical trials. We optimised our immunocytochemistry method and identified G449 dilutions at which endogenous CFTR levels were negligible in CF samples, thus enhancing detection of transgenic CFTR protein. After developing a transfection technique for brushed human nasal epithelial cells, we transfected non-CF and CF cells with a clinically relevant CpG-free plasmid encoding human CFTR.


RESULTS
The optimised immunocytochemistry method gave improved discrimination between CF and non-CF samples. Transfection of a CFTR expression vector into primary nasal epithelial cells resulted in detectable RNA and protein expression. CFTR protein was present in 0.05-10% of non-CF cells and 0.02-0.8% of CF cells.


CONCLUSIONS
We have developed a sensitive, clinically relevant immunocytochemical assay for CFTR protein and have used it to detect transgene-expressed CFTR in transfected human primary airway epithelial cells.
Davidson
Heather
H
Medical Genetics, School of Molecular and Clinical Medicine, University of Edinburgh, Western General Hospital, Edinburgh EH42XU, UK. H.Davidson@ed.ac.uk
Wilson
Abigail
A
Gray
Robert D
RD
Horsley
Alex
A
Pringle
Ian A
IA
McLachlan
Gerry
G
Nairn
Angus C
AC
Stearns
Cordelia
C
Gibson
James
J
Holder
Emma
E
Jones
Lisa
L
Doherty
Ann
A
Coles
Rebecca
R
Sumner-Jones
Stephanie G
SG
Wasowicz
Marguerite
M
Manvell
Michelle
M
Griesenbach
Uta
U
Hyde
Stephen C
SC
Gill
Deborah R
DR
Davies
Jane
J
Collie
D David S
DD
Alton
Eric W F W
EW
Porteous
David J
DJ
Boyd
A Christopher
AC
D000906:Antibodies / Q000276:immunology; D002478:Cells, Cultured; D003550:Cystic Fibrosis / Q000235:genetics / Q000378:metabolism / Q000473:pathology / Q000628:therapy; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D004847:Epithelial Cells / Q000378:metabolism* / Q000473:pathology; D015316:Genetic Therapy / Q000379:methods; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods*; D008856:Microscopy, Fluorescence; D009297:Nasal Mucosa / Q000378:metabolism / Q000473:pathology; D015203:Reproducibility of Results; D020545:Respiratory Mucosa / Q000378:metabolism / Q000473:pathology; D014162:Transfection; D019076:Transgenes
D000906:Antibodies; C505032:CFTR protein, human; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D049452:Green Fluorescent Proteins