Adenovirus 5-fiber 35 chimeric vector mediates efficient apical correction of the cystic fibrosis transmembrane conductance regulator defect in cystic fibrosis primary airway epithelia.
In vivo gene transfer to the human respiratory tract by adenovirus serotype 5 (Ad5) vectors has revealed their limitations related to inefficient gene transfer, host antiviral response, and innate adenoviral toxicity. In the present work, we compared the cytotoxicity and efficiency of Ad5 and a chimeric Ad5F35 vector with respect to CFTR gene transfer to cystic fibrosis (CF) and non-CF human airway epithelial cells. We found that high doses of Ad5 vector had an adverse effect on the function of exogenous and endogenous CFTR. Results obtained with Ad5 capsid mutants suggested that the RGD motifs on the penton base capsomers were responsible for the negative effect on CFTR function. This negative interference did not result from a lower level of biosynthesis and/or altered cellular trafficking of the CFTR protein, but rather from an indirect mechanism of functional blockage of CFTR, related to the RGD integrin-mediated endocytic pathway of Ad5. No negative interference with CFTR was observed for Ad5F35, an Ad5-based vector pseudotyped with fibers from Ad35, a serotype that uses another cell entry pathway. In vitro, Ad5F35 vector expressing the GFP-tagged CFTR (Ad5F35-GFP-CFTR) showed a 30-fold higher efficiency of transduction and chloride channel correction in CFTR-deficient cells, compared with Ad5GFP-CFTR. Ex vivo, Ad5F35-GFP-CFTR had the capacity to transduce efficiently reconstituted airway epithelia from patients with CF (CF-HAE) via the apical surface, restored chloride channel function at relatively low vector doses, and showed relatively stable expression of GFP-CFTR for several weeks.
Université Lyon I, Faculté de Médecine Claude Bernard and IFR Laennec, Laboratoire de Virologie et Pathologie Humaine, CNRS FRE 3011, 69372 Lyon, France.
Ophélia
O
Granio
Katherine J D
KJ
Ashbourne Excoffon
Petra
P
Henning
Patricia
P
Melin
Caroline
C
Norez
Gaëlle
G
Gonzalez
Philip H
PH
Karp
Maria K
MK
Magnusson
Nagy
N
Habib
Leif
L
Lindholm
Frédéric
F
Becq
Pierre
P
Boulanger
Joseph
J
Zabner
Saw-See
SS
Hong
D000256:Adenoviridae / Q000235:genetics*; D000328:Adult; D015153:Blotting, Western; D036022:Capsid Proteins / Q000502:physiology; D002478:Cells, Cultured; D002712:Chlorides / Q000378:metabolism; D003550:Cystic Fibrosis / Q000235:genetics / Q000378:metabolism / Q000628:therapy*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics* / Q000378:metabolism; D004705:Endocytosis; D018014:Gene Transfer Techniques; D015316:Genetic Therapy; D005822:Genetic Vectors; D016679:Genome, Viral; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D016023:Integrins / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020545:Respiratory Mucosa / Q000378:metabolism* / Q000473:pathology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019076:Transgenes / Q000502:physiology
C505032:CFTR protein, human; D036022:Capsid Proteins; D002712:Chlorides; D016023:Integrins; D012333:RNA, Messenger; C485184:enhanced green fluorescent protein; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D049452:Green Fluorescent Proteins