title: Cystic fibrosis transmembrane conductance regulator-mRNA delivery: a novel alternative for cystic fibrosis gene therapy.
abstract: BACKGROUND
  Cystic fibrosis (CF) is the most frequent lethal genetic disease in the Caucasian population. CF is caused by a defective gene coding for the cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP- and ATP-dependent Cl(-) channel and central regulatory protein in epithelia. CFTR influences the fluid composition of the mucus in the respiratory tract. The most common mutation inducing CF, ΔF508, impairs CFTR processing within the cell and thus prevents functional CFTR expression in the apical membrane. The present study aimed to investigate the functional restoration of CFTR in human CF airway epithelia after transfection with optimized wild-type (wt)CFTR-mRNA.
  
  
  METHODS
  We used primary cultured human nasal epithelial (HNE) cells and the human bronchial epithelial cell line CFBE41o(-) that stably expresses ΔF508-CFTR and carried out transepithelial Ussing chamber measurements after transfection with optimized wtCFTR-mRNA. We confirmed the data obtained using immunofluorescence and protein biochemical approaches.
  
  
  RESULTS
  Transfection of the CFBE41o(-) cells with wtCFTR-mRNA restored cAMP-induced CFTR currents similar to the values seen in control cells (16HBE14o(-)). Using immunofluorescence approaches, we demonstrated that a considerable amount of CFTR is located at the apical surface in the CF cells after transfection. Western blot analyses of wtCFTR-mRNA transfected CFBE41o(-) cells confirmed these findings. Furthermore, we demonstrated physiological relevance by using primary cultured HNE cells and showed an almost two-fold increase in the cAMP-stimulated CFTR current after transfection.
  
  
  CONCLUSIONS
  From these data, we conclude that CFTR-mRNA transfection could comprise a novel alternative for gene therapy to restore impaired CFTR function.
authors.affiliation: Institute of Animal Physiology, University of Muenster, Muenster, Germany.
authors.forename: Nadine
authors.initials: N
authors.lastname: Bangel-Ruland
authors.forename: Katja
authors.initials: K
authors.lastname: Tomczak
authors.forename: Elena
authors.initials: E
authors.lastname: Fernández Fernández
authors.forename: Geraldine
authors.initials: G
authors.lastname: Leier
authors.forename: Barbara
authors.initials: B
authors.lastname: Leciejewski
authors.forename: Carsten
authors.initials: C
authors.lastname: Rudolph
authors.forename: Joseph
authors.initials: J
authors.lastname: Rosenecker
authors.forename: Wolf-Michael
authors.initials: WM
authors.lastname: Weber
mesh_terms: D000818:Animals; D002460:Cell Line; D003550:Cystic Fibrosis / Q000235:genetics* / Q000628:therapy; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics* / Q000378:metabolism; D004847:Epithelial Cells / Q000378:metabolism; D005260:Female; D015870:Gene Expression; D018014:Gene Transfer Techniques; D015316:Genetic Therapy; D006801:Humans; D009865:Oocytes / Q000378:metabolism; D061251:Primary Cell Culture; D012333:RNA, Messenger; D014162:Transfection / Q000379:methods; D014982:Xenopus laevis
chemical_list: D012333:RNA, Messenger; C093349:cystic fibrosis transmembrane conductance regulator delta F508; D019005:Cystic Fibrosis Transmembrane Conductance Regulator
keywords: cystic fibrosis; functional restoration; inherited disease; ion transport