Abstract
Cystic fibrosis (CF) is the most common life-threatening inherited disease in the Caucasian population. It is caused by genetic defects in the cystic fibrosis transmembrane conductance regulator gene (CFTR), a cAMP regulated chloride-bicarbonate channel mainly located in the apical membrane of polarized epithelial cells. CFTR is proposed to regulate other proteins, including the epithelial sodium channel (ENaC). Recently, we successfully restored chloride current in CFTR deficient human airway epithelial cells using wtCFTR-mRNA transfection compared to non-CF cells showing similar values. The present study aimed to optimize the wtCFTR-mRNA transfection procedures in primary cultured human nasal epithelial (HNE) cells. Dose and time dependence experiments were performed. In addition, we investigated the possible impact of the wtCFTR-mRNA transfection on ENaC function in transepithelial measurements. We reduced the wtCFTR-mRNA dose stepwise and determined the minimal concentration of 0.6 μg/cm2, which is needed for the most efficient restoration of CFTR function. Furthermore, CFTR expression was evaluated 24, 48 and 72 h after transfection. Using the minimal concentration of 0.6 μg/cm2 wtCFTR-mRNA we confirmed a positive functional CFTR restoration over a period of 72 h. Biochemical analyses confirmed these findings. Furthermore, we could not find any significant effect on ENaC after the recovery of CFTR by wtCFTR-mRNA transfection. Our data show that wtCFTR-mRNA transfection is an encouraging alternative “gene” therapy in human primary culture.
Highlights
Cystic fibrosis (CF) is the most common life-threatening inherited disease in the Caucasian population
In Western blot experiments a specific band of vimentin was detected in the range of 57–60 kDa solely in the human umbilical vein endothelial cells (HUVEC) sample, while in the other samples, the human bronchial epithelial cell lines (16HBE14o- and CFBE41o-) and primary human nasal epithelial (HNE) cells any band was found
We found that the most efficient Cystic fibrosis transmembrane conductance regulator (CFTR) activation was reached using a Messenger ribonucleic acid (mRNA) dose of 0.6 μg/cm2 compared to non-transfected cells
Summary
Cystic fibrosis (CF) is the most common life-threatening inherited disease in the Caucasian population. It is caused by genetic defects in the cystic fibrosis transmembrane conductance regulator gene (CFTR), a cAMP regulated chloride-bicarbonate channel mainly located in the apical membrane of polarized epithelial cells. CFTR is proposed to regulate other proteins, including the epithelial sodium channel (ENaC). The disease is caused by genetic defects in the cystic fibrosis transmembrane conductance regulator gene (CFTR) that encodes for a cyclic adenosine monophosphate (cAMP) -regulated chloride channel. At the cell membrane CFTR exhibits its function as a chloride channel and it is proposed to regulate other membrane proteins, including the epithelial sodium channel (ENaC) [2]. Cancer immunotherapy is the only field in which clinical testing of mRNA is at an advanced stage [13]
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