Optimization of CFTR-mRNA transfection in human nasal epithelial cells
© The Author(s) 2016
Received: 30 April 2016
Accepted: 24 September 2016
Published: 4 October 2016
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.
KeywordsCystic fibrosis mRNA Transfection Primary cultured cells
Cystic fibrosis (CF) is a life-limiting autosomal recessive disorder in the Caucasian population, affecting around 70.000 individuals worldwide. 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. CFTR is expressed in the plasma membrane of secretory epithelia- such as airways, intestine, pancreas, testis and exocrine glands- as well as in some non-epithelial cells types . 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) . CFTR and ENaC play the most important role in maintaining homeostasis of airway surface liquid (ASL) by controlling the movement of water through the epithelium, thus regulating the hydration of the epithelial surface in many organs, but predominantly in the airways. It has been demonstrated that Na+ absorption is enhanced in CF airways and contributes to the pathogenesis of the disease . However, the interactions between CFTR and ENaC are still not fully understood and the mechanism (s) remain unknown . The defect in CF cells due to the impaired chloride transport and ion transport disturbances evokes abnormally viscous secretions in the airways causing obstructions that lead to bacterial infections , inflammation, tissue damage and destruction of the organ. Obstructive lung failure is currently the primary cause of morbidity and is responsible for 80 % of mortality .
The discovery of the CFTR gene in 1989  created new possibilities for a curative treatment targeting the basic defect rather than treating the symptoms of the CF disease . Due to the significant improvements, survival has been increased substantially  over the last few decades in the treatment of CF. Gene therapy has been pointed out as the forefront to overcome this difficult challenge . The potential of mRNA for therapeutic protein expression in vivo has been investigated as an alternative to DNA-based gene therapy. The preclinical exploration in the 1990s of the synthetic mRNA was initiated for diverse applications, including protein substitution for cancer and infectious diseases [11, 12]. Nevertheless, cancer immunotherapy is the only field in which clinical testing of mRNA is at an advanced stage .
Recently, we established a new strategy to deliver CFTR-mRNA directly to epithelial cells . We showed a proof-of-concept for mRNA-based functional restoration of impaired CFTR functions in the cell culture, either using human bronchial CF cells (CFBE41o-) that stably express the most common mutation F508del-CFTR or primary cultured human epithelial (HNE) cells . We showed that after mRNA transfection the CFBE41o- cells functionally act very similar after cAMP stimulation compared to healthy bronchial epithelia cells (16HBE41o-). Furthermore, the amount of functional CFTR molecules in the CF cells after mRNA transfection is even larger than in non-CF control cells . Using the CF cell line or HNE cells wtCFTR-mRNA transfection procedures were performed using a dose of 2.4 μg/cm2 and 24 h after transfection the analyses were carried out.
In order to give more physiological relevance we only used primary HNE cells in our study. In vitro cell culture models of human nasal epithelium based on primary culture technologies are known to be extremely useful for permeability and transport studies in healthy and disease tissues . Furthermore, cultured nasal cells are reliable models since they are known to express important biological features such as tight junctions, mucin secretion, cilia, transporters comparable to those found in vivo systems . Thus, the use of primary human nasal cell culture systems could accurately represent an alternative to in vivo situations. In addition, nasal epithelial cells could be used as a substitute for bronchial epithelial cells as they show identical morphologies with similar expression of receptors and responses to cytokine stimulation .
The major purpose of the present study was to optimize the dose of wtCFTR-mRNA as well as the determination of the time for a suitable CFTR expression in primary HNE cells. We determined the optimal mRNA concentration reducing the transfection dose needed for the successful recovery of the CFTR function. Furthermore, we performed time dependent studies with the minimal wtCFTR-mRNA concentration in order to evaluate the duration of the CFTR protein expression after mRNA transfection. In addition, after the efficient restoration of CFTR function, we investigated the possible impact of the CFTR-mRNA transfection on ENaC. Since primary cultured nasal epithelial cells show only a low chloride secretion but a positive amiloride-sensitive ENaC current, it is an excellent cell model for the investigation of CFTR and ENaC interactions . We concluded that mRNA delivery, termed “transcript therapy”  is an encouraging alternative in human primary culture offering a promising opportunity for the study of CF and the potentially clinical therapies in patients suffering from this incurable disease.
For in vitro transcription (IVT) we used the pSTI‐A120/hCFTR cDNA (construct provided by C. Rudolph, Maximilian University of Munich, Munich, Germany). The mRNA synthesis was performed as described previously . Briefly, the linearized plasmids were extracted with phenol/chloroform and precipitated with ethanol. The IVT reaction was carried out using the mMessage mMachine Kit (Ambion, Foster City, USA). The reaction was purified using the RNeasy plus Mini Kit (Quiagen, Hilden, Germany) and ethanol/ammonium acetate precipitation was done to achieve good quality. The concentration of mRNA was determined by absorbance measurement at 260 nm using a microvolume spectrophotometer (Nanodrop, Thermo Scientific, Wilmington, DE, USA). The integrity and size distribution of mRNA was determined by agarose gel electrophoresis and ethidium bromide staining. The generated mRNA was stored at −80 °C in nuclease-free water.
Primary cell culture of HNE cells was performed as described previously . Briefly, cells were isolated by enzymatic digestion for 24–72 h and afterwards seeded on collagen coated (0.15 mg/ml collagen type I; Biochrom AG, Berlin, Germany) Transwell permeable filters (diameter 6.5 mm, Costar 3470; Corning Inc., Lowell, MA, USA) for transepithelial measurements, on coated cell culture dishes (diameter 35 mm) for biochemistry analyses or on coated glass coverslips (diameter 12 mm) for optical fluorescence assays. The cells were cultured with serum-free F-12 Nutrient Mixture (Ham) (Invitrogen/Gibco, Kahlsruhe, Germany) supplemented with insulin (2 g/ml) (Invitrogen, Gibco), epidermal growth factor (12 ng/ml) (Sigma, Deisenhofen, Germany), endothelial cell growth supplement (7.5 g/ml) (Becton Dickinson GmbH, Heidelberg, Germany), triiodo-thyronine (3 nM) (Sigma), hydrocortisone (100 nM) (Sigma), gentamycin (10 g/ml) (Biochrom AG), penicillin/streptomycin (100 U/ml) (Invitrogen/Gibco), L-glutamine (2 mM) (Invitrogen/Gibco) and transferrin (4 g/ml) (Invitrogen/Gibco). Fibroblast contamination is reduced by the use of serum-free media and cluster formation is avoided by filtering through special cell strainers. In addition, epithelial purity of the HNE cells was confirmed using specific antibodies against vimentin and keratin. Cells were incubated in 95 % air and 5 % CO2 at 37 °C. Cells were seeded on the membrane and a confluent monolayer was obtained after 7 to 9 days.
Transfection efficiency and fluorescence optical analyses
The in vitro transfection efficiency of Lipofectamine TM 2000 (Invitrogen) was evaluated in HNE cells using the pEGFP-C1 plasmid (Clontech/Takara Bio Europe, Saint-Germain-en-Laye, France). Co-transfection of pEGFP-C1 and GFP- siRNA and H2O transfections were used as controls. Cells were seeded on coverslips 2 days before transfection and were cultivated in HNE culture medium. The day of transfection confluence of the cells was greater than 80 %. Three hours before transfection, cells were cultivated in HNE culture medium without antibiotics. Cells were transfected with 1 μg of pEGFP-C1 or co-transfected with 2.5 pmol/cm2 GFP-siRNA (negative control) or a respective amount of nuclease free water. After 48 h of incubation, cells were fixed with 0.05 % glutaraldehyde and autofluorescence was quenched with 0.1 % sodium borohydride. Analysis of the fluorescence intensities was determined using a fluorescence microscope (LSM 510 META; Carl Zeiss, Oberkochen, Germany). The images were recorded using the AxioCamMRm and the LSM 510 4.2 SP1 software (Carl Zeiss). The exposure time was adjusted manually for comparison of total fluorescence intensities. For compensation images, confluent regions of the cell layer were used for the analysis.
wtCFTR-mRNA transfection procedure
Dose dependence studies
Cells were transfected with different concentrations of wtCFTR-mRNA (2.4 μg/cm2, 1.2 μg/cm2, 0.6 μg/cm2, 0.3 μg/cm2) and experiments were performed 24 h after transfection.
Time dependence studies
HNE cells were transfected with the optimal concentration of 0.6 μg/cm2 and analyses were carried out 24, 48 and 72 h after transfection.
Modified Ussing chambers designed by Prof. Willy Van Driessche (KU Leuven, Belgium) were used to perform the transepithelial measurements. Ag/Ag electrodes were connected to Ringer solution (130 mM NaCl, 5 mM KCl, 1 mM CaCl2, 2 mM MgCl2, 5 mM glucose and 10 mM HEPES, pH 7.3, 37 °C) and the transepithelial potential (Vt) was clamped to zero. The two compartments of the Ussing chamber, apical and basolateral, were continuously perfused with cell culture Ringer solution. The short-circuit current (Isc), which reflects the transported net charges over the epithelium was constantly monitored (ImpsDsp 1.4; KU Leuven). The parameter was normalized to 1 cm2. After stabilization of the parameters, a cAMP (8-[4-chlorophenylthio (CTP)]-cAMP) (100 mM; Biolog, Bremen, Germany)/ IBMX (1 mM; ApplieChem GmbH, Darmstadt, Germany) cocktail was applied on the basolateral side to activate CFTR. Subsequently, CFTR was inhibited using the specific blocker CFTRinh172 (20 μM; Tocris Bioscience, Bristol, UK). In addition, the Na+ absorption through ENaC was assessed as short-circuit current in the presence and absence of amiloride (100 μM). Therefore, the effect of the wtCFTR-mRNA transfection on the amiloride current via ENaC was investigated. If not otherwise stated, all chemicals were obtained from Roth (Karlsruhe, Germany).
For Western Blot experiments HNE cells were detached from culture dishes using 400 μl of lysis buffer (1 mM Tris, 15 mM NaCl, 0.2 mM EDTA, 2 % Triton X-100) and 1 % protease inhibitor cocktail (Sigma). The extracts were homogenized with a sonifier ultrasonic cell disrupter (Branson, Danbury, CT, USA) and placed on ice for 10 min. The lysate was centrifuged at 4000 g for 30 min at 4 °C to pellet the cell debris and the supernatant was used. The concentration of the proteins was determined using the BCA test (Pierce, Rockford, IL, USA). 40 μg total protein were separated via SDS-PAGE (7.5 % acrylamide) and subsequently transferred to a polyvinylidene fluoride PVDF membrane using a semi-dry blotting system. Non-specific bindings sites were blocked for 2 h at RT with 5 % non-fat dry milk in Tris- buffered saline/Tween (TBS-T: 10nM Tris HCl, pH 7.4; 140 nM NaCl; 0.05 % Tween 20). CFTR protein was detected using an anti- CFTR antibody (ABR-01129; Dianova, Hamburg, Germany) in a 1: 500 concentration diluted in 5 % non-fat dry milk/TBS-T overnight at 4 °C continuously shaking. Vimentin and keratin proteins were detected using anti-vimentin and anti-keratin antibody, respectively (Dianova, Hamburg, Germany) in a 1: 500 concentration diluted in 5 % non-fat dry milk/TBS-T overnight at 4 °C continuously shaking. After 24 h, the membrane was washed in TBS-T three times for 10 min and incubated with the secondary antibody goat anti-mouse Ig G conjugated with horseradish peroxidase (HRP) (Dianova) diluted 1: 10,000 in 5 % non-fat dry milk/TBS-T for one hour at room temperature continuously shaking. Then, the membrane was washed again three times in TBS-T for 10 min and once in TBS. The detection was carried out with enhanced chemiluminiscence (ECL). To assure comparable protein amount and expression, anti-alpha-tubulin (Tubulin, alpha, DLN-09993; Dianova) was used for normalization. Image J, version 1.41 (Wayne Rasband, National Institutes of Health, Bethesda, MD, USA) was used for densitometry evaluation of the CFTR band intensity.
Analysis of fluorescence intensities
Analysis of total fluorescence intensities was performed using ImageJ, version 1.41. The plug-in for RGB (Red, Green, Blue) measurement analyses the intensity of each channel of an image and displays the average channel intensity [(R + G + B)/3]. The generated value was employed for analysis and was compared with that of other images. The average of non-transfected cells was set to 100 % and the transfected average was normalized to that value to be expressed as a multiple of the non-transfected value.
Data are presented as the arithmetic means (± SEM). Sets of data were compared with Student’s t-test. Differences were considered statistically significant when p < 0.05 (*) or p < 0.01 (**). In all experiments, n gives the number of replications. All statistical tests were performed using Origin, version 7.0 (Originallab Corporation, Northampton, USA).
Characterization of primary HNE cells
Dose response studies
Western blot analyses
Time response studies
Western blot analyses
Influence of wtCFTR-mRNA on ENaC
The discovery of the disease-causing CFTR gene in 1989  created new hopes for a curative treatment targeting the basic defect rather than treating CF disease manifestations . Since this CFTR identification, there has been significant efforts to develop gene therapy strategies for the correction of the mutation on cellular level. The delivery of a therapeutic nucleic acid (DNA or RNA) is a promising concept for an inherited single-gene defect such as CF, with the prospect of correcting many aspects of the complex pathology . In addition, one single therapy might be suitable to treat subjects with a wide variety of mutations, meaning that a single treatment strategy would be relevant to all patients. However, initially approaches, which involved direct administration to the airway of recombinant CFTR based on conventional viral DNA-delivery have not been successful for a number of reasons . Subsequently, the development of mRNA-based therapeutic approaches presents several important differences in comparison with other nucleic acid-based therapies  like the direct translation in the cytoplasm, the missing integration into the genome and therefore the avoidance of the potential risk of insertional mutagenesis . Therefore, as already described by our working group, we developed a new strategy using mRNA instead of DNA to correct CFTR function in the apical plasma membrane of human CF airway epithelia after wtCFTR-mRNA transfection in vitro . Although other organs are affected in CF as well, the lung is the major site of pathology and thus, has been the target in the majority of gene therapy trials. Consequently, most investigators have focused towards minimizing CF lung disease . In fact, the best target for the CF gene therapy in the airways are the ciliated epithelial cells . Although it is known that CFTR constitutes a low-abundance mRNA in airway epithelia  a minor level of CFTR gene transfer to the airway epithelia is sufficient to correct the Cl− transport in vitro and in vivo . Furthermore, only 10 % of normal cells are sufficient to normalize the main dysregulated parameters such as Cl− or Na+ conductance and IL8 secretion .
Dose and time dependence studies
We used primary cultured human epithelial cells to perform our mRNA transfection experiments in a lipid-based transfection reagent to produce overexpression of CFTR protein. We have found that CFTR-mRNA can be effectively delivered in these primary cultured cells and that the expressed proteins are functional. The aim of this study was to find the optimal mRNA dose that is needed for an efficient chloride secretion mediated by the CFTR channel and to study the persistence of the wtCFTR-mRNA transfection over a period of time. Therefore, we carried out Ussing chambers experiments and measured the transepithelial current (Isc) in transfected HNE cells and in non-transfected cells. We performed dose dependent studies, in which a stepwise dosage reduction was carried out, from 2.4 to 0.3 μg/cm2. In these measurements, we found that the most efficient CFTR activation was reached using a mRNA dose of 0.6 μg/cm2 compared to non-transfected cells. Furthermore, CFTR mediated current in time experiments studies confirmed that 0.6 μg/cm2 wtCFTR-mRNA transfection could be persistent over a period of 72 h.
Accordingly, we verified these findings using protein biochemistry analyses. First, we analysed the amount of CFTR using Western blot procedures. Thus, we showed that the HNE cells transfected with 0.6 μg/cm2 expressed nearly twice as much CFTR protein compared to control cells. These biochemical results correlate perfectly with the increase in the cAMP-induced current observed in the electrophysiological analyses. In time dependence studies, 24 after 0.6 μg/cm2 wtCFTR- mRNA transfection 1.5- fold more CFTR protein compared to non-transfected cells was shown. Here, the obtained results indicated that the functional CFTR protein can be maintained in the plasma membrane over a few days in HNE cells. Nonetheless, after 48 h less protein are detectable due to the potential degradation of the CFTR. CFTR expression using unmodified mRNA decreased within 3 days, requiring repeated application in high frequencies [33, 34]. Moreover, possible difficulties in long-term effect may also include mRNA stability and therefore the duration of the mRNA effect in the target cells, which could make frequent dosing necessary.
Does the boosted CFTR expression affect ENaC?
In CF disease, salt and fluid absorption is prompted by the loss of CFTR and the inappropriate regulation of ENaC . The consequence is an increase in water and sodium reabsorption from the airways, compromising the formation of a sufficient periciliary liquid layer and mucus clearance . In CF airways, ENaC is stimulated by the increase in cAMP concentrations when functional CFTR is lacking, suggesting that CFTR acts in decreasing ENaC activity [35–37]. To better understand CFTR/ENaC interactions as well as to determine the role of ENaC in wtCFTR-mRNA transfection experiments in primary cultured human epithelial cells, electrophysiological measurements in Ussing chambers were carried out to study ENaC activity in the presence of boosted CFTR expression. As expected, the control cells had the highest amiloride-sensitive current. It is well known that primary HNE cells have an amiloride-sensitive current [38, 39]. In comparison, in cells transfected with different doses of wtCFTR-mRNA the transepithelial ENaC current decreased stepwise, possibly showing a trend in the down-regulation of ENaC by CFTR (Fig. 8a). However, no significant statistical difference was found, and therefore no effects of the transfection on ENaC function or expression were confirmed. On the other side, in time dependence studies an increasing trend in the amiloride-sensitive current was found (Fig. 8b), although no statistical difference was found, too. In a certain way, these results indicate a down-regulation of ENaC by CFTR. Firstly, the inhibition of ENaC was shown by a decreasing tendency in the amiloride-sensitive current after overexpression of CFTR with different wtCFTR-mRNA doses. Secondly, the restoration of ENaC was measured by an increasing trend of ENaC current after wtCFTR-mRNA transfection in time dependence experiments. Further studies, not only functional electrophysiological measurements but also biochemical and immunological studies are necessary to confirm these findings. In summary, more than 20 years after the initial discovery that ENaC’s sensitivity to cAMP is CFTR dependent, the mechanism of the CFTR/ENaC interaction, and in particular the ENaC regulation by CFTR, still remains unclear. Undoubtedly, ENaC represents an attractive alternative target to improve airway surface hydration and mucus clearance in patients with CF independent of their CFTR genotype.
Different delivery methods
In CF pulmonary disease the opportunity to selectively target a drug to the lungs remains a fascinating option . In fact, local drug delivery may allow maximum pharmacological targeting, and thus therapeutic efficacy. As a consequence, researchers continue applied efforts to develop new inhalation devices and advanced drug delivery . CFTR-mRNA aerosol administration to airways of CF patients could be delivered as it was previously showed . Furthermore, another study successfully demonstrates the gene delivery with magnetized aerosol comprising iron oxide nanoparticles in lungs of mice . Therefore, administration of drugs via the inhalation route is of great interest in CF treatment . The main advantages of aerosol technologies are the limited systemic toxicity, direct drug action on target site and the suitability for home therapy . An important issue in gene delivery is the biocompability and biosafety of the nanocarrier used in the transfection procedure . Lipid based formulations like cationic lipids o cationic polymers have become a successful method to transfect cells and to reach adequate transfection efficiency in vitro [15, 47]. Here, this study demonstrates promising results using this lipid-based delivery by Lipofectamine TM 2000 transfection reagent in primary cultured cells. On the other side, since Lipofectamine presents a high cytotoxicity for the cell viability despite its robust and high transfection efficiency , it is not an appropriate carrier to assess potentially clinical in vivo therapies in treatment of CF. Therefore, we are looking for an alternative and stable formulation like biopolymers, e.g. chitosan, that could be effective to target intratracheal routes. Efficient transfection in a CF cell line may show the future use of this nanocarrier in the gene therapy approaches. Nevertheless, the search for an optimal transfection agent is still open in the hope of finding a solution to address the barriers associated by the lung.
Future aspects: clinical application/dosage form
One of the major milestones for a potential clinical application of the mRNA based gene therapy is to circumvent unwanted immune responses, instability and delivery barriers. Chemical modifications of the mRNA, like the inclusion of pseudouridine in the mRNA prevented activation pattern recognition receptors  and 2’-5’-oligoadenylate synthetase . Furthermore, these modifications can stabilize the mRNA against cleavage and ultimately improve expression rates . These variations present new alternative therapies to avoid side effects and therefore the clinical application is in sight . In addition, efficient pulmonary drug delivery has been mostly achieved through specific devices and particle engineering technologies . The face of the future in CF lung therapy is the development of easy to use dry powder for inhalation . In this context, biocompatible nanoparticles, such as chitosan nanoparticles, are very suitable candidates because they permit an efficient protection of the gene material and its delivery to airway epithelial cells. In addition, despite chitosan biodegradability, future lung deposition studies should achieve the issue of chitosan-wtCFTR-mRNA complexes fate after they have landed. Furthermore, other challenges concerning targeting the lung should be considered because it is an immunologically sensitive organ and the airway cells turn over . For instance, higher doses are generally required for the effective administration to the lung  and sustainable expression of the therapeutic gene is difficult to achieve. Therefore, the potential dosage of chitosan-wtCFTR-mRNA complexes could demand a repeated administration. Multi dose clinical trials could achieve improvement in lung function, long-term expression and subsequently, future clinical benefits for CF patients.
We have clearly demonstrated that wtCFTR-mRNA can be effectively delivered in primary cultured human nasal epithelial cells, and that the expressed proteins are functional. Moreover, we have shown that CFTR-mRNA can be reduced to minimal dose and is persistent for a time period longer than 24 h after transfection. Furthermore, we could not find any effect on ENaC activity after the reconstitution of CFTR by transfection. Our study establishes the efficient mRNA transfection using primary cultured cells, thus creating the more physiological relevant conditions for further approaches in the potentially development of therapeutic strategies for CF disease treatment.
Significance level of p p ≤ 0.05
Significance level of p ≤ 0.01
Cyclic adenosine monophosphate
Cystic Fibrosis Bronchial Epithelial 41o− Cells
Cystic fibrosis transmembrane conductance regulator
- cm2 :
- CO2 :
Epithelial sodium channel
Green fluorescence protein
- HNE cells:
Human nasal epithelial cells
Human umbilical vein endothelial cells
- ISC :
In vitro transcription
Minimum essential medium
Messenger ribonucleic acid
- Na+ :
- P. aeruginosa :
Polyacrylamide gel electrophoresis
Polymerase chain reaction
- Rt :
Transepithelial electrical resistance
Sodium dodecyl sulfate
Standard error of the mean
Tris buffered saline
Tris buffered saline- Tween
We acknowledge financial support from “Deutsche Förderungsgesellschaft zur Mukoviszidoseforschung e.V.”. We thank Cristin Brand for her technical support in the cell characterization assay and Lisa Träger for helpful discussions of the manuscript.
PhD fellowship from “Deutsche Förderungsgesellschaft zur Mukoviszidoseforschung e.V.” to EFF.
Availability of data and materials
Please contact author for data requests.
EFF designed and performed experiments, analysed data and wrote the paper. NBR and KT supervised the project, participated in the design of the study and helped to draft the manuscript. WMW conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Consent for publication
Ethics approval and consent to participate
We obtained the nasal specimens from non-CF patients undergoing nasal surgery. Typically the samples were nasal polyps or nasal turbinates of patients suffering from chronic sinusitis. The study was approved by the committees for human studies of the University of Muenster (Ethik Kommission Muenster). This study was conducted according to the principles expressed in the Declaration of Helsinki and all patients provided their informed consent.
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