Interaction between calcium-activated chloride channels and the cystic fibrosis transmembrane conductance regulator

We investigated interactions between cystic fibrosis conductance regulator (CFTR) and endogenous Ca2+-activated Cl- channels (CaCC) in bovine pulmonary artery endothelium (CPAE). CPAE cells, which do not express CFTR, were transiently transfected with wild-type (WT) CFTR and the deletion mutant deltaF508 CFTR. Currents through CaCC were significantly reduced after expression of WT CFTR. This inhibition was increased by stimulation (isobutylmethylxanthine, forskolin) of CFTR in cells expressing WT CFTR. There were no such effects when deltaF508 mutant CFTR, which is retained in the endoplasmic reticulum, was expressed. It is concluded that CFTR and CaCC are functionally coupled probably through a direct channel-channel interaction.     

SERCA pump inhibitors do not correct biosynthetic arrest of deltaF508 CFTR in cystic fibrosis

Deletion of phenylalanine 508 (deltaF508) accounts for nearly 70% of all mutations that occur in the cystic fibrosis transmembrane conductance regulator (CFTR). The deltaF508 mutation is a class II processing mutation that results in very little or no mature CFTR protein reaching the apical membrane and thus no cAMP-mediated Cl- conductance. Therapeutic strategies have been developed to enhance processing of the defective deltaF508 CFTR molecule so that a functional cAMP-regulated Cl- channel targets to the apical membrane. Sarcoplasmic/endoplasmic reticulum calcium (SERCA) inhibitors, curcumin and thapsigargin, have been reported to effectively correct the CF ion transport defects observed in the deltaF508 CF mice. We investigated the effect of these compounds in human airway epithelial cells to determine if they could induce deltaF508 CFTR maturation, and Cl- secretion. We also used Baby Hamster Kidney cells, heterologously expressing deltaF508 CFTR, to determine if SERCA inhibitors could interfere with the interaction between calnexin and CFTR and thereby correct the deltaF508 CFTR misfolding defect. Finally, at the whole animal level, we tested the ability of curcumin and thapsigargin to (1) induce Cl- secretion and reduce hyperabsorption of Na+ in the nasal epithelia of the deltaF508 mouse in vivo, and (2) induce Cl- secretion in intestine (jejunum and distal colon) and the gallbladder of the deltaF508 CF mouse. We conclude that curcumin and thapsigargin failed to induce maturation of deltaF508 CFTR, or induce Cl- secretion, as measured by biochemical and electrophysiologic techniques in a variety of model systems ranging from cultured cells to in vivo studies.     

VCP/p97 AAA-ATPase does not interact with the endogenous wild-type cystic fibrosis transmembrane conductance regulator

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel that is defective in cystic fibrosis. The most common mutation, DeltaF508 CFTR, is retained in the endoplasmic reticulum, retrotranslocated into the cytosol, and degraded by the proteasome. In a proteomics screen to identify DeltaF508 CFTR interacting proteins, we found that valosin-containing protein (VCP)/p97, a Type II AAA ATPase that is a component of the retrotranslocation machinery, binds DeltaF508 CFTR, and this interaction is stabilized by proteasomal inhibition. Since wild-type (WT) CFTR has been reported to be inefficiently processed during biogenesis with as much as 75% of the newly synthesized protein degraded by the proteasome, we examined the VCP interaction in Calu-3, T-84, and 16HBE, three epithelial cell lines that endogenously express WT CFTR. The results indicate that when WT CFTR processing is efficient, as demonstrated in Calu-3 cells, VCP does not interact. Interestingly, overexpression of recombinant WT CFTR in Calu-3 cells results in inefficient processing and VCP interaction, demonstrating that CFTR processing efficiency and the VCP interaction are tightly coupled. Furthermore, induction of ER stress and activation of the unfolded protein response result in inefficient processing of WT CFTR in Calu-3 cells and promote the WT CFTR-VCP interaction. The results support the hypothesis that components of the retrotranslocation machinery such as VCP do not interact with CFTR in epithelial cells that endogenously express WT CFTR, since under normal conditions the processing of the WT protein is efficient.     

Pseudomonas aeruginosa-induced apoptosis is defective in respiratory epithelial cells expressing mutant cystic fibrosis transmembrane conductance regulator

Chronic lung infection with Pseudomonas aeruginosa constitutes the most severe manifestation of cystic fibrosis, a scenario that results from defects in early clearance of the microbe. Early clearance involves epithelial cell ingestion of bacteria, rapid activation of nuclear factor-kappa B and cellular desquamation within minutes of P. aeruginosa infection, processes that are deficient in cells with mutant alleles of Cftr. Analyzing the effect of Cftr genotype on the apoptotic response of airway epithelial cells to P. aeruginosa, we found that human bronchial epithelial cells expressing Delta F508 cystic fibrosis transmembrane conductance regulator (CFTR) underwent significantly delayed apoptosis compared with cells expressing wild-type (WT) CFTR. Mice with a WT Cftr allele had apoptotic cells in their lungs after P. aeruginosa infections, whereas mice homozygous for the Delta F508 or G551D Cftr alleles showed little apoptosis in response to acute infection. Pseudomonal infection induced expression of CD95 and CD95 ligand, a response that was also delayed in cells homozygous for mutant Cftr alleles. Thus, WT CFTR expression promotes a rapid expression of CD95/CD95 ligand and apoptotic response to P. aeruginosa infection. Prompt apoptosis of infected epithelial cells may be critical for clearance of P. aeruginosa, and CFTR-associated defects in apoptosis may contribute to the pathogenesis of the lung disease in cystic fibrosis.     

Species-specific differences in mouse and human airway epithelial biology of recombinant adeno-associated virus transduction

Differences in airway epithelial biology between mice and humans have presented challenges to evaluating gene therapies for cystic fibrosis (CF) using murine models. In this context, recombinant adeno-associated virus (rAAV) type 2 and rAAV5 vectors have very different transduction efficiencies in human air-liquid interface (ALI) airway epithelia (rAAV2 approximately = rAAV5) as compared with mouse lung (rAAV5 > rAAV2). It is unclear if these differences are due to species-specific airway biology or limitations of ALI cultures to reproduce in vivo airway biology. To this end, we compared rAAV2 and rAAV5 transduction biology in mouse and human ALI cultures, and investigated the utility of murine deltaF508 cystic fibrosis transmembrane conductance regulator (CFTR) ALI epithelia to study CFTR complementation. Our results demonstrate that mouse ALI epithelia retain in vivo preferences for rAAV serotype transduction from the apical membrane (rAAV5 > rAAV2) not seen in human epithelia (rAAV2 approximately = rAAV5). Viral binding of rAAV2 and rAAV5 to the apical surface of mouse ALI airway epithelia was not significantly different, and proteasome-modulating agents significantly enhanced rAAV2 transduction to a level equivalent to that of rAAV5 in the presence of these agents, suggesting that the ubiquitin/proteasome pathway represents a more significant intracellular block for rAAV2 transduction of mouse airway epithelia. Interestingly, cAMP-inducible chloride currents were enhanced in deltaF508CFTR mouse ALI cultures, making this model incompatible with CFTR complementation studies. These studies emphasize species-specific differences in airway biology between mice and humans that significantly influence the use of mice as surrogate models for rAAV transduction and gene therapy for CF.     

Endogenous surface expression of ΔF508-CFTR mediates cAMP-stimulated Cl(-) current in CFTR(ΔF508/ΔF508) pig thyroid epithelial cells

The cystic fibrosis transmembrane conductance regulator (CFTR) is both an anion channel and a regulator of other transport proteins. Mutations in the?CFTR?gene underlie the human disease, cystic fibrosis. The most common?CFTR?mutation, ΔF508, produces a misfolded protein which traffics improperly. The availability of transgenic?CFTR(ΔF508/ΔF508) pigs allows measurement of the impact of ΔF508 in native tissue. Thyroid epithelia respond to cAMP-elevating agents by increasing anion transport, a process reliant on functional CFTR. To assess whether endogenous levels of ΔF508-CFTR mediate thyroid transport, primary thyroid epithelial cultures (pThECs) were grown from newborn?CFTR(+/+) (wild-type) and?CFTR(ΔF508/ΔF508) (ΔF) pig thyroids and the stimulated, secretory components of short-circuit current (I(sc)) compared. Surface biotinylation studies assessed the surface presentation of ΔF508-CFTR. Baseline?I(sc) levels of both wild-type and ΔF pThECs consisted of an amiloride-sensitive component. In ΔF pThECs, this mirrored previous measurements in?CFTR(-/-) (knockout) pThECs. Surprisingly, elevation of cAMP transiently increased?I(sc) to peak levels ～65% of those achieved by wild-type. In contrast, knockout pThECs were indifferent to cAMP activation. In ΔF pThECs, total ΔF508-CFTR expression was ～9% that of wild-type, consistent with misfolding and enhanced degradation. Surface biotinylation studies indicated that ～4% of the total ΔF508 resided at the surface and did not increase with cAMP elevation. The present findings show that low endogenous levels of pig ΔF508-CFTR can mediate substantial anion transport by thyroid epithelia. These data suggest that both wild-type and ΔF508-CFTR regulate additional thyroid transporters, and together co-ordinate the overall?I(sc) response.     

Partial CFTR genotyping and characterisation of cystic fibrosis patients with myocardial fibrosis and necrosis

Myocardial necrosis and fibrosis is a rare complication of cystic fibrosis (CF) causing sudden and unexpected death in infancy due to cardiac arrest. Characteristic morphological lesions are recognisable postmortem. The 18 CF patients with this complication had varied clinical features including mild pulmonary involvement, early onset severe pancreatic insufficiency, and profound electrocardiogram (ECG) changes. In this group of patients, 5 were deltaF508 homozygotes, 1 was deltaF508/ N1303K and 1 was a deltaF508/M compound heterozygote. A pair of affected siblings (deltaF508 homozygotes) were fully concordant for myocardial involvement and for the general course of the disease. The co-existence of a genetic predisposition to myocardial lesions resulting most probably from severe cystic fibrosis transmembrane (CFTR) genotypes (such as deltaF508/deltaF508, deltaF508/N1303K) and deficiency of certain trophic factors necessary for metabolism of the myocardium, are postulated to cause myocardial complications in CF leading to circulatory failure and early death.     

Targeted replacement of normal and mutant CFTR sequences in human airway epithelial cells using DNA fragments

Recent studies have reported that mutant genomic cystic fibrosis (CF) transmembrane conductance regulator ( CFTR ) sequences can be corrected in transformed CF airway epithelial cell lines by targeted replacement with small fragments of DNA with wild-type sequence. To determine if the observed genotype modification following small fragment homologous replacement (SFHR) was limited to transformed CF cell lines, further studies were carried out in both transformed and non-transformed primary normal airway epithelial cells. The endogenous genotype of these normal cell lines was modified following liposome or dendrimer transfection using DNA fragments with DeltaF508 CFTR sequence (488 nt, complementary single strands) designed to also contain a unique restriction enzyme cleavage site (Xho I). Replacement at the appropriate genomic locus by exogenous DeltaF508 CFTR DNA and its expression as mRNA was demonstrated by PCR amplification of genomic DNA and mRNA-derived cDNA as well as Xho I digestion of the PCR products. These studies show that SFHR occurs in both transformed and non- transformed primary human airway epithelial cells and indicate that single base substitution (the silent mutation giving rise to the Xho I site) and deletion or insertion of at least three consecutive bases can be achieved in both normal and CF epithelial cells. Furthermore, these studies reiterate the potential of SFHR as a strategy for a number of gene targeting applications, such as site-specific mutagenesis, development of transgenic animals, development of isogenic cell lines and for gene therapy.      

Cystic fibrosis genotype and bacterial infection: a possible connection

Patients with cystic fibrosis (CF) have repeated bacterial infection of the airways, which can lead to chronic infection. There is evidence that disease severity is determined by the genetic mutations present. This study aims to establish if CF genotype is related to the frequency and types of airway bacterial infection. Adult patients attending the regional CF unit are followed for two years and assigned to one of three groups depending on whether they are chronically infected with Burkholderia cepacia complex (BCC) organisms, Pseudomonas aeruginosa, or neither of these organisms. Genotype analysis is performed on all patients to determine which of the cystic fibrosis transmembrane regulator (CFTR) gene mutations are present. The numbers and types of organism with the CFTR mutations isolated from sputum are identified. Data are available on 59 patients: 15 colonised with BCC, 24 colonised with P. aeruginosa, and 20 not colonised with either organism. Twenty patients were homozygous for deltaF508, 25 were heterozygous, and the deltaF508 mutation was not present in the remaining 14 patients. Patients homozygous or heterozygous for the deltaF508 mutation had an increased likelihood of colonisation with BCC or P. aeruginosa, an increased number of positive sputum cultures and a higher frequency of multiple infecting organisms. Cystic fibrosis mutational analysis identified seven patients who had the R117H mutation. These patients were less likely to be colonised with BCC or P. aeruginosa. In conclusion, patients homozygous or heterozygous for the deltaF508 deletion are more likely to suffer airway colonisation with BCC or P. aeruginosa, with increased numbers of positive sputum cultures and infecting organisms. Those with the R117H mutation are less likely to be colonised by Gram-negative organisms.     

Disruption of cytokeratin-8 interaction with F508del-CFTR corrects its functional defect

We have previously reported an increased expression of cytokeratins 8/18 (K8/K18) in cells expressing the F508del mutation of cystic fibrosis transmembrane conductance regulator (CFTR). This is associated with increased colocalization of CFTR and K18 in the vicinity of the endoplasmic reticulum, although this is reversed by treating cells with curcumin, resulting in the rescue of F508del-CFTR. In the present work, we hypothesized that (i) the K8/K18 network may interact physically with CFTR, and that (ii) this interaction may modify CFTR function. CFTR was immunoprecipitated from HeLa cells transfected with either wild-type (WT) CFTR or F508del-CFTR. Precipitates were subjected to 2D-gel electrophoresis and differential spots identified by mass spectrometry. K8 and K18 were found significantly increased in F508del-CFTR precipitates. Using surface plasmon resonance, we demonstrate that K8, but not K18, binds directly and preferentially to the F508del over the WT human NBD1 (nucleotide-binding domain-1). In vivo K8 interaction with F508del-CFTR was confirmed by proximity ligation assay in HeLa cells and in primary cultures of human respiratory epithelial cells. Ablation of K8 expression by siRNA in F508del-expressing HeLa cells led to the recovery of CFTR-dependent iodide efflux. Moreover, F508del-expressing mice topically treated with K8-siRNA showed restored nasal potential difference, equivalent to that of WT mice. These results show that disruption of F508del-CFTR and K8 interaction leads to the correction of the F508del-CFTR processing defect, suggesting a novel potential therapeutic target in CF.     

Differential localization of the cystic fibrosis transmembrane conductance regulator in normal and cystic fibrosis airway epithelium.

Deletion of the amino acid residue Phe 508 of the cystic fibrosis transmembrane conductance regulator (CFTR) protein represents the most common mutation identified in cystic fibrosis (CF) patients. A monoclonal and a polyclonal antibody directed against different regions of CFTR were used to localize the CFTR protein in normal and CF airway epithelium derived from polyps of non-CF and CF subjects homozygous for the delta Phe 508 CFTR mutation. To identify the cellular and subcellular localization of CFTR, immunofluorescent light microscopy, confocal scanning microscopy, and immunogold transmission electron microscopy were performed on cryofixed tissue. A markedly different subcellular distribution was identified between normal and CF airway epithelial cells. In normal epithelium, labeling was restricted to the surface apical compartment of the ciliated cells. In contrast, in the epithelium from homozygous delta Phe 508 CF patients, CFTR markedly accumulated in the cytosol of all the epithelial cells. These findings are consistent with the concept that the CFTR delta Phe 508 mutation modifies the intracellular maturation and trafficking of the protein, leading to an altered subcellular distribution of the delta Phe 508 mutant CFTR.     

Cystic fibrosis F508del patients have apically localized CFTR in a reduced number of airway cells

Present state of knowledge, mostly based on heterologous expression studies, indicates that the cystic fibrosis transmembrane conductance regulator (CFTR) protein bearing the F508del mutation is misprocessed and mislocalized in the cytoplasm, unable to reach the cell surface. Recently, however, it was described that protein levels and localization are similar between F508del and wild-type CFTR in airway and intestinal tissues, but not in the sweat glands. In this study, we used immunocytochemistry with three different anti-CFTR antibodies to investigate endogenous CFTR expression and localization in nasal epithelial cells from F508del homozygous patients, F508del carriers, and non-CF individuals. On average, 300 cells were observed per individual. No significant differences were observed for cell type distributions among CF, carrier, and non-CF samples; epithelial cells made up approximately 80% to 95% of all cells present. CFTR was detected mostly in the apical region (AR) of the tall columnar epithelial (TCE) cells, ciliated or nonciliated. By confocal microscopy analysis, we show that the CFTR apical region-staining does not overlap with either anti-calnexin (endoplasmic reticulum), anti-p58 (Golgi), or anti-tubulin (cilia) stainings. The median from results with three antibodies indicate that the apical localization of CFTR happens in 22% of TCE cells from F508del homozygous patients with CF (n = 12), in 42% of cells from F508del carriers (n = 20), and in 56% of cells from healthy individuals (n = 12). Statistical analysis indicates that differences are significant among all groups studied and for the three antibodies (p < 0.05). These results confirm the presence of CFTR in the apical region of airway cells from F508del homozygous patients; however, they also reveal that the number of cells in which this occurs is significantly lower than in F508del carriers and much lower than in healthy individuals. These findings may have an impact on the design of novel pharmacological strategies aimed at circumventing the CF defect caused by the F508del mutation.     

Localisation of wild-type and DeltaF508-CFTR in nasal epithelial cells

Wild-type and the DeltaF508 mutation of the cystic fibrosis transmembrane conductance regulator (DeltaF508-CFTR) were localised by confocal imaging in DeltaF508/DeltaF508 native airway epithelial cells using a well-characterised CFTR antibody. Surface nasal epithelial cells from three control and three CF individuals were obtained from nasal brushings. Cells were fixed, permeabilised and incubated with first antibody for 18 h at 4 degrees C. Following labelling with second antibody, cells were viewed with the confocal microscope. Wild-type CFTR was localised predominantly apically, whereas DeltaF508-CFTR was located mainly inside the cell in a region close to the nucleus. Incubation of cells with MPB-07 (250 microM) at 37 degrees C for 2 h resulted in pronounced movement of DeltaF508-CFTR to the cell periphery, but did not change the localisation of wild-type CFTR. The results show that DeltaF508-CFTR is mislocalised in native nasal epithelial cells and that its distribution is altered in response to the new CFTR activator, MPB-07. The findings should lead to development of a rational drug treatment for CF patients carrying the DeltaF508 mutation.     

Cellular localization and activity of Ad-delivered GFP-CFTR in airway epithelial and tracheal cells

Cystic fibrosis is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, and the cellular trafficking of the CFTR protein is an essential factor that determines its function in cells. The aim of our study was to develop an Ad vector expressing a biologically active green fluorescent protein (GFP)-CFTR chimera that can be tracked by both its localization and chloride channel function. No study thus far has demonstrated a GFP-CFTR construct that displayed both of these functions in the airway epithelia. Tracheal glandular cells, MM39 (CFTRwt) and CF-KM4 (CFTRDeltaF508), as well as human airway epithelial cells from a patient with cystic fibrosis (CF-HAE) and from a healthy donor (HAE) were used for the functional analysis of our Ad vectors, Ad5/GFP-CFTRwt and Ad5/GFP-CFTRDeltaF508. The GFP-CFTRwt protein expressed was efficiently addressed to the plasma membrane of tracheal cells and to the apical surface of polarized CF-HAE cells, while GFP-CFTRDeltaF508 mutant was sequestered intracellularly. The functionality of the GFP-CFTRwt protein was demonstrated by its capacity to correct the chloride channel activity both in CF-KM4 and CF-HAE cells after Ad transduction. A correlation between the proportion of Ad5-transduced CF-KM4 cells and correction of CFTR function showed that 55 to 70% transduction resulted in 70% correction of the Cl- channel function. In reconstituted CF-HAE, GFP-CFTRwt appeared as active as the nontagged CFTRwt protein in correcting the transepithelial Cl- transport. We show for the first time a GFP-CFTR chimera that localized to the apical surface of human airway epithelia and restored epithelial chloride transport to similar levels as nontagged CFTR.     

Regulation of chemokine expression by NaCl occurs independently of cystic fibrosis transmembrane conductance regulator in macrophages

Chronic pulmonary inflammation and infection are the leading causes of morbidity and mortality in cystic fibrosis (CF). While the effect of mutation of the cystic fibrosis transmembrane conductance regulator (CFTR) on airways remains controversial, some groups have demonstrated increases in Na(+) and Cl(-) in CF airway surface liquid compared to normal airways. We investigated the consequences of NaCl on pro-inflammatory chemokine and cytokine production by macrophages. Stimulation of mouse macrophages with increasing amounts of NaCl induced macrophage inflammatory protein-2 (MIP-2) and tumor necrosis factor-alpha (TNF-alpha) production. Further, co-incubation of macrophages with NaCl in the presence of either lipopolysaccharide (LPS) or TNF-alpha synergistically increased MIP-2 production. Both the NaCl and NaCl plus LPS responses were partially dependent on endogenous production and autocrine signaling by TNF-alpha. To investigate the role of CFTR in MIP-2 production, we compared the responses of wild-type and DeltaF508 CF mouse macrophages to NaCl and LPS. The responses of macrophages from both strains were indistinguishable. In addition, CFTR mRNA was not expressed in macrophages. Taken together, these findings suggest that NaCl stimulates MIP-2 production by macrophages through a mechanism that is partially dependent on TNF-alpha but independent of macrophage CFTR expression.     

Mucin glycosylation and sulphation in airway epithelial cells is not influenced by cystic fibrosis transmembrane conductance regulator expression

Abnormalities in mucus properties and clearance make a major contribution to the pathology of cystic fibrosis (CF). Our aim was to test the hypothesis that the defects in CF mucus are a direct result of mutations in the CF transmembrane conductance regulator (CFTR) protein. We evaluated a single mucin molecule MUC1F/5ACTR that carries tandem repeat sequence from MUC5AC, a major secreted airway mucin, in a MUC1 mucin vector. To establish whether the presence of mutant or normal CFTR directly influences the O-glycosylation and sulphation of mucins in airway epithelial cells, we used the CFT1-LC3 (DeltaF508 CFTR mutant) and CFT1-LCFSN (wild-type CFTR corrected) human airway epithelial cell lines. MUC1F/5ACTR mucin was immunoprecipitated, centricon purified, and O-glycosylation was evaluated by Matrix-assisted laser desorption ionization and electrospray tandem mass spectrometry to determine the composition of different carbohydrate structures. Mass spectrometry data showed the same O-glycans in both CFTR mutant and wild-type CFTR corrected cells. Metabolic labeling assays were performed to evaluate gross glycosylation and sulphation of the mucins and showed no significant difference in mucin synthesized in six independent clones of these cell lines. Our results show that the absence of functional CFTR protein causes neither an abnormality in mucin O-glycosylation nor an increase in mucin sulphation.     

Demographics of the UK cystic fibrosis population: implications for neonatal screening

The objective was to determine the composition of the Cystic Fibrosis (CF) Population attending specialist UK CF centres in terms of age, gender, age at diagnosis, genotype and ethnicity. With the planned introduction of the national CF screening programme in the UK, cystic fibrosis transmembrane regulator (CFTR) mutations were compared between different ethnic groups enabling a UK-specific frequency of mutations to be defined. Data were analysed from the patient biographies held in the UK CF Database (see www.cystic-fibrosis.org.uk). The currently registered population of 5,274 CF patients is 96.3% Caucasian with a male preponderance that significantly increases with age. The majority of the 196 non-Caucasian CF patients are from the Indian Subcontinent (ISC), of which one in 84 UK CF patients are of Pakistani origin. The commonest CFTR mutation, deltaF508, is found in 74.1% of all CF chromosomes. In the Caucasian CF population, 57.5% are deltaF508 homozygotes but the UK ISC CF population with only 24.7%, has significantly fewer deltaF508 homozygotes patients (95% confidence interval (CI) 0.2-0.4). The distribution of Caucasian patients with deltaF508/deltaF508, deltaF508/Other and Other/Other does not fit the expected distribution with a Hardy-Weinberg model unless those patients without a detected mutation are excluded (P<0.001). The UK CF Database has shown the UK CF population to have distinct characteristics separate from the North American and European CF Registries. The ISC group contains many mutations not recognised by current genetic analysis, and one in four ISC patients have no CFTR mutations identified. The CFTR analysis proposed for the screening programme would detect 96% of patients registered in the database, but is unlikely to achieve the desired >80% detection rates in the ethnic minority groups. Screen-positive, non-Caucasian infants without an identifiable CFTR mutation should be referred for a sweat test and genetic counselling when serum trypsinogen concentrations remain elevated after birth.     

Suppression of CFTR premature termination codons and rescue of CFTR protein and function by the synthetic aminoglycoside NB54

Certain aminoglycosides are capable of inducing "translational readthrough" of premature termination codons (PTCs). However, toxicity and relative lack of efficacy deter treatment with clinically available aminoglycosides for genetic diseases caused by PTCs, including cystic fibrosis (CF). Using a structure-based approach, the novel aminoglycoside NB54 was developed that exhibits reduced toxicity and enhanced suppression of PTCs in cell-based reporter assays relative to gentamicin. We examined whether NB54 administration rescued CFTR protein and function in clinically relevant CF models. In a fluorescence-based halide efflux assay, NB54 partially restored halide efflux in a CF bronchial epithelial cell line (CFTR genotype W1282X/F508del), but not in a CF epithelial cell line lacking a PTC (F508del/F508del). In polarized airway epithelial cells expressing either a CFTR-W1282X or -G542X cDNA, treatment with NB54 increased stimulated short-circuit current (I (SC)) with greater efficiency than gentamicin. NB54 and gentamicin induced comparable increases in forskolin-stimulated I (SC) in primary airway epithelial cells derived from a G542X/F508del CF donor. Systemic administration of NB54 to Cftr-/- mice expressing a human CFTR-G542X transgene restored 15-17% of the average stimulated transepithelial chloride currents observed in wild-type (Cftr+/+) mice, comparable to gentamicin. NB54 exhibited reduced cellular toxicity in vitro and was tolerated at higher concentrations than gentamicin in vivo. These results provide evidence that synthetic aminoglycosides are capable of PTC suppression in relevant human CF cells and a CF animal model and support further development of these compounds as a treatment modality for genetic diseases caused by PTCs.