Assays of CFTR Function In Vitro, Ex Vivo and In Vivo.

Cystic fibrosis, a multi-organ genetic disease, is characterized by abnormal function of the cystic fibrosis transmembrane conductance regulator (CFTR) protein, a chloride channel at the apical membrane of several epithelia. In recent years, therapeutic strategies have been developed to correct the CFTR defect. To evaluate CFTR function at baseline for diagnosis, or the efficacy of CFTR-restoring therapy, reliable tests are needed to measure CFTR function, in vitro, ex vivo and in vivo. In vitro techniques either directly or indirectly measure ion fluxes; direct measurement of ion fluxes and quenching of fluorescence in cell-based assays, change in transmembrane voltage or current in patch clamp or Ussing chamber, swelling of CFTR-containing organoids by secondary water influx upon CFTR activation. Several cell or tissue types can be used. Ex vivo and in vivo assays similarly evaluate current (intestinal current measurement) and membrane potential differences (nasal potential difference), on tissues from individual patients. In the sweat test, the most frequently used in vivo evaluation of CFTR function, chloride concentration or stimulated sweat rate can be directly measured. Here, we will describe the currently available bio-assays for quantitative evaluation of CFTR function, their indications, advantages and disadvantages, and correlation with clinical outcome measures.

Functional Assays Are Essential for Interpretation of Missense Variants Associated with Variable Expressivity.

Missense DNA variants have variable effects upon protein function. Consequently, interpreting their pathogenicity is challenging, especially when they are associated with disease variability. To determine the degree to which functional assays inform interpretation, we analyzed 48 CFTR missense variants associated with variable expressivity of cystic fibrosis (CF). We assessed function in a native isogenic context by evaluating CFTR mutants that were stably expressed in the genome of a human airway cell line devoid of endogenous CFTR expression. 21 of 29 variants associated with full expressivity of the CF phenotype generated <10% wild-type CFTR (WT-CFTR) function, a conservative threshold for the development of life-limiting CF lung disease, and five variants had moderately decreased function (10% to ∼25% WT-CFTR). The remaining three variants in this group unexpectedly had >25% WT-CFTR function; two were higher than 75% WT-CFTR. As expected, 14 of 19 variants associated with partial expressivity of CF had >25% WT-CFTR function; however, four had minimal to no effect on CFTR function (>75% WT-CFTR). Thus, 6 of 48 (13%) missense variants believed to be disease causing did not alter CFTR function. Functional studies substantially refined pathogenicity assignment with expert annotation and criteria from the American College of Medical Genetics and Genomics and Association for Molecular Pathology. However, four algorithms (CADD, REVEL, SIFT, and PolyPhen-2) could not differentiate between variants that caused severe, moderate, or minimal reduction in function. In the setting of variable expressivity, these results indicate that functional assays are essential for accurate interpretation of missense variants and that current prediction tools should be used with caution.

Hypothesis: Possible respiratory advantages for heterozygote carriers of cystic fibrosis linked mutations during dusty climate of last glaciation.

This paper puts forward a new hypothesis to interpret the high carrier frequency of CFTR mutations in individuals of European descent. The proposed heterozygote advantage factor is related to the specific climate conditions in Europe during the last 50 ky that might have heavily compromised the respiratory function of our ancestors in Eurasia. A large part of the last 50 ky was cold, and the coldest period was the Last Glacial Maximum (LGM) (26.5 to 19 kya). The global climate was dry with a dust-laden atmosphere (20 to 25 times more dust than the present level). High levels of atmospheric dust started more than 40 kya and ended less than 10 kya. Secretion of airway fluid is usually related to the submucosal tissue hydration, while salt reabsorption relies on activation of CFTRs that allow ENaCs to absorb salt and water. The water loss by evaporation depends on the air humidity and flow rate. Salt accumulation in the mucus is normally prevented by reabsorption of Na(+) and Cl(-) by epithelial cells if the presence of functional CFTRs is normal. If one gene for CFTR is mutated, the number of functional CFTRs is reduced and this limits the capacity of salt reabsorption by epithelial cells. This means that evaporation makes the airway fluid more hypertonic, and osmotic forces bring more water from the interstitial space, thus leading to a new balance in mucosal fluid traffic. Increased osmolarity and volume of airway fluid can be more moveable in cases when evaporation and dust exposure is increased. If both CFTR genes are mutated, low number of functional CFTRs diminishes salt resorption of epithelial cells. Salt accumulated in the mucous fluid within respiratory ducts, as previously described. The hypertonic ductal content forces more water and some electrolytes to enter the airway fluid from the interstitial fluid, and evaporation leads to further concentration of thick immobile mucus. The proposed interpretation is that CFTR mutations have spread among our ancestors that roamed the central Eurasia after the LGM. The heterozygote individuals might have benefitted from the limited water resorption in their respiratory mucosa that allowed improved airway cleansing.

Heterogeneity of CFTR modulator-induced sweat chloride concentrations in people with cystic fibrosis.

BACKGROUND: Sweat chloride (SC) concentrations in people with cystic fibrosis (PwCF) reflect relative CF transmembrane conductance regulator (CFTR) protein function, the primary CF defect. Populations with greater SC concentrations tend to have lesser CFTR function and more severe disease courses. CFTR modulator treatment can improve CFTR function within specific CF genotypes and is commonly associated with reduced SC concentration. However, SC concentrations do not necessarily fall to concentrations seen in the unaffected population, suggesting potential for better CFTR treatment outcomes. We characterized post-modulator SC concentration variability among CHEC-SC study participants by genotype and modulator. METHODS: PwCF receiving commercially approved modulators for ≥90 days were enrolled for a single SC measurement. Clinical data were obtained from chart review and the CF Foundation Patient Registry (CFFPR). Variability of post-modulator SC concentrations was assessed by cumulative SC concentration frequencies. RESULTS: Post-modulator SC concentrations (n = 3787) were collected from 3131 PwCF; most (n = 1769, 47 %) were collected after elexacaftor/tezacaftor/ivacaftor (ETI) treatment. Modulator use was associated with lower SC distributions, with post-ETI concentrations the lowest on average. Most post-ETI SC concentrations were <60 mmol/L (79 %); 26 % were <30 mmol/L. Post-ETI distributions varied by genotype. All genotypes containing at least one F508del allele had individuals with post-ETI SC ≥60 mmol/L, with the largest proportion being F508del/minimal function (31 %). CONCLUSIONS: Post-modulator SC concentration heterogeneity was observed among all genotypes and modulators, including ETI. The presence of PwCF with post-modulator SC concentrations within the CF diagnostic range suggests room for additional treatment-associated CFTR restoration in this population.

Establishment of a conditionally reprogrammed primary eccrine sweat gland culture for evaluation of tissue-specific CFTR function.

BACKGROUND: Sweat chloride concentration is used both for CF diagnosis and for tracking CFTR modulator efficacy over time, but the relationship between sweat chloride and lung health is heterogeneous and informed by CFTR genotype. Here, we endeavored to characterize ion transport in eccrine sweat glands (ESGs). METHODS: First, ESGs were microdissected from a non-CF skin donor to analyze individual glands. We established primary cultures of ESG cells via conditional reprogramming for functional testing of ion transport by short circuit current measurement and examined cell composition by single-cell RNA-sequencing (scRNA-seq) comparing with whole dissociated ESGs. Secondly, we cultured nasal epithelial (NE) cells and ESGs from two people with CF (pwCF) to assess modulator efficacy. Finally, NEs and ESGs were grown from one person with the CFTR genotype F312del/F508del to explore genotype-phenotype heterogeneity. RESULTS: ESG primary cells from individuals without CF demonstrated robust ENaC and CFTR function. scRNA-seq demonstrated both secretory and ductal ESG markers in cultured ESG cells. In both NEs and ESGs from pwCF homozygous for F508del, minimal baseline CFTR function was observed, and treatment with CFTR modulators significantly enhanced function. Notably, NEs from an individual bearing F312del/F508del exhibited significant baseline CFTR function, whereas ESGs from the same person displayed minimal CFTR function, consistent with observed phenotype. CONCLUSIONS: This study has established a novel primary culture technique for ESGs that allows for functional ion transport measurement to assess modulator efficacy and evaluate genotype-phenoytpe heterogeneity. To our knowledge, this is the first reported application of conditional reprogramming and scRNA-seq of microdissected ESGs.

Characterizing CFTR modulated sweat chloride response across the cf population: Initial results from the CHEC-SC study.

BACKGROUND: CHEC-SC is an ongoing epidemiologic study characterizing modulator-induced sweat chloride (SC) responses across the CF population, with interim results available prior to the availability of triple combination modulator therapy. METHODS: Eligible participants had been prescribed a modulator for ≥90 days with re-enrollment allowed upon establishment of a new modulator. Pre-modulator SC values were obtained from chart review; post-modulator sweat was collected and analyzed locally. SC changes were descriptively summarized with biologic sex effects adjusted for age, weight, and CFTR genotype. Heterogeneity in ivacaftor SC response was characterized in relation to published CFTR functional responses. RESULTS: 1848 participants provided 2004 SC measurements, 26.2% on ivacaftor, 39.1% on lumacaftor/ivacaftor, and 34.7% on tezacaftor/ivacaftor. Average SC changes for all modulators were consistent with those reported in previous clinical studies, with greater variation in SC response observed among rarer mutations and notable shifts in the proportion with SC <60mmol/L independent of the magnitude of SC change. Ivacaftor induced in vitro CFTR functional change was significantly correlated with ivacaftor-modulated SC response (Pearson correlation= ‒0.52, 95% CI: ‒0.773, ‒0.129). Average SC change from ivacaftor to tezacaftor/ivacaftor was ‒4.9 mmol/L (n=17,95% CI:‒9.3, ‒0.5) and differed from those switching from lumacaftor/ivacaftor (10.0 mmol/L, n=139, 95% CI:7.8,12.3). Sex at birth was not associated with SC response. CONCLUSIONS: CHEC-SC is the largest study characterizing modulator-induced SC changes across the CF population. There was a strong association between ivacaftor induced in vitro CFTR function and SC response across a genotypically heterogenous cohort. Biological sex was not associated with SC response.

Effects of lumacaftor-ivacaftor therapy on cystic fibrosis transmembrane conductance regulator function in F508del homozygous patients with cystic fibrosis aged 2-11 years.

Rationale: Lumacaftor/ivacaftor was approved for the treatment of patients with cystic fibrosis who are homozygous for F508del aged 2 years and older following positive results from phase three trials. However, the improvement in CFTR function associated with lumacaftor/ivacaftor has only been studied in patients over 12 years of age, while the rescue potential in younger children is unknown. Methods: In a prospective study, we aimed to evaluate the effect of lumacaftor/ivacaftor on the CFTR biomarkers sweat chloride concentration and intestinal current measurement as well as clinical outcome parameters in F508del homozygous CF patients 2-11 years before and 8-16 weeks after treatment initiation. Results: A total of 13 children with CF homozygous for F508del aged 2-11 years were enrolled and 12 patients were analyzed. Lumacaftor/ivacaftor treatment reduced sweat chloride concentration by 26.8 mmol/L (p = 0.0006) and showed a mean improvement in CFTR activity, as assessed by intestinal current measurement in the rectal epithelium, of 30.5% compared to normal (p = 0.0015), exceeding previous findings of 17.7% of normal in CF patients homozygous for F508del aged 12 years and older. Conclusion: Lumacaftor/ivacaftor partially restores F508del CFTR function in children with CF who are homozygous for F508del, aged 2-11 years, to a level of CFTR activity seen in patients with CFTR variants with residual function. These results are consistent with the partial short-term improvement in clinical parameters.

Protospacer modification improves base editing of a canonical splice site variant and recovery of CFTR function in human airway epithelial cells.

Canonical splice site variants affecting the 5' GT and 3' AG nucleotides of introns result in severe missplicing and account for about 10% of disease-causing genomic alterations. Treatment of such variants has proven challenging due to the unstable mRNA or protein isoforms that typically result from disruption of these sites. Here, we investigate CRISPR-Cas9-mediated adenine base editing for such variants in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. We validate a CFTR expression minigene (EMG) system for testing base editing designs for two different targets. We then use the EMG system to test non-standard single-guide RNAs with either shortened or lengthened protospacers to correct the most common cystic fibrosis-causing variant in individuals of African descent (c.2988+1G>A). Varying the spacer region length allowed placement of the editing window in a more efficient context and enabled use of alternate protospacer adjacent motifs. Using these modifications, we restored clinically significant levels of CFTR function to human airway epithelial cells from two donors bearing the c.2988+1G>A variant.

Changes in the R-region interactions depend on phosphorylation and contribute to PKA and PKC regulation of the cystic fibrosis transmembrane conductance regulator chloride channel.

The CFTR chloride channel is regulated by phosphorylation at PKA and PKC consensus sites within its regulatory region (R-region) through a mechanism, which is still not completely understood. We used a split-CFTR construct expressing the N-term-TMD1-NBD1 (Front Half; FH), TMD2-NBD2-C-Term (Back Half; BH), and the R-region as separate polypeptides (Split-R) in BHK cells, to investigate in situ how different phosphorylation conditions affect the R-region interactions with other parts of the protein. In proximity ligation assays, we studied the formation of complexes between the R-region and each half of the Split-CFTR. We found that at basal conditions, the density of complexes formed between the R-region and both halves of the split channel were equal. PKC stimulation alone had no effect, whereas PKA stimulation induced the formation of more complexes between the R-region and both halves compared to basal conditions. Moreover, PKC + PKA stimulation further enhanced the formation of FH-R complexes by 40% from PKA level. In cells expressing the Split-R with the two inhibitory PKC sites on the R-region inactivated (SR-S641A/T682A), density of FH-R complexes was much higher than in Split-R WT expressing cells after PKC or PKC + PKA stimulation. No differences were observed for BH-R complexes measured at all phosphorylation conditions. Since full-length CFTR channels display large functional responses to PKC + PKA in WT and S641A/T682A mutant, we conclude that FH-R interactions are important for CFTR function. Inactivation of consensus PKC site serine 686 (S686A) significantly reduced the basal BH-R interaction and prevented the PKC enhancing effect on CFTR function and FH-R interaction. The phospho-mimetic mutation (S686D) restored basal BH-R interaction and the PKC enhancing effect on CFTR function with enhanced FH-R interaction. As the channel function is mainly stimulated by PKA phosphorylation of the R-region, and this response is known to be enhanced by PKC phosphorylation, our data support a model in which the regulation of CFTR activation results from increased interactions of the R-region with the N-term-TMD1-NBD1. Also, serine S686 was found to be critical for the PKC enhancing effect which requires a permissive BH-R interaction at basal level and increased FH-R interaction after PKC + PKA phosphorylation.

Cystic fibrosis transmembrane conductance regulator function, not TAS2R38 gene haplotypes, predict sinus surgery in children and young adults with cystic fibrosis.

BACKGROUND: Chronic rhinosinusitis symptomatology begins in early childhood individuals with cystic fibrosis (CF). Cystic fibrosis transmembrane conductance regulator (CFTR) function contributes to sinus development and disease. Genetic variants of the bitter taste receptor TAS2R38 have been suggested to contribute to sinus disease severity in individuals without CF. Our objective was to explore whether functional TAS2R38 haplotypes and CFTR function are associated with sinus disease or the need for sinus surgery in individuals with CF. METHODS: We conducted a retrospective study using prospectively collected data from the CF Twin-Sibling Study. The function of CFTR was assessed via chloride conductance. Genotyping of the TAS2R38 gene identified patients who were homozygous for the functional haplotype, heterozygous, or homozygous for nonfunctional haplotypes. Clustered multivariate logistic regression was performed, controlling for sex and family relationship. RESULTS: A total of 1291 patients were evaluated. Patients with ≤1% CFTR function were 1.56 times more likely to require sinus surgery than those with >1% CFTR function (p = 0.049). CFTR function did not correlate significantly with the presence of sinus disease (p = 0.30). In addition, there were no statistically significant differences in diagnosis of sinus disease or need for sinus surgery between patients with functional and nonfunctional TAS2R38 haplotypes. CONCLUSION: CFTR function correlates with need for sinus surgery, whereas TAS2R38 function does not appear to contribute to sinus disease or the need for sinus surgery in patients with CF.

Phenotyping of Rare CFTR Mutations Reveals Distinct Trafficking and Functional Defects.

Background. The most common CFTR mutation, F508del, presents with multiple cellular defects. However, the possible multiple defects caused by many rarer CFTR mutations are not well studied. We investigated four rare CFTR mutations E60K, G85E, E92K and A455E against well-characterized mutations, F508del and G551D, and their responses to corrector VX-809 and/or potentiator VX-770. Methods. Using complementary assays in HEK293T stable cell lines, we determined maturation by Western blotting, trafficking by flow cytometry using extracellular 3HA-tagged CFTR, and function by halide-sensitive YFP quenching. In the forskolin-induced swelling assay in intestinal organoids, we validated the effect of tagged versus endogenous CFTR. Results. Treatment with VX-809 significantly restored maturation, PM localization and function of both E60K and E92K. Mechanistically, VX-809 not only raised the total amount of CFTR, but significantly increased the traffic efficiency, which was not the case for A455E. G85E was refractory to VX-809 and VX-770 treatment. Conclusions. Since no single model or assay allows deciphering all defects at once, we propose a combination of phenotypic assays to collect rapid and early insights into the multiple defects of CFTR variants.

Ivacaftor in cystic fibrosis with residual function: Lung function results from an N-of-1 study.

BACKGROUND: Ivacaftor shows benefit in patients with cystic fibrosis (CF) and CFTR mutations associated with residual CF transmembrane conductance regulator (CFTR) function. Here we further assess the effect of ivacaftor in such patients using an N-of-1 study design. METHODS: Patients aged ≥12 years with CF with clinical or molecular evidence of residual CFTR function were randomized to 1 of 4 treatment sequences for two 4-week, double-blind crossover cycles (each divided into 2 weeks of ivacaftor treatment and placebo) followed by 8 weeks of open-label ivacaftor treatment. The primary endpoint was absolute change from cycle baseline of percent predicted forced expiratory volume in 1 s (ppFEV1) after 2 weeks of treatment with ivacaftor relative to placebo. RESULTS: Absolute change (SD) from study baseline in ppFEV1 favored ivacaftor by 2.3 (1.0) percentage points (95% credible interval, 0.4-4.1) after 2 weeks of treatment. Absolute mean change (SD) from open-label baseline (defined as day 1 of the open-label ivacaftor treatment period) in ppFEV1 after 8 weeks of treatment was 4.7 (4.2) percentage points (P<.0001). Safety of ivacaftor was consistent with that observed in prior studies. CONCLUSIONS: Ivacaftor improved lung function during the double-blind and open-label treatment periods in patients with CF and CFTR mutations associated with residual CFTR function (ClinicalTrials.gov, NCT01685801).

Nasal potential difference in suspected cystic fibrosis patients with 5T polymorphism.

BACKGROUND: 5T polymorphism is a CFTR mutation with unclear clinical consequences: the phenotype varies from healthy individuals to Cystic Fibrosis (CF). The aim of this study was to evaluate if nasal potential difference (NPD) and sweat testing correlate with symptoms and CF diagnosis in 5T patients. METHODS: 86 patients with 5T who had undergone NPD measurement, were included (6 homozygous (5T/5T), 41 with a PI-CF causing mutation in trans (5T/PI-CF), 11 with a PS-CF causing mutation in trans (5T/PS-CF) and 28 without a known mutation in trans (5T/?). Data including age, phenotype, sweat chloride and follow up were collected. RESULTS: 33% of the 5T/5T patients had abnormal NPD results, compared to 70% in 5T/PI-CF; 33% in 5T/PS-CF and 29% in 5T/?. The percentage of high or borderline sweat chloride was highest in 5T/PI-CF, and 5T/?, compared to 5T/5T and 5T/PS-CF (91, 96, 80, and 63%, respectively). TGm (number of TG repeats in intron 8) analysis was performed in 21 5T/PI-CF patients. TG11 was associated with lower sweat chloride, lower percentage of abnormal NPD and less progression of symptoms compared to TG12 and TG13. CONCLUSION: There is much variation in clinical status among 5T patients. All patients in this study with 5T/PS CF, all patients with both normal NPD and sweat test, and most patients with TG11 were stable or improving over time. Therefore, NPD measurement and TGm status aid to assess if a patient is at high risk for developing CF or CFTR-related disease and if specific follow up in a CF center is required.

Functional Profiling of CFTR-Directed Therapeutics Using Pediatric Patient-Derived Nasal Epithelial Cell Models.

Functional profiling of CFTR-directed therapeutics offers the potential to provide significant benefits to young people with cystic fibrosis (CF). However, the development of 2D airway epithelial cell models for individual response tests in CF children remains a central task. The objective of this study was to determine the utility of EpiXTM technology for expansion of nasal epithelial cells for use in electrophysiological CFTR function measurements. An initial harvest of as few as 20,000 cells was sufficient to expand up to 50 million cells that were used to generate air-liquid interface (ALI) cultures for ion transport studies with the Ussing assay. CFTR function was assessed by measuring responses to forskolin and the CFTR potentiator VX-770 (ivacaftor) in ALI cultures generated from passage 3 and 4 cells. Short-circuit current (Isc) measurements of blocked CFTR currents (ΔICFTRinh) discriminated CFTR function between healthy control (wild type, WT) and patients with intermediate (F508del/R117H-7T: 56% WT) and severe (F508del/F508del: 12% WT) CF disease. For the mixed genotypes, CFTR activity for F508del/c.850dupA was 12% WT, R334W/406-1G>A was 24% WT, and CFTRdele2,3(21 kb)/CFTRdele2,3(21 kb) was 9% WT. The CFTR correctors VX-809 (lumacaftor) and VX-661 (tezacaftor) significantly increased CFTR currents for F508del/R117H to 73 and 67% WT, respectively. Cultures with the large deletion mutation CFTRdele2,3(21 kb) unexpectedly responded to VX-661 treatment (20% WT). Amiloride-sensitive sodium currents were robust and ranged between 20-80 µA/cm2 depending on the subject. In addition to characterizing the electrophysiological profile of mutant CFTR activity in cultures for five genotypes, our study exemplifies the promising paradigm of bed-to-bench side cooperation and personalized medicine.

Individualized medicine using intestinal responses to CFTR potentiators and correctors.

Cystic fibrosis transmembrane conductance regulator (CFTR) modulators that target the mutant CFTR protein are being introduced for treatment of cystic fibrosis. Stratification of subjects based on their CFTR genotype has been proven essential to demonstrate clinical efficacy of these novel treatments. Despite this stratification, considerable heterogeneity between subjects receiving CFTR modulators is still observed which remains largely uncharacterized. The CFTR genotype, and additional genetic and environmental factors that impact either tissue-specific CFTR protein characteristics or the pharmacokinetic properties of treatments will likely determine the individual response to therapy. The development of intestinal biomarkers for CFTR modulators may help to better quantitate individual responses to treatment, with potential to optimize treatments for subjects with limited responses, and the selection of responsive subjects that currently do not receive treatments. Here, recent advances concerning the use of intestinal biomarkers for CFTR modulator treatments are reviewed, with a focus on biomarkers of CFTR function in ex vivo rectal biopsies and in vitro cultured primary intestinal organoids. Their potential value is considered in the context of the current unmet needs for better treatments for the majority of subjects with CF, and individual biomarkers that enable the prediction of long term therapeutic responses to CFTR modulators. Pediatr Pulmonol. 2016;51:S23-S34. © 2016 Wiley Periodicals, Inc.

Elevated Mirc1/Mir17-92 cluster expression negatively regulates autophagy and CFTR (cystic fibrosis transmembrane conductance regulator) function in CF macrophages.

Cystic fibrosis (CF) is a fatal, genetic disorder that critically affects the lungs and is directly caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, resulting in defective CFTR function. Macroautophagy/autophagy is a highly regulated biological process that provides energy during periods of stress and starvation. Autophagy clears pathogens and dysfunctional protein aggregates within macrophages. However, this process is impaired in CF patients and CF mice, as their macrophages exhibit limited autophagy activity. The study of microRNAs (Mirs), and other noncoding RNAs, continues to offer new therapeutic targets. The objective of this study was to elucidate the role of Mirs in dysregulated autophagy-related genes in CF macrophages, and then target them to restore this host-defense function and improve CFTR channel function. We identified the Mirc1/Mir17-92 cluster as a potential negative regulator of autophagy as CF macrophages exhibit decreased autophagy protein expression and increased cluster expression when compared to wild-type (WT) counterparts. The absence or reduced expression of the cluster increases autophagy protein expression, suggesting the canonical inverse relationship between Mirc1/Mir17-92 and autophagy gene expression. An in silico study for targets of Mirs that comprise the cluster suggested that the majority of the Mirs target autophagy mRNAs. Those targets were validated by luciferase assays. Notably, the ability of macrophages expressing mutant F508del CFTR to transport halide through their membranes is compromised and can be restored by downregulation of these inherently elevated Mirs, via restoration of autophagy. In vivo, downregulation of Mir17 and Mir20a partially restored autophagy expression and hence improved the clearance of Burkholderia cenocepacia. Thus, these data advance our understanding of mechanisms underlying the pathobiology of CF and provide a new therapeutic platform for restoring CFTR function and autophagy in patients with CF.

Improved repeatability of nasal potential difference with a larger surface catheter.

OBJECTIVE: To increase the power of nasal potential difference (NPD) as a biomarker of CFTR function, improvement of its repeatability is needed. We evaluated the improvement in repeatability resulting from measuring NPD (1) over a larger surface area and (2) at a fixed location. METHODS: To assess repeatability, NPD was measured on two occasions with a new method using a larger surface catheter at fixed locations on the nasal floor (LSC-floor(5cm) and LSC-floor(3cm)) or at the most negative basal potential (LSC-floor(max)); with a sidehole catheter on the nasal floor at 5 cm) from the nasal margin (SHC-floor(5cm)) or at the most negative potential (SHC-floor(max)); and with an endhole catheter below the inferior surface of the lower turbinate at the most negative potential (EHC-turb(max)). RESULTS: The within-subject standard deviation (S(w)) for repeated measurements of the total chloride response in the controls was smallest with the LSC-floor at a fixed location (LSC-floor(5cm) 3.1 mV; 95% CI 2.3-4.6 mV) and highest with the SHC-floor (SHC-floor(max) 14.6 mV; 95% CI 10.9-22.2 mV) or the EHC-turbinate (EHC-turb(max) 12.5 mV; 95% CI 10.7-23.0 mV) at the most negative basal potential. Measuring with the LSC-floor at the maximal potential increased the Sw (LSC-floor(max) 8.8 mV, 95% CI 6.0-16.1 mV, p=0.009 vs LSC-floor(5cm)), while measuring with the SHC-floor at a fixed location slightly decreased the Sw (SHC-floor(5cm) 9.8 mV, 95% CI 8.9-20.6 mV, p=0.06 vs SHC-floor(max)). In patients with cystic fibrosis, the S(w) was comparable, between 2.2 mV and 4.3 mV. Sample size calculations for trials using NPD to assess changes in ion transport showed that the number of subjects to be included could be approximately halved measuring with the larger surface catheter at a fixed location vs SHC or EHC at fixed locations. CONCLUSION: Measuring the NPD at a fixed location and over a larger surface resulted in increased repeatability and thereby also power as a biomarker of CFTR modulation.

Increased efficacy of VX-809 in different cellular systems results from an early stabilization effect of F508del-CFTR.

Cystic fibrosis (CF), the most common recessive autosomal disease among Caucasians, is caused by mutations in the gene encoding the CF transmembrane conductance regulator (CFTR) protein. The most common mutation, F508del, leads to CFTR impaired plasma membrane trafficking. Therapies modulating CFTR basic defect are emerging, such as VX-809, a corrector of F508del-CFTR traffic which just succeeded in a Phase III clinical trial. We recently showed that VX-809 is additive to two other correctors (VRT-325 and compound 4a). Here, we aimed to determine whether the differential rescuing by these compounds results from cell-specific factors or rather from distinct effects at the early biogenesis and/or processing. The rescuing efficiencies of the above three correctors were first compared in different cellular models (primary respiratory cells, cystic fibrosis bronchial epithelial and baby hamster kidney [BHK] cell lines) by functional approaches: micro-Ussing chamber and iodide efflux. Next, biochemical methods (metabolic labeling, pulse-chase and immunoprecipitation) were used to determine their impact on CFTR biogenesis / processing. Functional analyses revealed that VX-809 has the greatest rescuing efficacy and that the relative efficiencies of the three compounds are essentially maintained in all three cellular models tested. Nevertheless, biochemical data show that VX-809 significantly stabilizes F508del-CFTR immature form, an effect that is not observed for C3 nor C4. VX-809 and C3 also significantly increase accumulation of immature CFTR. Our data suggest that VX-809 increases the stability of F508del-CFTR immature form at an early phase of its biogenesis, thus explaining its increased efficacy when inducing its rescue.

Novel opportunities for CFTR-targeting drug development using organoids.

Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. CFTR mutations lead to production of non-functional CFTR, reduced amounts of normal functioning CFTR or misfolded CFTR with defects in trafficking or function. For decades, CF treatment has been focused on the symptoms of CF, but pharmacotherapy using small molecules that target the basic defect of CF, the mutant CFTR protein, is now possible for a limited amount of subjects with CF. This raises the exciting possibility that the majority of people with CF may receive effective treatment targeting the different CFTR mutants in the future. We recently described a functional CFTR assay using rectal biopsies from subjects with CF that were cultured in vitro into self-organizing mini-guts or organoids. We here describe how this model may assist in the discovery of new CFTR-targeting drugs, the subjects that may benefit from these drugs, and the mechanisms underlying variability in CFTR genotype-phenotype relations.