Function Of Cystic Fibrosis Transmembrane Conductance Regulator Protein Research Articles

In Utero Mapping and Development Role of CFTR in Lung and Gastrointestinal Tract of Cystic Fibrosis Patients.

In cystic fibrosis (CF) the ability of the CF transmembrane conductance regulator (CFTR) protein to mediate chloride and water transport is disrupted. While much progress has been made in CF research leading to effective treatments to improve CFTR function, including small molecule modulators, patients present with varying disease manifestations and responses to therapy. For many CF-affected organs, disease onset is known to occur during in utero development before treatments can be administered and progresses over time leading to irreversible damage to these organs. Thus, the role of functional CFTR protein, in particular, during early development needs to be further elucidated. Studies have detected CFTR proteins at very early gestational stages and revealed temporally and spatially variable CFTR expression patterns in fetuses, suggesting a potential role of CFTR in fetal development. However, the actual mechanisms of how defective CFTR in CF results in fetal morphogenetic abnormalities are yet to be established. This review aims to summarize fetal CFTR expression patterns specifically in the lung, pancreas, and gastrointestinal tract (GIT), as compared to adult patterns. Case studies of structural abnormalities in CF fetuses and newborns and the role of CFTR in fetal development will also be discussed.

S945L-CFTR molecular dynamics, functional characterization and tezacaftor/ivacaftor efficacy in vivo and in vitro in matched pediatric patient-derived cell models.

Cystic Fibrosis (CF) results from over 400 different disease-causing mutations in the CF Transmembrane Conductance Regulator (CFTR) gene. These CFTR mutations lead to numerous defects in CFTR protein function. A novel class of targeted therapies (CFTR modulators) have been developed that can restore defects in CFTR folding and gating. This study aimed to characterize the functional and structural defects of S945L-CFTR and interrogate the efficacy of modulators with two modes of action: gating potentiator [ivacaftor (IVA)] and folding corrector [tezacaftor (TEZ)]. The response to these modulators in vitro in airway differentiated cell models created from a participant with S945L/G542X-CFTR was correlated with in vivo clinical outcomes of that participant at least 12 months pre and post modulator therapy. In this participants' airway cell models, CFTR-mediated chloride transport was assessed via ion transport electrophysiology. Monotherapy with IVA or TEZ increased CFTR activity, albeit not reaching statistical significance. Combination therapy with TEZ/IVA significantly (p = 0.02) increased CFTR activity 1.62-fold above baseline. Assessment of CFTR expression and maturation via western blot validated the presence of mature, fully glycosylated CFTR, which increased 4.1-fold in TEZ/IVA-treated cells. The in vitro S945L-CFTR response to modulator correlated with an improvement in in vivo lung function (ppFEV1) from 77.19 in the 12 months pre TEZ/IVA to 80.79 in the 12 months post TEZ/IVA. The slope of decline in ppFEV1 significantly (p = 0.02) changed in the 24 months post TEZ/IVA, becoming positive. Furthermore, there was a significant improvement in clinical parameters and a fall in sweat chloride from 68 to 28 mmol/L. The mechanism of dysfunction of S945L-CFTR was elucidated by in silico molecular dynamics (MD) simulations. S945L-CFTR caused misfolding of transmembrane helix 8 and disruption of the R domain, a CFTR domain critical to channel gating. This study showed in vitro and in silico that S945L causes both folding and gating defects in CFTR and demonstrated in vitro and in vivo that TEZ/IVA is an efficacious modulator combination to address these defects. As such, we support the utility of patient-derived cell models and MD simulations in predicting and understanding the effect of modulators on CFTR function on an individualized basis.

Rate of Lung Function Decline in People with Cystic Fibrosis Having a Residual Function Gene Mutation.

Cystic fibrosis (CF) is an autosomal recessive disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. Approximately 5% of people with CF have residual function (RF) CFTR mutations that result in partially retained CFTR activity. Published literature on disease trajectory among those with RF mutations is limited. In this retrospective study, we characterized lung function decline across different age groups in CFTR modulator-untreated people with CF heterozygous for F508del and an RF mutation (F/RF). Rate of decline in percent predicted forced expiratory volume in 1s (ppFEV1) was analyzed using data from the US CF Foundation Patient Registry (2006-2014) in F/RF (all), F/RF (excluding R117H), and F508del homozygous (F/F) cohorts. Annual rates of ppFEV1 decline were estimated over 2-year periods based on calendar year. Subgroup analyses by age [6-12 (children), 13-17 (adolescents), 18-24 (young adults), and ≥ 25years (adults)] were performed. The estimated annualized rate of ppFEV1 decline was - 0.70percentage points per year (95% CI -1.09, -0.30) in the F/RF (all) cohort (N = 1242) versus -1.91 percentage points per year (95% CI -2.01, -1.80) in the F/F cohort (N = 11,916) [difference, 1.29 percentage points per year (95% CI 0.88, 1.70); P < 0.001]. In the F/RF (all) cohort, all age groups demonstrated lung function decline ranging from -0.30 to -1.38. In the F/RF (excluding R117H) cohort, the rate of decline was -1.05 percentage points per year (95% CI -1.51, -0.60) [difference versus F/F cohort, 0.95 percentage points per year (95% CI 0.48, 1.41; P < 0.001); not statistically significant in children and young adults]. Progressive lung function decline was observed in people with F/RF genotypes across all assessed age groups, reinforcing the importance of early intervention and clinical monitoring to preserve lung function in all people with CF.

Effectiveness and Safety of Cystic Fibrosis Transmembrane Conductance Regulator Modulators in Children With Cystic Fibrosis: A Meta-Analysis.

Background and AimCystic fibrosis (CF) is a genetic disease that is difficult to treat and caused by dysfunction of the cystic fibrosis transmembrane conductance regulator (CFTR) protein. Small molecules have been used to treat the symptom caused by CFTR mutations by restoring CFTR protein function. However, the data on children with CF are scarce. This meta-analysis aimed to evaluate the effectiveness and safety of this therapy in children diagnosed with CF.Materials and MethodsRelevant studies were identified through searching medical databases before April 1, 2022. The primary outcomes of ppFEV1, lung clearance index2.5 (LCI2.5), sweat chloride concentration (SwCI), and Cystic Fibrosis Questionnaire-Revised (CFQ-R) score were pooled and analyzed. The secondary outcomes were nutritional status (weight, BMI, stature, and their z-score) and adverse events under therapy.ResultsA total of twelve studies were included. Compared with the placebo group, the pooled outcome of the ppFEV1, LCI2.5, SwCI, and CFQ-R score were improved by 7.91 {[95% confidence interval (CI), 3.71–12.12], –1.00 (95% CI, –1.38 to –0.63), –35.22 (95% CI, –55.51 to –14.92), and 4.45 (95% CI, 2.31–6.59), respectively}. Compared with the placebo group, the pooled result of the change in weight was improved by 1.53 (95% CI, 0.42–2.63). All the aforementioned results were also improved in single-arm studies. No clear differences in adverse events were found between CFTR modulator therapy and the placebo group.ConclusionCFTR modulators could improve multiaspect function in children with CF and result in comparable adverse events.

Cirrhosis and Portal Hypertension in Compound Heterozygous People With Cystic Fibrosis Harboring One F508del CFTR Gene Mutation

Cirrhosis and Portal Hypertension in Compound Heterozygous People With Cystic Fibrosis Harboring One F508del CFTR Gene Mutation

CFTR Modulators in People with Cystic Fibrosis: Real-World Evidence in France.

Cystic fibrosis (CF) is a rare genetic multisystemic disease, the manifestations of which are due to mutations in the gene encoding the CF transmembrane conductance regulator (CFTR) protein and can lead to respiratory insufficiency and premature death. CFTR modulators, which were developed in the past decade, partially restore CFTR protein function. Their clinical efficacy has been demonstrated in phase 3 clinical trials, particularly in terms of lung function and pulmonary exacerbations, nutritional status, and quality of life in people with gating mutations (ivacaftor), homozygous for the F508del mutation (lumacaftor/ivacaftor and tezacaftor/ivacaftor), and in those with at least one F508del mutation (elexacaftor/tezacaftor/ivacaftor). However, many questions remain regarding their long-term safety and effectiveness, particularly in patients with advanced lung disease, liver disease, renal insufficiency, or problematic bacterial colonization. The impact of CFTR modulators on other important outcomes such as concurrent treatments, lung transplantation, chest imaging, or pregnancies also warrants further investigation. The French CF Reference Network includes 47 CF centers that contribute patient data to the comprehensive French CF Registry and have conducted nationwide real-world studies on CFTR modulators. This review seeks to summarize the results of these real-world studies and examine their findings against those of randomized control trials.

CFTR Modulators and Lung Transplantation: Factors to Consider - A UK Transplant Centre Experience

<h3>Purpose</h3> Cystic fibrosis transmembrane conductance regulator (CFTR) modulators are drugs that act on the defective CFTR protein in people with cystic fibrosis (pwCF), resulting in improved CFTR protein function and thus improved CF symptoms. Evidence is increasing of their positive impact on extra-pulmonary features of CF, which remains problematic in pwCF post-lung transplantation (LTx). Elexacaftor/tezacaftor/ivacaftor (ele/tez/iva) was licensed in the UK in August 2020. It has been debated since if ele/tez/iva has a role post-LTx for ongoing non-pulmonary CF symptoms. We aim to highlight the practical issues faced when considering this drug in the post-LTx CF population in a single large UK LTx unit. <h3>Methods</h3> Retrospective analysis of electronic records of pwCF post-LTx with ongoing extra-pulmonary manifestations considered for initiation of ele/tez/iva August 2020-2021, including demographics, potential benefits, contraindications and outcomes. <h3>Results</h3> 5 pwCF with LTx have been discussed with their CF physicians to consider initiating ele/tez/iva (some had more than one problem):- sinus (n = 2); bowels (n = 2); weight (n = 2); other (n = 2). All were at least 12 months post-LTx (median 42 months; range 21-191 months). Discussions were initiated by the patient in 1 case, the CF team in 2, and transplant team in 2. 3 were not initiated - 1 due to potential side effects on mental health and 2 were contraindicated with deranged liver tests and ongoing bowel symptoms. 2 were initiated on ele/tez/iva. Of these, one is on tacrolimus, needed no immunosuppression adjustment, but stopped due to drop in FEV1 (CT and biopsy negative for features of rejection) which recovered. The other is on sirolimus, needed significant reduction in immunosuppression (9mg to 4mg) and remains on ele/tez/iva. <h3>Conclusion</h3> Extra-pulmonary manifestations of CF are varied and continue post-LTx. With increasing evidence supporting using CFTR modulators to manage extra-pulmonary symptoms, it is important to establish the role these drugs can have in the post-LTx CF population, but also the potential risks. Our experience so far shows mixed results and that discussion between CF and transplant physicians, with patient involvement is vital to ensure correct decisions are taken. Randomised controlled trials are needed to ensure modulator use in pwCF post-LTx is supported by robust evidence.

Efficacy and Safety of Triple Combination Cystic Fibrosis Transmembrane Conductance Regulator Modulators in Patients With Cystic Fibrosis: A Meta-Analysis of Randomized Controlled Trials

Background: Cystic fibrosis is a rare, recessive, progressive genetic disease caused by dysfunction of the cystic fibrosis transmembrane conductance regulator (CFTR) protein. Small molecules have recently been developed to treat the molecular consequences of CFTR mutations and restore CFTR protein function. However, the data on triple combination therapy (mainly from Vertex Pharmaceuticals, which is most tested in clinical trials) are limited. This meta-analysis was aimed to assess the efficacy and safety of this therapy according to different mutation genotypes and comparators. Methods: Relevant publications were identified through searching several medical databases before 31 December 2021. The primary outcomes of ppFEV1, sweat chloride concentration and Cystic Fibrosis Questionnaire-Revised (CFQ-R) score were pooled and analyzed. The secondary outcomes were adverse events in triple combination therapy. Results: Six randomized controlled trials were eligible for analysis. The total outcome of the ppFEV1 change was higher with triple combination therapy than triple placebo or active control (mean difference, MD, 13.6% and 8.74%, respectively). The pooled result of sweat chloride concentrations with triple combination therapy was lower than that of triple placebo or active control (MD, −44.13 and −39.26, respectively). The pooled estimate of the CFQ-R score was higher with triple combination therapy than triple placebo or active control (MD, 19.8% and 14.63%, respectively). No clear differences in adverse events were found between triple combination therapy and the control (placebo or active control). Conclusion: CFTR modulators in triple combination achieve better clinical results than placebo and active control, and result in comparable adverse events. Systematic Review Registration: https://www.crd.york.ac.uk/prospero/display_record.php?ID=CRD42021293402, identifier PROSPERO 2021 CRD42021293402.

Therapeutic Approaches for Patients with Cystic Fibrosis Not Eligible for Current CFTR Modulators.

Cystic fibrosis is a severe autosomal recessive disorder caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene encoding the CFTR protein, a chloride channel expressed in many epithelial cells. New drugs called CFTR modulators aim at restoring the CFTR protein function, and they will benefit many patients with cystic fibrosis in the near future. However, some patients bear rare mutations that are not yet eligible for CFTR modulators, although they might be amenable to these new disease-modifying drugs. Moreover, more than 10% of CFTR mutations do not produce any CFTR protein for CFTR modulators to act upon. The purpose of this review is to provide an overview of different approaches pursued to treat patients bearing mutations ineligible for CFTR modulators. One approach is to broaden the numbers of mutations eligible for CFTR modulators. This requires developing strategies to evaluate drugs in populations bearing very rare genotypes. Other approaches aiming at correcting the CFTR defect develop new mutation-specific or mutation-agnostic therapies for mutations that do not produce a CFTR protein: readthrough agents for nonsense mutations, nucleic acid-based therapies, RNA- or DNA-based, and cell-based therapies. Most of these approaches are in pre-clinical development or, for some of them, early clinical phases. Many hurdles and challenges will have to be solved before they can be safely translated to patients.

Triple Therapy for Cystic Fibrosis Phe508del–Gating and –Residual Function Genotypes

BackgroundElexacaftor–tezacaftor–ivacaftor is a small-molecule cystic fibrosis transmembrane conductance regulator (CFTR) modulator regimen shown to be efficacious in patients with at least one Phe508del allele, which indicates that this combination can modulate a single Phe508del allele. In patients whose other CFTR allele contains a gating or residual function mutation that is already effectively treated with previous CFTR modulators (ivacaftor or tezacaftor–ivacaftor), the potential for additional benefit from restoring Phe508del CFTR protein function is unclear.MethodsWe conducted a phase 3, double-blind, randomized, active-controlled trial involving patients 12 years of age or older with cystic fibrosis and Phe508del–gating or Phe508del–residual function genotypes. After a 4-week run-in period with ivacaftor or tezacaftor–ivacaftor, patients were randomly assigned to receive elexacaftor–tezacaftor–ivacaftor or active control for 8 weeks. The primary end point was the absolute change in the percentage of predicted forced expiratory volume in 1 second (FEV1) from baseline through week 8 in the elexacaftor–tezacaftor–ivacaftor group.ResultsAfter the run-in period, 132 patients received elexacaftor–tezacaftor–ivacaftor and 126 received active control. Elexacaftor–tezacaftor–ivacaftor resulted in a percentage of predicted FEV1 that was higher by 3.7 percentage points (95% confidence interval [CI], 2.8 to 4.6) relative to baseline and higher by 3.5 percentage points (95% CI, 2.2 to 4.7) relative to active control and a sweat chloride concentration that was lower by 22.3 mmol per liter (95% CI, 20.2 to 24.5) relative to baseline and lower by 23.1 mmol per liter (95% CI, 20.1 to 26.1) relative to active control (P<0.001 for all comparisons). The change from baseline in the Cystic Fibrosis Questionnaire–Revised respiratory domain score (range, 0 to 100, with higher scores indicating better quality of life) with elexacaftor–tezacaftor–ivacaftor was 10.3 points (95% CI, 8.0 to 12.7) and with active control was 1.6 points (95% CI, −0.8 to 4.1). The incidence of adverse events was similar in the two groups; adverse events led to treatment discontinuation in one patient (elevated aminotransferase level) in the elexacaftor–tezacaftor–ivacaftor group and in two patients (anxiety or depression and pulmonary exacerbation) in the active control group.ConclusionsElexacaftor–tezacaftor–ivacaftor was efficacious and safe in patients with Phe508del–gating or Phe508del–residual function genotypes and conferred additional benefit relative to previous CFTR modulators. (Funded by Vertex Pharmaceuticals; VX18-445-104 ClinicalTrials.gov number, NCT04058353.)

CFTR Modulator Therapy and Its Impact on Lung Transplantation in Cystic Fibrosis.

Cystic fibrosis (CF) is the most common autosomal recessive disorder in Caucasian people and is caused by mutations in the gene encoding for the CF transmembrane conductance regulator (CFTR) protein. It is a multisystem disorder; however, CF lung disease causes most of its morbidity and mortality. Although survival for CF has improved over time due to a multifaceted symptomatic management approach, CF remains a life-limiting disease. For individuals with progressive advanced CF lung disease (ACFLD), lung transplantation is considered the ultimate treatment option if compatible with goals of care. Since 2012, newer drugs, called CFTR modulators, have gradually become available, revolutionizing CF care, as these small-molecule drugs target the underlying defect in CF that causes decreased CFTR protein synthesis, function, or stability. Because of their extremely high efficacy and overall respectable tolerability, CFTR modulator drugs have already proven to have a substantial positive impact on the lives of individuals with CF. Individuals with ACFLD have generally been excluded from initial clinical trials. Now, however, these drugs are being used in clinical practice in selected individuals with ACFLD, showing promising results, although randomized controlled trial data for CFTR modulators in this subgroup of patients are lacking. Such data need to be gathered, ideally in randomized controlled trials including patients with ACFLD. Furthermore, the efficacy and tolerability of the newer modulator therapies in individuals with ACFLD need to be monitored, and their impact on lung disease progression and the need for lung transplantation as the ultimate therapy call for an objective evaluation in larger patient cohorts. As of today, guidelines for referral and listing of lung transplant candidates with CF have not incorporated the status of the new CFTR modulator therapies in the referral and listing process. The purpose of this review article, therefore, is threefold: first, to describe the effects of new therapies, with a focus on the subgroup of individuals with ACFLD; second, to provide an update on the recent outcomes after lung transplantation for individuals with CF; and third, to discuss the referral, evaluation, and timing for lung transplantation as the ultimate therapeutic option in view of the new treatments available in CF.

Effect of Alpha-1 Antitrypsin on CFTR Levels in Primary Human Airway Epithelial Cells Grown at the Air-Liquid-Interface.

The cystic fibrosis transmembrane conductance regulator (CFTR) gene is influenced by the fundamental cellular processes like epithelial differentiation/polarization, regeneration and epithelial–mesenchymal transition. Defects in CFTR protein levels and/or function lead to decreased airway surface liquid layer facilitating microbial colonization and inflammation. The SERPINA1 gene, encoding alpha1-antitrypsin (AAT) protein, is one of the genes implicated in CF, however it remains unknown whether AAT has any influence on CFTR levels. In this study we assessed CFTR protein levels in primary human lung epithelial cells grown at the air-liquid-interface (ALI) alone or pre-incubated with AAT by Western blots and immunohistochemistry. Histological analysis of ALI inserts revealed CFTR- and AAT-positive cells but no AAT-CFTR co-localization. When 0.5 mg/mL of AAT was added to apical or basolateral compartments of pro-inflammatory activated ALI cultures, CFTR levels increased relative to activated ALIs. This finding suggests that AAT is CFTR-modulating protein, albeit its effects may depend on the concentration and the route of administration. Human lung epithelial ALI cultures provide a useful tool for studies in detail how AAT or other pharmaceuticals affect the levels and activity of CFTR.

Genetics of CFTR and male infertility.

Cystic fibrosis (CF) is a rare autosomal-recessive disorder manifested as multisystem organ dysfunction. The cystic fibrosis transmembrane conductance regulator (CFTR) protein functions as an ion transporter on the epithelium of exocrine glands, regulating secretion viscosity. The CFTR gene, encoded on chromosome 7, is required for the production and trafficking of the intact and functional CFTR protein. Literally thousands of human CFTR allelic mutations have been identified, each with varying impact on protein quality and quantity. As a result, individuals harboring CFTR mutations present with a spectrum of symptoms ranging from CF to normal phenotypes. Those with loss of function but without full CF may present with CFTR-related disorders (CFTR-RDs) including male infertility, sinusitis, pancreatitis, atypical asthma and bronchitis. Studies have demonstrated associations between higher rates of CFTR mutations and oligospermia, epididymal obstruction, congenital bilateral absence of the vas deferens (CBAVD), and idiopathic ejaculatory duct obstruction (EDO). Genetic variants are detected in over three-quarters of men with CBAVD, the reproductive abnormality most classically associated with CFTR aberrations. Likewise, nearly all men with clinical CF will have CBAVD. Current guidelines from multiple groups recommend CFTR screening in all men with clinical CF or CBAVD though a consensus on the minimum number of variants for which to test is lacking. CFTR testing is not recommended as routine screening for men with other categories of infertility. While available CFTR panels include 30 to 96 of the most common variants, complete gene sequencing should be considered if there is a high index of suspicion in a high-risk couple (e.g., partner is CFTR mutation carrier). CF treatments to date have largely targeted end-organ complications. Novel CFTR-modulator treatments aim to directly target CFTR protein dysfunction, effectively circumventing downstream complications, and possibly preventing symptoms like vasal atresia at a young age. Future gene therapies may also hold promise in preventing or reversing genetic changes that lead to CF and CFTR-RD.

Lumacaftor/ivacaftor in cystic fibrosis: effects on glucose metabolism and insulin secretion

PurposeThe question whether the new cystic fibrosis transmembrane conductance regulator (CFTR) modulator drugs aimed at restoring CFTR protein function might improve glucose metabolism is gaining attention, but data on the effect of lumacaftor/ivacaftor treatment (LUMA/IVA) on glucose tolerance are limited. We evaluated the variation in glucose metabolism and insulin secretion in CF patients homozygous for Phe508del CFTR mutation after one-year treatment with LUMA/IVA in comparison to patients with the same genotype who did not receive such treatment.MethodsWe performed a retrospective case–control study on 13 patients with a confirmed diagnosis of CF, homozygous for the Phe508del CFTR mutation, who received LUMA/IVA for one year (cases) and 13 patients with identical genotype who did not receive this treatment (controls). At the beginning and conclusion of the follow-up, all subjects received a modified 3 h OGTT, sampling at baseline, and at 30 min intervals for plasma glucose, serum insulin, and c-peptide concentrations to evaluate glucose tolerance, and quantify by modeling beta-cell insulin secretion responsiveness to glucose, insulin clearance and insulin sensitivity.ResultsLUMA/IVA did not produce differences in glucose tolerance, insulin secretory parameters, clearance and sensitivity with respect to matched controls over one-year follow-up.ConclusionWe found no evidence of improvements in glucose tolerance mechanisms in patients with CF after one-year treatment with LUMA/IVA.

Entering the era of highly effective modulator therapies.

Since the discovery of the gene responsible for cystic fibrosis (CF) in 1989, hopes have been pinned on a future with novel therapies tackling the basis of the disease rather than its symptoms. These have become a reality over the last decade with the development through to the clinic of CF transmembrane conductance regulator (CFTR) modulators. These are oral drugs which improve CFTR protein function through either increasing the time the channel pore is open (potentiators) or facilitating its trafficking through the cell to its location on the cell membrane (correctors). The first potentiator, ivacaftor, is now licensed and available clinically in many parts of the world. It is highly effective with impressive clinical impact in the lungs and gastrointestinal tract; longer-term data from patient registries show fewer exacerbations, a slower rate of lung function loss and reduced need for transplantation in patients receiving ivacaftor. However, as a single drug, it is suitable for only a small minority of patients. The commonest CFTR mutation, F508del, requires both correction and potentiation for clinical efficacy. Two dual-agent drugs (lumacaftor/ivacaftor and tezacaftor/ivacaftor) have progressed through to licensing, although their short term impact is more modest than that of ivacaftor; this is likely due to only partial correction of protein misfolding and trafficking. Most recently, triple compounds have been developed: two different corrector molecules (elexacaftor and tezacaftor) which, by addressing different regions in the misfolded F508del protein, more effectively improve trafficking. In addition to large improvements in clinical outcomes in people with two copies of F508del, the combination is sufficiently effective that it works in patients with only one copy of F508del and a second, nonmodulator responsive mutation. For the first time, we thus have a drug suitable for around 85% of people with CF. Even more gains are likely to be possible when these drugs can be used in younger children, although more sensitive outcome measures are needed for this age group. Special consideration is needed for people with very rare mutations; those with nonmodulatable mutation combinations will likely require gene or messenger RNA-based therapeutic approaches, many of which are being explored. Although this progress is hugely to be celebrated, we still have more work to do. The international collaboration between trials networks, pharma, patient organizations, registries, and people with CF is something we are all rightly proud of, but innovative trial design and implementation will be needed if we are to continue to build on this progress and further develop drugs for people with CF.

Emerging technologies for cystic fibrosis transmembrane conductance regulator restoration in all people with CF.

Although effective cystic fibrosis transmembrane conductance regulator (CFTR) modulator therapy has the potential to change the lives of many patients with cystic fibrosis (CF), it is unlikely that these drugs will be a game changing therapy for all. There are about 10% of patients with CF who don't produce a mutant protein tomodulate, potentiate, or optimize and for these patients such therapies are unlikely to be of significant benefit. There is a need to develop new therapeutic approaches that can work for this patient population and can advance CF therapies. These new therapies will be genetic-based therapies and each approach will result in functional CFTR protein inpreviously affected CF cells. In this review we will examine the potential of RNA therapies, gene transfer therapies, and gene editing therapies for the treatment of CF as well as the challenges that will need to be facedas we harness the power of these emerging therapies towards a one-time cure.

Identification of Compounds That Promote Readthrough of Premature Termination Codons in the CFTR.

Cystic fibrosis (CF) is caused by a mutation of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene, which disrupts an ion channel involved in hydration maintenance via anion homeostasis. Nearly 5% of CF patients possess one or more copies of the G542X allele, which results in a stop codon at residue 542, preventing full-length CFTR protein synthesis. Identifying small-molecule modulators of mutant CFTR biosynthesis that affect the readthrough of this and other premature termination codons to synthesize a fully functional CFTR protein represents a novel target area of drug discovery. We describe the implementation and integration for large-scale screening of a homogeneous, 1536-well functional G542X-CFTR readthrough assay. The assay uses HEK 293 cells engineered to overexpress the G542X-CFTR mutant, whose functional activity is monitored with a membrane potential dye. Cells are co-incubated with a CFTR amplifier and CFTR corrector to maximize mRNA levels and trafficking of CFTR to the cell surface. Compounds that allow translational readthrough and synthesis of functional CFTR chloride channels are reflected by changes in membrane potential in response to cAMP stimulation with forskolin and CFTR channel potentiation with genistein. Assay statistics yielded Z′ values of 0.69 ± 0.06. As further evidence of its suitability for high-throughput screening, we completed automated screening of approximately 666,000 compounds, identifying 7761 initial hits. Following secondary and tertiary assays, we identified 188 confirmed hit compounds with low and submicromolar potencies. Thus, this approach takes advantage of a phenotypic screen with high-throughput scalability to identify new small-molecule G542X-CFTR readthrough modulators.

Mechanism-Based Personalized Medicine for Cystic Fibrosis by Suppressing Pseudo Exon Inclusion

Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene that compromise its chloride channel activity. Here, we present a therapeutic strategy to ameliorate RNA splicing deficiency of CFTR with a small molecule. The 3,849+ 10 kb C>T is the most common splicing mutation in CF, creating a pseudo exon with premature stop codon. We reveal that the 3,849+ 10 kb C>T-induced CFTR pseudo exon is regulated by phosphorylation of serine/arginine-rich splicing factors, and their functional inhibition by a CDC-like kinase inhibitor restores normal splicing of CFTR. Subsequent screening of our focused chemical library identified CaNDY as a rectifier of the aberrant splicing. CaNDY treatment restored normal splicing of CFTR with the 3,849+ 10 kb C>T in CF patient cells and functional CFTR protein expression in the CF model cells. Our findings open the door for mechanism-based personalized medicine for pseudo-exon-type genetic diseases.

Correcting airways in cystic fibrosis

Gene Therapy Cystic fibrosis is caused by inactivating mutations in the cystic fibrosis transmembrane conductance regulator ( CFTR ) gene. Deletion of phenylalanine-508 is the most common mutation. Vaidyanathan et al. used CRISPR-associated protein 9 (Cas9)–mediated CFTR editing and adeno-associated virus delivery to correct the phenylalanine-508 deletion in upper-airway basal stem cells from 10 patients with cystic fibrosis. They achieved 30 to 50% gene correction and improved CFTR protein function. The corrected cells successfully engrafted within a clinically approved scaffold. This finding offers possibilities for clinical development of upper airway implants to treat respiratory failure, which is the biggest cause of mortality in cystic fibrosis patients. Cell Stem Cell 10.1016/j.stem.2019.11.002 (2019).

Positional effects of premature termination codons on the biochemical and biophysical properties of CFTR.

Biochemical and biophysical characterizations of three nonsense mutations of cystic fibrosis transmembrane conductance regulator (CFTR) associated with a severe form of cystic fibrosis (CF) reveal the importance and heterogenous effects of the position of the premature termination codon (PTC) on the CFTR protein function. Electrophysiological studies of W1282X-CFTR, whose PTC is closer to the C-terminus of CFTR, suggest the presence of both C-terminus truncated CFTR proteins that are poorly functional and read-through, full-length products. For G542X- and E60X-CFTR, the only mechanism capable of generating functional proteins is the read-through, but the outcome of read-through products is highly variable depending on the interplay between the missense mutation caused by the read-through and the structural context of the protein. Pharmacological studies of these three PTCs with various CFTR modulators suggest position-dependent therapeutic strategies for these disease-inflicting mutations. About one-third of genetic diseases and cancers are caused by the introduction of premature termination codons (PTCs). In theory, the location of the PTC in a gene determines the alternative mechanisms of translation, including premature cessation or reinitiation of translation, and read-through, resulting in differential effects on protein integrity. In this study, we used CFTR as a model system to investigate the positional effect of the PTC because of its well-understood structure-function relationship and pathophysiology. The characterization of three PTC mutations, E60X-, G542X- and W1282X-CFTR revealed heterogenous effects of these PTCs on CFTR function. The W1282X mutation results in both C-terminus truncated and read-through proteins that are partially or fully functional. In contrast, only the read-through protein is functional with E60X- and G542X-CFTR, although abundant N-terminus truncated proteins due to reinitiation of translation were detected in E60X-CFTR. Single-channel studies of the read-through proteins of E60X- and G542X-CFTR demonstrated that both mutations have a single-channel amplitude similar to wild type (WT), and good responses to high-affinity ATP analogues, suggesting intact ion permeation pathways and nucleotide binding domains (NBDs), albeit with reduced open probability (Po ). The comparison of the Po of these mutations with the proposed missense mutations revealed potential identities of the read-through products. Importantly, a majority of the functional protein studied responds to CFTR modulators like GLPG1837 and Lumacaftor. These results not only expand current understanding of the molecular (patho)physiology of CFTR, but also infer therapeutic strategies for different PTC mutations at large.