CFTR Modulators: Shedding Light on Precision Medicine for Cystic Fibrosis

1Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene cause cystic fibrosis (CF) – the most common life-limiting autosomal recessive inherited disorder in Caucasian people. The mutated gene translates into a defective CFTR protein with loss-of-activity. CFTR encodes an anion channel expressed in several cell types that: (1) transports chloride across the apical membrane; (2) modulates the activity of other ion channels, thereby regulating fluid and electrolyte balance in the mucosal membranes; and (3) secretes bicarbonate, which is crucial for pH regulation, host defense and protection against noxious stimuli. As a protein of the ATP-binding cassette (ABC) family, CFTR is comprised of two transmembrane domains (TMDs), two nucleotide-binding domains (NBDs) and a unique regulatory domain (RD) (Figure 1). Cyclic AMP (cAMP)-dependent protein kinase A (PKA), protein kinase C (PKC) and ATP control its activity. The most prevalent CFTR mutation was discovered almost 30 years ago; it was the deletion of a phenylalanine at position 508 (ΔF508), which affects ∼80% of CF patients worldwide. The mutation ΔF508 reduces thermal and kinetic stability of NBD1 and precludes interdomain interactions. Hence, the endoplasmic reticulum (ER) retains misfolded ΔF508-CFTR, which forms only a partially glycosylated protein, and the proteasome promptly degrades it.

2CF is a systemic disease that affects ∼85,000 people and presents heterogeneous distribution globally (Figure 2, Table 1). According to the latest registry reports, 38,985 CF patients are in Europe, 28,676 in the United States, 4,128 in Canada, 3,294 in Australia and 3,511 in Brazil (Figure 3). The major cause of morbidity and mortality in CF is the respiratory disorder caused by the lack of CFTR at the plasma membrane (PM), which decreases the anion permeability in airway cells and leads to a progressive pathophysiological cascade (Figure 4): (1) A faulty conductance of other ions, such as excessive sodium transport mediated by epithelial Na+ channel (ENaC), since it is negatively regulated by CFTR. Controversially, some reports have shown that CFTR loss-of-activity reduces chloride conductance without increasing sodium absorption in CF epithelia. (2) The imbalance of ion regulation depletes water content and/or decreases pH in the airway surface liquid. (3) Dehydration and/or acidification of epithelial lining, as well as increased mucin polymer cross-links, raise the amount and viscosity of mucus in gel phase, making the mucus tenacious and difficult to remove by ciliary beating. (4) Accumulation of mucus leads to obstruction of the airways, inflammation and bronchiectasis. (5) Frequently, pathogens colonize the airways and increase the recruitment of inflammatory cells. Furthermore, oxygen depletion below the sputum-air interface favors biofilm formation. (6) The destruction of airway and lung parenchyma epithelial cells causes tissue remodeling, reduction of gas exchange area and impairment of lung function. (7) As the disease progresses, the patient succumbs to death due to respiratory failure.

3In addition to the substantial impact of CFTR dysfunction in the upper and lower respiratory tract, CF patients also experience clinical manifestations related to the reproductive, gastrointestinal and glandular systems (Table 2). Male infertility is present in 98% of cases, and about 85% of CF patients have pancreatic insufficiency, often associated to malnutrition.

4Salt loss syndrome also affects CF patients, since chloride reabsorption via CFTR in the sweat glands is compromised, increasing chloride elimination (>60 mmol.L-1), as well as of other ions. In fact, the iontophoretic sweat test is considered the ‘gold standard’ for the diagnosis of CF. It precedes only the confirmation of a diagnosis by genetic testing. In addition, sweat chloride measurement is a biomarker of CFTR activity and response for new treatments.

5Multifactorial improvements have increased the survival of CF patients (Figure 5): early diagnosis, genotype–phenotype detection, nutritional support, more efficient and effective pulmonary interventions, multidisciplinary professional monitoring, establishment of CF specialist centers and, most recently, the development of precision medicine.

6In the last decades, many countries have adopted newborn screening programs. Newborns are diagnosed through the measurement of immunoreactive trypsinogen. This early diagnosis and the confirmation of the disease through the sweat test are important for a better prognosis. Thereafter, identifying which are the mutations in the CFTR gene is crucial, since different ethnic or regional populations can have a different spectrum of CFTR variants, each leading to different degrees of disease severity.

7A multidisciplinary team of health professionals should perform periodic monitoring of the disease progression and make any necessary adjustments in treatments in order for patients to achieve the best clinical outcomes. CF patients show weight loss and impaired growth due to pancreatic insufficiency and intestinal malabsorption. Supplementations with pancreatic enzymes and fat-soluble vitamins have improved their nutritional status. Lung injuries are the most common clinical characteristic due to mucociliary clearance impairment in the airways. Production of very viscous mucus impairs the ciliary beating, and treatments with mucolytic (e.g., dornase-alpha) or hydrator (e.g., mannitol, hypertonic saline) are helpful in eliminating the retained mucus. Antibiotic therapy is also essential, since the mucus becomes trapped in the respiratory tract, causing recurrent and persistent infections by a group of opportunistic pathogens: Burkholderia cepacia, Haemophilus influenzae, Pseudomonas aeruginosa (hallmark of CF), Staphylococcus aureus and Stenotrophomonas maltophilia. It is noteworthy that during infection by P. aeruginosa, the bacterial population can segregate and evolve differently according to the lung microenvironment, leading to differences in the bacterial characteristics, such as nutritional requirements, defense and microbial resistance. This may explain, partly, why this pathogen is so prevalent and difficult to eradicate from CF patient lungs.

8Non-pharmacological treatments, such as aerobic exercise, physiotherapy, feeding and physiological supports, are also important to high-quality care and better outcomes. As the disease progresses, patients require continuous therapy with oxygen, and in the end-stage lung disease, the only alternative is lung transplantation, which still presents a high risk of cellular rejection.

9Over the past decades, all the above-mentioned improvements have lengthened the life expectancy of CF patients. In 1960, CF patients only lived through childhood. Nowaday, they live to see their forties (Figure 5), and those born more recently are expected to make it to their fifties. However, CF patients still present even more reduced life expectancy – between 20 and 30 years – in some regions of the world, including Brazil and the African continent.

10Several new complications associated with increased survival (Table 2), which were rare or not previously observed, pose new challenges for CF scholars. CF-related diabetes, metabolic bone disease and multidrug-resistant pulmonary pathogens are some comorbidities in older CF patients. Although still uncommon, adult CF patients have a higher risk of gastrointestinal cancer compared to an age-matched non-CF population, which may be explained by the fact that CFTR is a tumor suppressor gene. Another severe complication is the allergic bronchopulmonary aspergillosis, which is correlated to accelerated decline of lung function in adult CF patients.

11Despite the increase in life expectancy, CF patients still present a limited quality of life due to the high costs and the burden of treatments needed. In particular, CF patients living in developing countries tend to have worse clinical outcomes compared to those who live in developed countries, since lower-income countries have scarce financial resources to optimize therapies, including the application of precision medicine. Precision medicine (or personalized treatment) is a new approach that takes into account the genetic variances of each individual. Therefore, CF patients with different CFTR mutations may require different treatments to quell their debilitating symptoms. The knowledge obtained about the molecular scenario in which CFTR mutants are degraded has contributed to understanding the different defects caused by different mutants, and thus to developing drugs that rectify the primary defect of specific mutations.

12CFTR biogenesis is a cellular process that involves several steps: post-transcriptional splicing, protein translation, folding at the ER, glycosylation at the Golgi apparatus, trafficking to the apical membrane, endosomal recycling and retrieval. The cellular and transcellular protein trafficking involves multiple quality control systems to compensate the limited fidelity of each system. Therefore, a protein homeostasis (proteostasis) network carefully checks CFTR maturation pathway. Among the quality control systems, ubiquitin-proteasome pathway destroys the largest fraction of misfolded CFTR, and aggresomes degrade by autophagy CFTR molecules that proteasomes cannot degrade. In addition, lysosomes eliminate non-native CFTR that escapes from ER-associated degradation (ERAD). The mutant ΔF508 fails to achieve the native conformation and ERAD machinery arrests CFTR, thereby precluding the protein from being delivered to the PM.

13CFTR folding is facilitated by many molecular chaperones and co-chaperones, which form a CFTR interactome; however, unstable and misfolded CFTR remains bound to chaperones, promoting premature degradation of CFTR (Figure 6). The heat shock protein (Hsp)27 (or HspB1) binds ΔF508 with small ubiquitin-like modifier (SUMO)-2, directing it to be degraded via SUMOylation. Hsp27 also prevents protein aggregation during stress. Hsp40 (or DnaJ) sequesters misfolded CFTR for ERAD and it works as a co-chaperone for Hsp70. Hsp70 is a core ER chaperone and its prolonged association with CFTR mutant results in CFTR ubiquitination and degradation by the 26S proteasome. Hsp70 connects to heat shock cognate (Hsc)70, forming the first ER checkpoint that retains the mutant ΔF508. Hsc70 couples to its co-chaperone, the carboxyl terminus of Hsc70-interacting protein (CHIP), and leads CFTR mutant to degradation. Hsp90 helps wild type (wt)-CFTR to achieve the complete folding, but it also forms a ‘chaperones trap’ with Hsp40/Hsp70 that targets ΔF508 for proteasome degradation. The ATPase activity of Hsp90 is regulated by its co-chaperone, the activator of 90kDa Hsp APTase homolog 1 (AHSA1 or Aha1). Calnexin is an ER transmembrane chaperone that acts as second checkpoint, assessing CFTR folding status and sending the misfolded protein for degradation.

14In addition to the molecular chaperones, inhibition of the histone deacetylase (HDAC) or the vasolin-containing protein (VCP or p97) rescues the trafficking and gating of ΔF508-CFTR. VCP and HDAC6 translocate misfolded proteins to proteasomes and aggresomes, respectively. VCP is associated to ubiquitinated CFTR, whereas derlin-1 recognizes misfolded, non-ubiquitinated CFTR and initiates its dislocation and degradation in the early course of CFTR biogenesis. The CFTR-associated ligand (CAL) is a PDZ domain-containing protein, located primarily at Golgi apparatus, which modulates surface expression of CFTR. CAL forms a complex with syntaxin (STX)6 that promotes CFTR degradation in lysosomes. STX8 is an endosomal protein that impairs CFTR trafficking and inhibits its chloride channel activity by soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) machinery. Vasoactive intestinal peptide (VIP) is a neuropeptide that promotes interaction between CFTR and Na+/H+ exchanger regulatory factor 1 (NHERF-1). NHERF-1 is a CFTR-binding protein that regulates CFTR distribution and function at the PM. Moreover, a system of peripheral proteins quality control (PPQC) removes CFTR from the PM if that is recognized as improperly folded.

15In healthy conditions, the proteostasis network maintains a healthful proteome by integrating transcription, translation, folding and trafficking systems. However, a decline in the cellular proteostasis capacity occurs with aging, as well as protein misfolding/aggregation disorders cause an imbalance in the expression and/or binding of proteostasis components with client proteins, which accentuates the importance of a healthy quality control system. CFTR protein must fold into well-defined three-dimensional structure to attain stability and functionality. The protein lack or its incorrect folding prevents CFTR from performing its normal function and cells may respond to that by maintaining an activated heat shock response and leading to a maladaptive stress response. This effort to neutralize misfolding protein accumulation causes an additional stress to cells that may result in the exacerbation of disease symptoms. These findings were observed in cell lines and primary epithelium from CF and other misfolding protein diseases: alpha-1-antitrypsin, Alzheimer’s disease and Niemann-Pick type C1 disease. Another important determinant in inherited disorders is that the disease severity depends of the genetic background of each individual and not solely on the defect of a particular gene. CFTR gene is located at the chr7q31.2 and a genome-wide association analysis identified five loci that display significant relevance in the variable manifestation and progression of lung disease in CF (chr3q29, chr5p15.3, chr6p21.3, chr11p12-p13 and chrXq22-q23).

16The ΔF508 is the most prevalent CFTR mutation with about 60% of CF patients’ chromosomes worldwide presenting this mutant (Figure 3). The remaining 40% present other CFTR mutations and there have been nearby 2,000 mutations reported in the Cystic Fibrosis Mutation Database (CFTR1 database)1. CFTR mutants can reduce protein expression, function, stability or a combination of these, and to help in understanding the nature of such gene and protein variability, CFTR defects have been classified into six classes (Figure 7).

17Distribution of CFTR mutants into classes may contribute to the application of precision medicine, since similar strategies might rescue CFTR from similar defects. However, the classification has a few caveats: (1) Numerous mutations have not been characterized, with respect to which group they should be allocated. The mutations’ characteristics for a subset of known mutations can be found at the Clinical and Functional Translation of CFTR (CFTR2 database)2. (2) At first glance, CFTR mutations in the same group show similar characteristics, but they may respond differently to the same treatment. (3) Several mutations (e.g., ΔF508) present pleiotropic defects, which means they could fit in more than one class. The major characteristic of ΔF508 is the incomplete folding of the protein caused by NBD1 instability (class II), but this mutation also affects channel gating (class III) and cell surface residence time (class VI). Based on this limitation, new classifications are under debate, including a scheme that would be composed of the traditional classes I, II, III/IV, V, VI and their 26 combinations, totaling 31 CFTR mutations classes.

18Monotherapies could be efficient in overcoming the molecular defect of some CFTR mutations; however, given the complexity of pleiotropic CFTR variants, combination of treatments may be required to rectify their defects and thus achieve therapeutic levels in the patients. Libraries of compounds have been screened by high-throughput screening (HTS) to identify more efficacious and non-cytotoxic drugs for application of precision medicine. Among these new pharmacological treatments, ‘CFTR modulators’ are small molecules that target specific defects caused by mutations in the CFTR gene and they are classified into five main groups: read-through agents, correctors, potentiators, stabilizers and amplifiers.

19Read-through agents could benefit CF patients bearing class I mutations, since the presence of a premature stop codon (class Ia) precludes protein synthesis of full-length CFTR. Class Ia mutations represent about 9% of the mutants causing CF and they are present in more than 50% of Israeli CF patients. In this line, ribosomal ‘over-reading’ of a premature stop codon should permit the continuing translation to the normal end of the transcript. This read-through effect was first observed with aminoglycoside antibiotics, such as gentamicin and tobramycin, which are commonly used for eradication of P. aeruginosa. Both antibiotics promoted expression of full-length CFTR at the PM and restored partially its chloride secretion in cell lines and transgenic mice. Despite these preclinical results, CF patients bearing nonsense mutations did not present CFTR activity after nasal application of aminoglycosides. Furthermore, high systemic levels or long-term use of gentamycin has potential toxic effects in CF patients.

20Ataluren (formerly PTC124) is among the new drugs discovered by HTS. This drug suppressed the human G542X nonsense mutant in transgenic mice, restoring CFTR functional expression at the PM. Thereafter, this drug resulted in some improvement in forced expiratory volume in 1 s (FEV1) of CF patients bearing nonsense mutations in phase II trials. However, these findings were inconclusive in the first long-term phase III trial, with only a subgroup of patients who were not receiving chronic inhaled aminoglycosides presenting a slight effect in FEV1. Ataluren has also shown limited premature stop codon suppression in CF rectal organoids. Moreover, other read-through agents have shown promising results in preclinical experiments, including synthetic aminoglycosides, ataluren derivatives, and the US Food and Drug Administration (FDA)-approved herbal agent, escin.

21For other class I mutations (class Ib), such as frameshifts caused by small deletions or insertions during protein synthesis, very little has been achieved in treatments yet.

22Correctors are small-molecules that enhance the conformational stability of CFTR, resulting in greater efficacy of protein folding and rescuing the trafficking of the mature CFTR to the PM. CF patients bearing class II mutations, including ΔF508, could benefit from correctors treatment, since these CFTR mutants fail to reach complete folding and the ER machinery targets the protein to be degraded.

23New treatments may target the defective CFTR structure directly by binding to the mutated protein (pharmacological chaperone) and/or indirectly by modulating CFTR interactome (proteostasis regulator). Some reports have shown that correctors act either as pharmacological chaperones or as proteostasis regulators. Based on the possible mechanism of action as pharmacological chaperones, correctors have been classified into three groups: 1) correctors that stabilize the interactions between NBD1 and intracellular loops 1 and 4 (e.g., C3, C18 and VX-809); 2) correctors that restore NBD2 stability and its interfaces with other CFTR domains (e.g., C4); and 3) correctors that directly stabilize NBD1.

24Lumacaftor (formerly VX-809) restored ΔF508-CFTR expression and function in human bronchial epithelial (HBE) cells. The higher efficacy of lumacaftor, compared to other correctors (C3 and C4), seems to be due to its effect on the early CFTR synthesis. Despite progress in vitro, lumacaftor treatment by itself showed a significant decrease only in sweat chloride levels and no improvements in FEV1 in a phase II trial with ΔF508-homozygous patients. Lumacaftor presented the most variable effects in primary HBE cells and rectal organoids bearing ΔF508 in only one allele or other CFTR mutants in both alleles, with some mutants being ‘un-rescuable’ (e.g., N1303K). Furthermore, a synonymous mutation changing ATC to ATT at position 507 (I507) alters mRNA and protein structure, consequently, affecting the efficacy of correctors to rescue ΔF508-CFTR. Aiming at greater effects, combinations of correctors have been evaluated and have been shown to enhance the rescue of CFTR bearing ΔF508, as well as other class II mutants, compared to monotherapies. Some correctors could also increase CFTR maturation of class V mutants caused by amino acid substitution, such as A455E. In addition, VIP rescued functional expression of ΔF508-CFTR by stimulating both PKA- and PKC-dependent pathways in nasal and bronchial epithelial cells. Many newly discovered correctors are being investigated, including VX-661 that presented more a favorable pharmacokinetic profile than lumacaftor and is in phase III trials to ΔF508-homozygous and -heterozygous patients (NCT02347657, NCT02392234 and NCT02412111).

25Besides the correctors, drugs that modulate proteostasis have been evaluated to restore CFTR functional expression at the PM, since wt-CFTR and ΔF508 present a rather different interactome during their processing and trafficking. Cysteamine, a proteostasis regulator approved by the FDA for nephropathic cystinosis, has shown promise for treating CF. Cysteamine (reduced form of cystamine), in association with epigallocatechin gallate, restored beclin 1-dependent autophagy protein levels and depleted sequestrosome 1/p62, thereby correcting autophagy flux, and rescuing CFTR trafficking, function and stability at the PM. These findings were observed in lungs from CftrΔF508 mice, CFBE41o- cell line expressing ΔF508-CFTR, and primary nasal epithelial cells freshly harvested from ΔF508-homozygous patients. Recently, a combination of cysteamine and epigallocatechin gallate decreased sweat chloride levels, as well as inflammatory biomarkers levels in the sputum, and tended to improve FEV1 in ΔF508-homozygous and -heterozygous (with a class II mutant in the second allele) patients in a phase II trial. Several other proteostasis regulators have been investigated to rescue CFTR, including sildenafil analogs, oubain, roscovitine, suberoylanilide hydroxamic acid and latonduine analogs. Silencing of RPL12, a ribosomal stalk protein, also rescued ΔF508-CFTR and presented a synergistic effect with lumacaftor, restoring the mutant function to about 50% of the wt-CFTR in primary HBE cells bearing ΔF508 in both alleles.

26Potentiators are drugs that increase channel open probability, improving CFTR channel activity. Potentiators could benefit CF patients bearing class III and IV mutations, since CFTR is present at the PM, but it exhibits no gating or reduced activity. Furthermore, patients bearing class I or II mutations for which protein synthesis or trafficking were rescued, but not proper channel activity, may benefit from this additional approach.

27Ivacaftor (formerly VX-770) rescued CFTR channel gating in HBE cells bearing G551D mutation through a nonconventional ATP-independent mechanism. Ivacaftor also potentiated channel activity of CFTR bearing other class III or IV mutants in Fisher rat thyroid cells and rescued forskolin-induced swelling in rectal organoids bearing CFTR mutants with residual function. A subsequent series of phase II and III clinical trials showed that ivacaftor reduced sweat chloride levels and improved FEV1 in CF patients bearing G551D or one of other eight class III mutants (G178R, S549N, S549R, G551S, G1244E, S1251N, S1255P, and G1349D) in at least one allele. For R117H, a residual function mutant, ivacaftor decreased sweat chloride levels of all patients, but only individuals older than 18 years and with a polythymidine tract variant of 5T presented improvements in FEV1. Currently, ivacaftor is available for CF patients bearing the aforementioned mutations.

28Monotherapy with ivacaftor or lumacaftor was unsuccessful in improving FEV1 of ΔF508-homozygous patients in phase II trials. A closer investigation of the pleiotropic defects caused by ΔF508 in CFTR reveals that combinations of drugs with different mechanism of actions will be required to obtain more efficient rescue of the mutated protein that may ultimately result in therapeutic levels in the patients. Toward this goal, phase II and III trials tested the effects of co-administration of lumacaftor/ivacaftor. This approach reduced pulmonary exacerbations, slightly decreased sweat chloride levels and induced significant, but modest, improvements in FEV1 of ΔF508-homozygous patients. Recently, co-administration of lumacaftor/ivacaftor was licensed for ΔF508-homozygous patients.

29Some flavonoids also increase channel open probability of CFTR mutants. In particular, genistein and curcumin have shown synergy with lumacaftor to enhance forskolin-induced swelling in rectal organoids bearing CFTR mutants ΔF508, G551D and S1251N. Ivacaftor has shown clinical benefits when administered by itself, but its effects could be even greater when used with other compounds for CFTR bearing G551D or R117H. In addition, new potentiators are under investigation to restore channel activity of CFTR mutants, including QBW251 (NCT02190604) and GLPG1837 (NCT02690519 and NCT02707562), as well as dual activity compounds that act as both correctors and potentiators, such as aminoarylthiazoles and rattlesnake phospholipase A2.

30Stabilizers are agents that anchor CFTR channel at the PM and decrease protein degradation rate, thereby correcting the instability of class VI mutants. Low-temperature incubation of cells bearing ΔF508 rescue CFTR to the PM (rΔF508); however, the protein still presents reduced half-life due to both increased endocytosis and decreased recycling. Lumacaftor also did not confer long-term stability of wt-CFTR for the mutant ΔF508.

31New treatments must rectify the intrinsic protein instability to rescue the steady state levels and augment CFTR residence time at the PM. In this line, hepatocyte growth factor (HGF) activated Rac1 signaling and promoted ΔF508 stabilization favoring interaction of CFTR and NHERF-1. Lumacaftor also promoted interaction of CFTR and NHERF-1, but co-administration of HGF/lumacaftor further increased the rescue of ΔF508-CFTR and enhanced protein anchoring at the PM, compared to lumacaftor alone. Administration of VIP or activation of EPAC1, an exchange protein directly activated by cAMP, also stabilized CFTR at the PM by promoting its interaction with NHERF-1 and by decreasing its endocytosis rate.

32In addition, inhibition of S-nitrosoglutathione reductase enhanced ΔF508 maturation and stability by preventing interaction of CFTR with Hsp70/Hsp90 organizing protein. Cavosonstat (formerly N91115) increased S-nitrosoglutathione levels in pre-clinical studies and it is the first CFTR stabilizer in phase II trials being evaluated for ΔF508-homozygous patients using the combination lumacaftor/ivacaftor (NCT02589236) or for those patients bearing gating mutants and receiving ivacaftor (NCT02724527).

33Antisense oligonucleotides-based therapy is an emerging approach to correct class V mutations caused by alternative splicing that generate aberrant mRNA variants. About 11% of mutations-causing CF occur by incorrect splicing and this approach has shown to modulate the splicing and restore normal full-length CFTR transcript, as well as rescue functional CFTR protein.

34A synthetic RNA oligonucleotide, QR-010, is being evaluated in two phase I trials to assess: (1) the effects of inhaled single and multiple doses in ΔF508-homozygous patients (NCT02532764), and (2) the effects of intranasal administration in ΔF508-homozygous and -heterozygous patients (NCT02564354).

35From the first pathological description of the disease until the discovery of the CFTR gene and its correlation with CF, therapeutic interventions were only aimed at reducing clinical symptoms and end-organ complications. Nowadays, based on the accumulated knowledge about CFTR protein (synthesis, folding, trafficking and degradation), many studies have sought more specific treatments to restore expression, function and stability of CFTR mutants, thereby overcoming the primary molecular defect that causes CF. As such, CFTR modulators provided new perspectives and advances in the treatment of patients bearing common and rare CFTR mutations.

36Many clinical trials have completed the tests of safety and efficacy of CFTR modulators (Table 3). The first breakthrough in personalizing CF treatments came with the approval of ivacaftor (KalydecoTM from Vertex Pharmaceuticals) for patients bearing G551D-CFTR in at least one allele, initially licensed by the FDA and later by the European Medicines Agency (EMA). Clinical studies showed that ivacaftor treatment improves FEV1 and almost normalizes sweat chloride levels, as well as reduces frequency of infections caused by P. aeruginosa, enhances nutritional status, insulin secretion and quality of life. Acute administration of ivacaftor also corrects smooth muscle abnormalities, improving airway distensibility and vascular tone. Clinical benefits obtained by ivacaftor treatment have proven durable effects, with no new safety concerns. Ivacaftor was later licensed for patients bearing one of the other eight gating mutations and a mutant with residual function in at least one allele, increasing to 5–7% the number of individuals worldwide who may benefit from this pharmaceutical treatment. Despite these optimistic results, patients using ivacaftor should continue with conventional therapies, which have also been optimized, to prevent pulmonary exacerbations and complications.

37Ataluren (TranslarnaTM from PTC Therapeutics) was approved by the EMA for individuals who have Duchenne muscular dystrophy caused by nonsense mutation. After the inconclusive results from the first phase III trial, a second one is ongoing to evaluate the effects of ataluren in CF patients bearing nonsense mutations and not receiving inhaled aminoglycosides (NCT02139306).

38Recently, the FDA and the EMA licensed the combination lumacaftor/ivacaftor (OrkambiTM from Vertex Pharmaceuticals) for ΔF508-homozygous patients, adding a new pharmaceutical treatment to 40–45% of CF patients worldwide. Results from a phase II trial evaluating this combination in ΔF508-homozygous patients encouraged the pursuance of two long-term phase III trials (24 weeks) involving more than 1,100 people. Co-administration of lumacaftor/ivacaftor proved clinical effectiveness, despite fairly small improvement in FEV1. A longer-term (96 weeks) phase III trial showed that co-administration of lumacaftor/ivacaftor presents sustained benefit with decreased pulmonary exacerbations, reduced rate of lung function decline and improved nutritional status of ΔF508-homozygous patients. Although it is still a glimmer of light at the end of the tunnel, this combination provides proof of concept that pleiotropic CFTR mutants can be rescued at therapeutic levels, representing a new hope to restore a healthy life to patients.

39Intriguingly, studies using cell lines bearing ΔF508-CFTR showed that: (1) Chronic ivacaftor exposure (>1 μM) reduced CFTR correction obtained by lumacaftor treatment; however, exposure to a lower concentration (<1 μM) of ivacaftor did not inhibit the functional correction obtained by lumacaftor. Therefore, co-administration of lumacaftor/ivacaftor may present a dose-dependent inhibitory effect. Addition of C4 also reversed the negative effects of ivacaftor on lumacaftor-corrected CFTR. (2) P. aeruginosa infection reduces CFTR chloride secretion stimulated by lumacaftor and/or ivacaftor treatments. (3) In cells previously treated with lumacaftor, the PPQC system still recognizes the CFTR delivered at the PM as improperly folded and removes the protein from the cell surface. Taken together, the experimental results indeed may explain the modest findings in the clinical trials and highlight the importance of better evaluating drug-drug and drug-protein interactions for future drug combinations.

40The introduction of KalydecoTM and OrkambiTM to the market add feasible treatment for about 50% of all CF patients worldwide (Table 4). Unfortunately, there is still an unmet need for the other half of the CF population, who are ΔF508-heterozygous and bear a wide range of other variants (so-called orphan mutants). The patient registries and the clinical studies network have optimized the clinical phase of drug development due to heterogeneous distribution of CFTR variants. Randomized controlled trials are suitable for common mutations, but many CFTR mutants are rare or even unique, requiring different approaches. As such, modified single-patient (‘N-of-1’) is an alternative trial design in which single patients serve as their own control by measuring outcome parameters after multiple cycles of on-off treatment.

41Rectal organoids have also shown to be a good model for identifying drug-responsive individuals with rare CFTR genotypes. In addition, both KalydecoTM and OrkambiTM are in ongoing clinical trials to: (1) evaluate the longer-term safety and efficacy of the treatments; (2) increase the panel of mutations that may benefit from these drugs; and (3) evaluate the safety and efficacy in younger patients. Since the progressive damage caused by CF starts during babyhood, earlier treatments are critical to offering the best chances of improving long-term outcomes.

42In an attempt to achieve greater clinical outcomes, two new classes of CFTR modulators are under investigation in association with OrkambiTM: (1) PTI-428 is an amplifier that doubled the activity of the combination lumacaftor/ivacaftor in HBE cells bearing ΔF508-CFTR in both alleles; and (2) cavosonstat is a stabilizer that improved ΔF508-CFTR stability at the PM after co-administration of lumacaftor/ivacaftor. Moreover, other correctors and potentiators are advancing in triple combination for treatment of ΔF508-homozygous and -heterozygous patients: GLPG1837/GLPG2222/GLPG2665, VX-152/VX-661/ivacaftor and VX-440/VX-661/ivacaftor.

43An important limitation that has to be addressed regarding these new pharmaceutical treatments is the high cost (over US$250,000 per patient per year), which renders difficult their acquisition for CF patients living in low-income countries. Investments in the identification of drugs already available and able to overcome the primary molecular defect of CF could be another way, cheaper and fast, to optimize treatments.

44Soon after the CFTR gene was discovered and cloned, gene therapy was proposed to cure lung disease in CF. The idea of introducing a copy of wt-CFTR in airway cells to re-establish the production of functional protein seemed graceful and ‘simple’. Although CF is a ‘simple’ monogenic disorder, most approaches (viral and non-viral) failed to cross the trapped mucus, thereby resulting in inefficient transduction of CFTR gene, and some of them promoting immune response activation. Nevertheless, researchers have sought more efficient gene delivery systems. Poly (β-amino esters)-based biodegradable polymers were able to deliver a plasmid that encodes full-length CFTR in murine lungs and CFBE41o- cell lines expressing wt-CFTR or ΔF508, as well as to penetrate in freshly expectorated mucus from CF patients. Furthermore, repeated nebulization of pGM169/GL67A, a gene-liposome complex, showed feasible, safe and reproducible results in murine lungs, as well as stabilized lung function of CF patients, thereby presenting a significant, albeit modest, benefit in FEV1 in a phase II trial (NCT01621867). These systems may also be an interesting approach to delivery of CFTR modulators, bringing some benefits in usefully exploited patients’ treatment: (1) non-invasive administration route by inhalation/nebulization; (2) delivering directly to a specific lung region or defined cell type; (3) reduced systemic side effects and no serum proteins sequestered; and (4) sustained release, which decrease times and dose of therapies to maintain the beneficial effects.

45Plasma membrane proteins belong to some of the largest families, encoding ion channels, transporters, aquaporins and ATP-powered pumps. Forty-nine proteins constitute the ABC transporters family in the human genome and among these proteins, CFTR is unique in possessing a RD and in functioning as a chloride channel. Mutations in ABC transporters have been linked to diseases and, since these transporters share a similar domain organization and structure, some CFTR modulators have been evaluated to rescue protein trafficking and function of other ABC transporters. Here are some examples: ABCA4, ABCB1 (or P-glycoprotein), ABCC6 (or multidrug resistance-associated protein 6), ABCC8 (or SUR1), and ABCG2. These findings show that CFTR modulators could be a promising treatment for several diseases caused by mutations in other ABC transporters.

46Since the discovery that CFTR loss-of-activity causes CF, the biological understanding of CFTR processing has advanced steadily and opened new perspectives for more sophisticated treatments, which act directly on the molecular defect that causes the disease. The presence of CFTR modulators in the market has affected positively the clinical outcomes of CF patients, representing a new dawn in their lives. Although there is still a long way to go in completely restoring a healthy life for all CF patients, research from the bench to the clinic is moving forward at an accelerated pace toward precision medicines, which would enable “the highest attainable standard of health,” as enshrined in the constitution of the World Health Organization.

47The author confirms being the sole contributor of this work and approved it for publication.

48The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.