CFTR Modulators: The Changing Face of Cystic Fibrosis in the Era of Precision Medicine

1In Memoriam: The author dedicates this review article to his youngest brother, Nilo Lopes Pacheco, who fought against CF for 26.5 years.

2Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene cause cystic fibrosis (CF)—the most common life-threatening autosomal recessive disease in Caucasian populations. CFTR encodes a cAMP-dependent, phosphorylation-activated anion channel that transports chloride and bicarbonate across the apical plasma membrane (PM) of epithelial cells. Furthermore, CFTR modulates the activity of other ion channels, such as the epithelial sodium channel (ENaC). The absence or dysfunction of the CFTR protein at the PM leads to an impaired transepithelial balance of ions and fluid in cells of the sweat glands, airways, intestine, and pancreas, among other organs. Although CF is a multi-organ disease, the respiratory disorder represents the major cause of morbidity and mortality of these patients. A vicious cycle of mucus buildup in the airways, chronic inflammation, and recurrent infections leads to epithelial damage, tissue remodeling and progressive deterioration of lung function, ultimately resulting in respiratory failure (refer to Figure 4 in ).

3Archeological estimates indicate that the most prevalent CF-causing mutation, the deletion of a phenylalanine at position 508 (F508del), originated in Western Europe during the Early Bronze Age. Although there are some archaic references regarding “children whose brow had salty taste when kissed and prematurely died”, CF remained uncharacterized until the 1930s. The first pathological description of the disease came in 1938 when Dorothy Anderson recognized CF as a separate entity from celiac syndrome after autopsy studies of malnourished infants, being then known as “cystic fibrosis of the pancreas”. Another critical discovery was reported by Paul di Sant' Agnese in 1953 when he noticed that CF patients demonstrated an abnormal excess of salt in the sweat during a heat wave in New York. Nevertheless, the decreased chloride transport and increased sodium reabsorption was described as a basic defect in CF epithelia in the 1980s by experiments using sweat duct cells. Such findings served as basis for the sweat chloride test extensively used nowadays in CF diagnosis. Finally, the correlation between CF and the CFTR gene was discovered in 1989 when the gene was cloned by using chromosome walking and jumping, and linkage disequilibrium analysis. Soon after, some reports demonstrated that CFTR-dependent chloride secretion could be restored by transfecting cells derived from CF patients with wild type (WT)-CFTR cDNA.

4The extensive knowledge obtained over this research path has enabled the early diagnosis and the discovery of more efficient and sophisticated therapies, resulting in increased life expectancy. In fact, the mean age of survival of CF has risen from early childhood in the 1960s to 40–50 years currently in several countries, although it still is much lower in certain regions worldwide. The “backbone” of CF treatment is symptomatic, focusing on the compensation of pancreatic insufficiency and intestinal malabsorption with pancreatic enzymes, fat-soluble vitamins, and high-calorie ingestion, as well as slowing lung function deterioration with physical and inhaled therapies to enhance airway clearance, anti-inflammatory drugs, and antibiotic therapy to eradicate infections. At late stages of disease, lung transplantation remains the only feasible intervention, although still presenting a risk of cellular rejection. Furthermore, several comorbidities that were rare or not previously observed, including CF-related diabetes, metabolic bone and kidney disorders, and certain types of cancer, have become increasingly common as CF patients' lives have lengthened. A tremendous effort has been made to continue optimizing the therapeutic regimens and the multidisciplinary healthcare in order to further enhance CF patients' life expectancy. Quality of life has also improved but patients are still subjected to substantial clinical, psychosocial and economic burdens.

5Novel therapeutic approaches acting more upstream on the pathogenesis cascade have emerged, including precision medicine with the discovery of drugs (termed CFTR modulators) that rectify the underlying defects of certain CFTR mutations. To date, four CFTR modulators have reached the market for the treatment of patients carrying specific CF-causing mutations and these breakthroughs have paved the way for the development of novel pharmacotherapies, which are currently under experimental and clinical investigations. This review provides a summary of recent developments in CFTR-directed therapeutics and sheds light on barriers that must be overcome for precision medicine efficiently to reach all individuals with CF.

6CFTR is a long gene located on the long arm of chromosome 7, specifically in 7q31.2 (Figure 1). It is composed of 27 coding exons, spanning approximately 190 kb of human genomic DNA that is transcribed into a CFTR mRNA of 6.2 kb. Over 2,000 CFTR gene variants have been reported in the Cystic Fibrosis Mutation Database. Many of them might be a variation in the DNA sequence eliciting neither a defect in CFTR mRNA or protein nor clinical symptoms. To date, 432 of these variants have been annotated in the Clinical and Functional Translation of CFTR, of which 352 variants have confirmed disease liability and 46 variants have demonstrated variable clinical consequences. Many other variants are still uncharacterized, and in silico tools may be useful to predict the cellular and molecular consequences caused by these alterations, while they are not confirmed in cell models. Some variants may also be pathogenic when two or more mutations are in cis, i.e., complex allele, thus contributing to the variable clinical phenotypes and responsiveness to CFTR modulator therapies.

7The CFTR mRNA translates into a 1,480-amino acid protein. Soon after co- and post-translational folding, and core glycosylation in the endoplasmic reticulum (ER), CFTR protein traffics to the Golgi complex, where it is fully glycosylated. Thereafter, it is exported to the apical PM of epithelial cells, where it functions as a chloride and bicarbonate channel.

8The CFTR protein has five distinct domains: two transmembrane domains (TMD1 and TMD2), two nucleotide-binding domains (NBD1 and NBD2), and a regulatory domain (RD). Each TMD contains six segments that completely cross the phospholipid bilayer and together they form the channel pore through which anions may flow. The TM segments are joined by three extracellular loops and two intracellular loops in each TMD, and they have critical roles in CFTR biogenesis and in the post-translational folding in order to establish interdomain interactions required to achieve conformational stability. Furthermore, the fourth extracellular loop possesses two N-linked glycosylation sites (N894 and N900), which are assessed by the ER quality control during the protein folding process. These glycans are modified upon traversing the Golgi complex and may interact with extracellular macromolecules when the protein is located at the PM. The NBDs and the RD are exposed to the cytosol and are rich in charged residues. The NBDs present highly conserved sequence for ATP binding and hydrolysis, while the RD is highly disordered and has multiple consensus sequences containing serines and threonines for phosphorylation by protein kinase A (PKA) and protein kinase C (PKC). ATP and cAMP-dependent PKA and PKC phosphorylation induce alterations in CFTR protein conformation, thus allowing anion conductance through the pore. Among the 48 members of the human ATP-binding cassette (ABC) transporter family, CFTR (also known as ABCC7) is the unique one that possesses a RD and functions as an anion channel. Novel insights into channel opening and closure mechanisms have recently been elucidated with electron cryomicroscopy of the phosphorylated and dephosphorylated protein state. While these structures have not played a role in the development of CFTR modulators to date, such findings will be greatly useful for the identification of hotspots for drug-binding and for the application of rational design of next-generation modulator drugs based on the CFTR structure.

9CF affects over 90,000 individuals and they are heterogeneously distributed worldwide (Figure 2). The F508del is the most prevalent CF-causing mutation, affecting approximately 82% of the CF population (Figures 3A, B). This mutation leads to CFTR protein misfolding that is arrested by the ER quality control, thus precluding its processing and trafficking to the PM, being instead targeted and prematurely degraded by proteasomes. Nevertheless, a small fraction of the mutant protein may evade the quality control checkpoints and reach the PM; however, it still presents a defective gating and a considerable reduction in protein stability.

10The F508del mutation accounts for approximately 70% of CF alleles and other CFTR mutations are responsible for the remaining ones. Notably, most are rare or demonstrate a certain frequency in specific regions, and only six mutations present a prevalence ≥1% while approximately 50 mutations have a prevalence ≥0.1% considering the whole CF population (Figure 3C).

11CFTR mutations may impair mRNA and protein expression, function, stability or a combination of these, and as such they have been stratified into different classes according to the primary molecular defect. The classification has historically been evolving according to the gained knowledge, and the current scheme is composed of six classes (Figure 4), although a seventh class has been proposed to separately consider large deletions that may abrogate production of CFTR mRNA. Another system has also been proposed to take into account the pleiotropic defects of many CFTR mutations, including the F508del. Although limitations in the classification system are evident, it has been useful in understanding the distinct cellular and molecular defects of different CFTR mutations as well as in the development of pharmacotherapies for specific defects (Figure 5).

12Mutations in classes I, II, and III are usually associated with a classical and more severe disease, while those in classes IV, V, and VI are related to milder (or atypical) phenotypes. Individuals with CF may nevertheless carry different CFTR mutations on the two alleles, leading to thousands of possible combinations of CF genotypes. Noteworthy, CFTR mutations may differently respond to the same intervention (e.g., correction by low temperature or by chemical compounds), even for those classified as belonging to the same defect class. Therapeutic responses may also differ between individuals carrying the same CF genotypes. In fact, several other factors exert influence on disease severity beyond CFTR mutations, such as gene modifiers and epigenetic factors, social and economic status, patient's lifestyle, and adherence to therapies. Primary, reprogrammed and engineered human cell models have become important tools to identify novel pharmacotherapies. The effects of certain therapies may also be exploited at an individual level in ex vivo patient-derived specimens, such as primary bronchial/nasal epithelial cells, and intestinal/respiratory organoids. As these cell models recapitulate several features of the parental organ, they are useful to understand the impact of genetic factors on individual disease and predict clinical efficacy of therapies.

13Numerous libraries of compounds have been screened by distinct high-throughput screening (HTS) methods and using several cell models. These experimental approaches have been contributing to the identification of small-molecules from different chemical series (Figure 6). Notably, as cell background may significantly influence the pharmacological rescue of CFTR mutants, most promising hits should be further validated in well-differentiated cells in order to identify non-cytotoxic and effective compounds that might result in optimal therapeutic benefits in the clinical scenario.

14CFTR modulator drugs enhance or even restore the expression, function, and stability of a defective CFTR by distinct manners, and they have been classified into five main groups depending on their effects on CFTR mutations: potentiators, correctors, stabilizers, read-through agents, and amplifiers. To date, four CFTR-directed modulators have reached the market for the treatment of CF patients carrying specific CFTR mutations. Several clinical trials have been completed (Table 1) and many others are ongoing (Table 2) in extension/observational studies or to evaluate the safety and efficacy of novel modulators. As the experimental and clinical research in the CF field is moving at an accelerated pace, the most recent advances in precision medicine have been summarized below in order to update information previously published in the 2016 Review in Frontiers in Pharmacology.

15Around 5% of CF-causing mutations lead to impaired CFTR channel gating or conductance as primary defects (Classes III and IV, Figure 4). The R117H, R334W, R347P, and G551D are among the most common mutations that cause such abnormalities and they are found in 1.3%, 0.3%, 0.4%, and 2.1% of CF alleles, respectively. Potentiators are compounds that restore or even enhance the channel open probability, thus allowing for CFTR-dependent anion conductance.

16Initial studies demonstrated an enhancement of the open probability of certain CFTR variants in cells by using either chemically modified analogs of ATP or compounds that increase intracellular cAMP levels. However, their use in the clinics is limited due to potential modulation of multiple signaling pathways and physiological functions. Genistein is an isoflavone that binds to and inhibits protein-tyrosine kinase, resulting in increased intracellular cAMP levels, which leads to potentiation of CFTR activity in cell models. However, clinical benefits were not clearly demonstrated when it was used in combination with 4-phenylbutyrate (NCT00590538). Other potentiators have been identified by cell-based HTSs but did not reach the clinical investigation for CF, such as the phenylglycine molecule PG-01 and VRT-532.

17Ivacaftor (VX-770; Vertex Pharmaceuticals) is a potentiator identified by HTS that partially restored CFTR activity in G551D-expressing cell lines and in primary bronchial epithelial cells. Although the mechanisms of action of ivacaftor are not entirely elucidated, it was demonstrated to mediate CFTR channel potentiation in a phosphorylation-dependent and ATP-independent manner. It binds and potentiates CFTR function by promoting decoupling between ATP hydrolysis and gating cycles. Several studies have been proposing the putative binding site for ivacaftor by distinct methods. Some initial reports indicated that ivacaftor might bind to a region of amino acids at the NBD1/2 interface and the coupling helix of intracellular loop 1 and/or the ‘ball-and-socket’ joint close to intracellular loop 4. More recent studies provided direct evidence of the binding site of ivacaftor at the interface between TMDs by using electron cryomicroscopy and electrophysiological assays. As chemical interactions are dynamic and the protein undergoes multiple conformational changes during channel opening and closure, some binding sites might also differ depending on the protein conformational state.

18In 2012, both the U.S. Food and Drug Administration (FDA) and the European Medicines Agency (EMA) approved the use of ivacaftor (Kalydeco®, Vertex Pharmaceuticals) for CF patients aged ≥6 years carrying at least one G551D mutation after a phase III trial showing that ivacaftor has successfully improved clinical outcomes (sweat chloride concentration, percent predict forced expiratory volume in 1 sec [ppFEV1], and others). A similar effectiveness was also observed in patients carrying one G551D mutation who had a more severe impairment in lung function. Ivacaftor is on the market for over 7 years, and it is transforming patients' lives with sustained and long-term benefits, including reduction in sweat chloride to normal levels, slower deterioration of lung function, reduction in the number of pulmonary exacerbation episodes, less frequent detection of Pseudomonas aeruginosa and other common pathogens, better body mass index (BMI), exercise capacity and well-being, and quality of life. Furthermore, observational studies have demonstrated that ivacaftor improved pancreatic function and mucociliary clearance. Reduction in the levels of blood inflammatory biomarkers, and smooth muscle and lung structure abnormalities have also been observed.

19A subsequent series of experimental and clinical studies led to the extended approval of ivacaftor to treat CF patients carrying other gating mutations. Thereafter, another extension has been approved to include CFTR mutations with residual function based on in vitro studies, totalizing 38 eligible CF-causing mutations and increasing the number of patients who may benefit from ivacaftor treatment (Table 3). Notably, the membrane CFTR conductance depends on the number of functional channels, the average open probability and conductance of each CFTR channel (Figure 5). Therefore, some splicing mutations have shown to respond to ivacaftor treatment due to an improvement of the last two determinants, even though presenting less protein abundance at the PM. More recently, ivacaftor treatment has been extended for CF patients aged ≥6 months carrying at least one of the listed mutations (Table 3). In an animal model of CF, intrauterine and postnatal administration of ivacaftor also demonstrated to protect ferrets (G551D/G551D genotype) against development of multi-organ disease, indicating that starting the treatment in newly diagnosed children may increase the chances of improving long-term outcomes or even preventing the appearance of certain symptoms. Long-term use of ivacaftor has also been associated with a decreased need for lung transplant and improved survival. Despite all demonstrated benefits, patients receiving ivacaftor may eventually experience pulmonary exacerbations, thus requiring hospitalizations and worsening quality of life. Furthermore, ivacaftor treatment does not improve the rate of complete lung function recovery after an exacerbation episode. Lung function also declines in the long term, albeit at a slower rate.

20Several novel potentiators have demonstrated promising effects and are currently under experimental and clinical investigations. The potentiators VX-561 (deutivacaftor or formerly CTP-656; Vertex Pharmaceuticals) is a deuterated form of ivacaftor that demonstrated an enhanced stability in vitro and in healthy volunteers compared to ivacaftor, which would allow CF patients to take it once daily rather than twice as it has been the case with the ivacaftor. A phase II trial is in progress to comparatively evaluate the effects of VX-561 and ivacaftor in CF patients who have the following CFTR gating mutations in at least one allele: G178R, S549N, S549R, G551D, G551S, G1244E, S1251N, S1255P, or G1349D (NCT03911713).

21The molecules ABBV-974, ABBV-2451, and ABBV-3067 (formerly GLPG-1837, GLPG-2451, and GLPG-3067, respectively) have been developed by Abbvie/Galapagos. ABBV-974 and ABBV-2451 have demonstrated greater and similar CFTR potentiation, respectively, in comparison with ivacaftor in R117H-, G178R-, S549N-, and G551D-expressing cells. Interestingly, the simultaneous administration of ABBV-974 or ABBV-2451 with ivacaftor did not further enhance CFTR function, indicating that these molecules may act by the same mechanism of action. In fact, recent reports demonstrated that ABBV-974 and ivacaftor share a common binding site at the interface between the two TMDs near the kink of helix 8 (Figure 7). In a phase IIa trial, patients were subjected to a one-week ivacaftor washout before receiving three consecutive and increasing doses of ABBV-974. Although some adverse effects were observed in a dose-dependent manner, treatment was able to decrease sweat chloride concentration and improve ppFEV1 in CF patients carrying G551D-CFTR in at least one allele.

22The molecules FDL176 (Flatley Discovery Lab) and PTI-808 (dirocaftor; Proteostasis Therapeutics) are other potentiators being evaluated in early clinical trials (NCT03173573 and NCT03251092). The potentiator QBW251 (Novartis) was tested in both healthy volunteers and CF patients in a phase I/II clinical trial (NCT02190604). However, no significant improvements were observed in lung function of CF patients and adverse effects were also reported in many participants, resulting in early termination of this study. Recent studies have also demonstrated that co-administration of potentiators with complementary mechanisms of action may be an interesting approach for those CF-causing mutations in which CFTR gating/conductance is not completely restored by single potentiators.

23Most CF patients carry a mistrafficking CFTR mutation, since F508del is the most prevalent CF-causing mutation (Class II, Figure 4). Other common mutations that cause such abnormality are the G85E, I507del, R560T, and N1303K (found in 0.4%, 0.5%, 0.2%, and 1.6% CF alleles, respectively). Correctors are compounds that rescue folding, processing and trafficking to the PM of a CFTR mutant. While these compounds may act by distinct mechanisms, they usually enhance protein conformational stability during the ER folding process.

24New pharmacotherapies may target the defective CFTR structure directly by binding to the misfolding protein (termed as pharmacological chaperones) and/or indirectly by modulating its interaction with protein homeostasis (termed as proteostasis regulators), thus enabling the passage of CFTR protein through the ER quality control checkpoints. Studies using genome- and proteome-wide association analyses have identified proteostasis components that could be targeted to rescue CFTR in F508del-expressing cells. Furthermore, the recent identification of differences in WT- and F508del-CFTR interactomes unveiled several targets that could be exploited to rescue F508del and potentially other misfolding CFTR mutants.

25Initial studies were focused on certain proteostasis regulators, such as 4-phenylbutyrate and miglustat. Although these molecules demonstrated an increase in functional expression of F508del-CFTR in experimental models, only modest to no efficacy was found in early clinical studies. Several studies have also demonstrated a rescue of CFTR processing and trafficking to the PM in F508del-expressing cells by knocking down expression of certain proteostasis components, such as Aha1, an Hsp90 cochaperone, HDAC7, Hsp27, and CFTR-associated ligand (CAL), among others.

26Advances in HTS technologies have been facilitating the fast-tracking of novel chemical compounds that act as proteostasis regulators and/or pharmacological chaperones. A classification of pharmacological chaperones has been proposed based on their molecular targets in the CFTR structure: class 1 correctors stabilize NBD1-TMD1 and/or NBD1-TMD2 interfaces; class 2 correctors stabilize NBD2 and its interfaces with other CFTR domains; and class 3 correctors directly stabilize NBD1. This classification may be useful to evaluate corrector combinations with complementary mechanisms of action or targeting different defects in the mutant CFTR structure.

27Lumacaftor (VX-809; Vertex Pharmaceuticals) is a first-generation corrector that demonstrated remarkable rescue of CFTR folding and function in F508del-expressing cell lines and primary bronchial epithelial cells. Other first-generation correctors had been identified previously but they were not suitable for the clinical practice due to a significant cytotoxicity and reduced efficacy. Some reports have suggested that lumacaftor has a putative binding site at the interface of NBD1 with the intracellular loop 4 in TMD2. Other studies have also indicated that lumacaftor acts on TMD1 and promote interactions between NBD1 and the intracellular loop 1 in TMD1. While the identification of single putative binding site for small molecules, such as lumacaftor and ivacaftor, is important to unravel the mechanism(s) of action, multiple binding sites on the CFTR protein remain a possibility. Notably, lumacaftor was demonstrated to rescue ABCA4 trafficking mutants, another ABC transporter that shares approximately 45% homology with CFTR in NBDs.

28Despite promising results in vitro, lumacaftor alone failed to demonstrate any improvement in lung function in F508del-homozygous patients in a phase IIa clinical study. A significant, albeit modest, improvement in lung function (3%–4% in ppFEV1) was found only when lumacaftor was co-administered with ivacaftor in a following clinical trial. Nevertheless, treatment with lumacaftor alone or in combination with ivacaftor was not able to improve ppFEV1 of F508del-heterozygous patients. Certain adverse effects, such as chest tightness and dyspnea, have also been reported by both F508del-homozygous and -heterozygous patients in the early stage of the treatment.

29In 2015, the FDA and the EMA approved the co-treatment with lumacaftor/ivacaftor (Orkambi®, Vertex Pharmaceuticals) for F508del-homozygous patients aged ≥12 years. Initial clinical trials usually tend to exclude patients with more severe disease, as they might present a more accelerated rate of decline in lung function. Nevertheless, F508del-homozygous patients with a more severe impairment of lung function were demonstrated to benefit from co-treatment initiation with lumacaftor/ivacaftor at a lower dose. Co-treatment with lumacaftor/ivacaftor was also demonstrated to benefit F508del-homozygous patients with distinct lung function impairment in a pooled analysis by subgrouping patients based on the FEV1 baseline. Although the temporal process of recovery from pulmonary exacerbations may differ in each patient, co-treatment with lumacaftor/ivacaftor reduced the number of pulmonary exacerbation episodes even in patients without early lung function improvement. Furthermore, longer-term use of lumacaftor/ivacaftor has demonstrated continued benefits in patients, including improvement in BMI, reduction in the incidence of pulmonary exacerbations and hospitalizations, and slower deterioration of lung function. Moreover, an observational study measured certain CFTR biomarkers — nasal potential differences and intestinal current measurements — and demonstrated that co-treatment with lumacaftor/ivacaftor results in partial correction of F508del-CFTR function to similar levels of the lower range of CFTR activity usually observed in patients with residual function mutations. More recently, co-treatment with lumacaftor/ivacaftor has been extended for F508del-homozygous patients aged ≥2 years after phase III clinical trials demonstrating safety and efficacy in younger patients. A clinical trial has also evaluated the effects of lumacaftor/ivacaftor in patients carrying A455E in at least one allele (NCT03061331), since this mutation has demonstrated an increase in CFTR PM abundance and function after corrector treatments in cell models.

30Lumacaftor has demonstrated different efficiency and potency in rescuing other CFTR mutations causing protein misfolding. In cell lines, lumacaftor was able to rescue CFTR function for E92K, L1077P, and M1101K, although it had no effect on G85E, R560S, and N1303K. The correction effects were also variable in patients-derived specimens carrying the F508del mutation in one allele and a minimal function mutation in trans (i.e., in the second allele); the mutants A561E, Y1092X, and W1282X demonstrated a response to lumacaftor treatment, but no effect was found for E60X, 394delTT, 711-1G>T, G542X, 1717-1G>A, and N1303K, among others. Even for F508del-homozygous patients, co-treatment with lumacaftor/ivacaftor resulted in significant but variable clinical responsiveness. The routine use of patient-specific biomarkers could be an effective approach to pre-clinically evaluate the safety and efficacy of certain pharmacotherapies and predict effectiveness at an individual level.

31Notably, interaction between lumacaftor and ivacaftor revealed to negatively impact the rescue of F508del-CFTR protein. Chronic ivacaftor exposure (>1 µM) reduces lumacaftor-rescued CFTR in F508del-expressing cells, although lower concentrations of ivacaftor (≤1 µM) may prevent such negative effect. Lumacaftor also triggers cytochrome P450 3A4 activation, resulting in reduced plasma concentration of ivacaftor. Such findings might partially explain the modest efficacy observed in co-treatment with lumacaftor/ivacaftor in clinical trials. It also highlights the relevance of better evaluating drug-drug and drug-protein interactions for combined therapies. Recent studies have searched for potentiators of F508del-CFTR that do not interfere with lumacaftor actions. Other studies have also identified genes of which either silencing or inhibition induce further lumacaftor-stimulated F508del-CFTR rescue, including RNF5, FAU, and RFFL. Suppression of RPL12, a component of 60S subunit P stalk, was also demonstrated to rescue folding and function of F508del and other CFTR mutants by modulating ribosome velocity. CFTR rescue was increased when lumacaftor was concurrently administered.

32Individuals with CF are susceptible to infection by distinct opportunistic pathogens, including Aspergillus spp., Burkholderia cepacia, Haemophilus influenzae, Pseudomonas aeruginosa, Staphylococcus aureus, and Stenotrophomonas maltophilia. Some studies have demonstrated that CFTR modulator therapies may reduce P. aeruginosa infection in clinics. Co-administration of lumacaftor/ivacaftor in vitro also reduced pro-inflammatory responses induced by P. aeruginosa exoproducts in well-differentiated human bronchial epithelial cells (F508del/F508del genotype). Nevertheless, P. aeruginosa infection was demonstrated to reduce lumacaftor- and lumacaftor/ivacaftor-stimulated CFTR activity in F508del-expressing cells. On the other hand, exposure of cells to supernatant from mucopurulent material derived human CF lung resulted in greater rescue of F508del-CFTR by lumacaftor. As both inflammation and infection may influence responses to modulator therapies, further research is needed to better understand such differences as well as to evaluate the effect of these therapies on infection caused by other pathogens.

33Tezacaftor (VX-661; Vertex Pharmaceuticals) is a second-generation corrector developed based on lumacaftor structure but demonstrating better pharmacokinetic properties and fewer adverse effects. In clinical trials, co-treatment with tezacaftor/ivacaftor demonstrated comparable therapeutic outcomes (sweat chloride concentration, ppFEV1, and others) to those with lumacaftor/ivacaftor in F508del-homozygous patients. For F508del-heterozygous patients with a residual function mutation in trans, co-treatment with tezacaftor/ivacaftor was more effective, demonstrating even better improvements in ppFEV1 in comparison to treatment with ivacaftor only. In 2018, the FDA and the EMA approved the co-treatment with tezacaftor/ivacaftor (Symdeko® or Symkevi®, Vertex Pharmaceuticals) for patients aged ≥12 years who are F508del-homozygous or F508del-heterozygous with a residual function mutation in trans (Table 3). In following phase III clinical trials, co-treatment of tezacaftor/ivacaftor was demonstrated to reduce sweat chloride concentration and preserve lung function with relatively low respiratory symptom burden. Such findings served as basis for the extended approval of tezacaftor/ivacaftor for patients aged ≥6 years. Extension clinical trials are ongoing to evaluate the longer-term effects of tezacaftor/ivacaftor (NCT03537651) and the safety and efficacy in younger children.

34As the dual combinations lumacaftor/ivacaftor and tezacaftor/ivacaftor demonstrated only modest efficacy in F508del-homozygous patients, Vertex Pharmaceuticals performed additional HTSs to identify next-generation correctors that act by different mechanisms and could therefore yield additive/synergistic effects in triple-combination regimens. Tezacaftor/ivacaftor was selected as the backbone for the triple combination based on the more favorable pharmacological properties, including lower cytochrome P450 3A activation. Four novel correctors — VX-152, VX-440, VX-445, and VX-659 — demonstrated a pronounced improvement of CFTR activity when co-administered with tezacaftor/ivacaftor in human bronchial epithelial cells (F508del/F508del genotype). In early-stage clinical studies, all four triple combinations presented evidence of therapeutic benefits to relatively similar degrees (NCT02951195, NCT02951182, NCT3029455, and NCT03227471). Nevertheless, two compounds (VX-445 [or elexacaftor] and VX-659 [or bamocaftor]) demonstrated more suitable pharmacological properties and safety profiles for long-term use, making them better candidates for subsequent clinical studies. Both triple combinations — tezacaftor/ivacaftor/VX-445 and tezacaftor/ivacaftor/VX-659 — were safe, with most adverse effects being mild to moderate, and led to a reduction of sweat chloride concentration and incidence of pulmonary exacerbations in phase II and III trials. A significant increase in ppFEV1 was also found after triple-combination regimens in F508del-homozygous patients (up to 11.0% for the combination with VX-445 and 9.7% for the combination with VX-659 compared to tezacaftor/ivacaftor only) and F508del-heterozygous patients with a minimal function mutation in trans (up to 14.3% for the combination with VX-445 and 13.3% for the combination with VX-659 compared to placebo). In fact, the magnitude of therapeutic responses with triple-combination regimens was even greater than the benchmark achieved by ivacaftor alone in patients with a G551D-CFTR mutation (up to 10.6% in ppFEV1) or other gating mutations. The triple combination tezacaftor/ivacaftor/VX-445 (Trikafta™, Vertex Pharmaceuticals) has been recently approved by the FDA for the treatment of CF patients aged ≥12 years with the mutation F508del in at least one allele. Furthermore, other clinical trials are in progress to evaluate the long-term effects of this triple combination in patients who are F508del-homozygous or F508del-heterozygous with a minimal function mutation in trans (NCT03525574, NCT04043806, NCT04058366) as well as the safety and efficacy in younger patients (NCT03691779, NCT04183790). Another clinical study is also ongoing to evaluate the safety and efficacy of this triple-combination regimen in F508del-heterozygous patients with a gating or residual function mutation in trans (NCT04058353).

35Other compound combinations have been evaluated in order to enhance the correction of F508del-CFTR, but only modest effects were observed in several experimental studies. A rational design for combinatory corrector therapy resulted in the discovery of small-molecule from different chemical series — 6258, 3151, and 4172 — that target defects at NBD1, NBD2, and TMD interfaces. Although the individual administration of these compounds was demonstrated to modestly rescue the functional expression of F508del-CFTR, the combination of three compounds resulted in greater effects than lumacaftor alone. The effects of this triple combination on F508del-CFTR reached ~50-100% of WT-level correction in cell lines, patient-derived specimens, and in mouse nasal epithelia. Moreover, this triple combination significantly increased protein folding efficacy of rare mutations across different CFTR domains.

36Several novel correctors are being developed in collaboration between pharmaceutical companies and academic laboratories. The molecules FDL169 and FLD1737 have been investigated by Flatley Discovery Lab. FDL169 has demonstrated a rescue of CFTR PM expression in F508del-expressing cells with similar efficacy as lumacaftor but with no additive effects when they were co-administered, suggesting that these correctors may act by the same mechanisms. Nevertheless, ivacaftor had lesser inhibitory effect on FDL169 activity than on lumacaftor. The safety profile of FDL169 is under clinical investigation alone and in combination with the potentiator FDL176 (NCT02768297, NCT03093714, NCT03756922). Riociguat (Bayer), a soluble guanylate cyclase stimulator, was demonstrated to improve CFTR function in experimental studies. Although no safety concerns were identified in F508del-homozygous patients in a phase II trial (NCT02170025), the clinical development of this molecule has been terminated for CF.

37The molecules ABBV-2222, ABBV-2737, ABBV-2851, ABBV-3221, and ABBV-3748 (formerly GLPG-2222, GLPG-2737, GLPG-2851, GLPG-3221, and GLPG-3748, respectively) are among the most promising correctors developed by Abbvie/Galapagos. ABBV-2222 (or galicaftor) has a similar chemical structure to lumacaftor and tezacaftor but was reported to be more potent. It was also highly functional in primary bronchial epithelial cells from a F508del-homozygous patient. ABBV-2737 was demonstrated to rescue functional expression of CFTR in F508del-expressing cells, and such effects were enhanced when it was co-administered with lumacaftor or ABBV-2222. ABBV-3221 also rescued CFTR function in F508del-expressing cells with a greater effect in combination with ABBV-2222 and ABBV-974. In phase IIa clinical trials, ABBV-2222 reduced sweat chloride concentrations but did not improve ppFEV1 in F508del-homozygous patients or F508del-heterozygous patients with a gating mutation and receiving ivacaftor. Based on previous studies evaluating drugs with a similar mechanism of action in F508del-homozygous patients, a substantial improvement in lung function would be unusual for a single corrector. Nevertheless, such approach allows a better evaluation of the safety profile for the following studies with combined therapies. In this line, other clinical studies are in progress to evaluate the safety and efficacy of ABBV-2222 alone and in combination with ABBV-2737 and ABBV-2451 (NCT03540524) or with ABBV-3067 (NCT03969888). ABBV-2737 was also investigated for F508del-homozygous patients and on stable treatment with lumacaftor/ivacaftor in a phase IIa trial. A small improvement in ppFEV1 (3.6%) and a significant reduction in sweat chloride concentration were found in patients that received ABBV-2737 compared to placebo cohort.

38The molecule PTI-801 (posenacaftor; Proteostasis Therapeutics) — a third-generation corrector — has demonstrated higher efficacy compared to first- and second-generation correctors (e.g., lumacaftor and tezacaftor) with additive/synergistic effects when they were co-administered in vitro. PTI-801 was also less sensitive to the ivacaftor-mediated decrease on CFTR function. In a phase I trial, PTI-801 was demonstrated to significantly reduce sweat chloride concentration, and improve ppFEV1 and BMI in F508del-homozygous patients receiving lumacaftor/ivacaftor therapy. Another phase I clinical trial is in progress in healthy volunteers and CF patients (NCT03140527). The corrector VX-121 (Vertex Pharmaceuticals) is also in early-stage clinical trials to evaluate the effects in CF patients receiving ivacaftor or tezacaftor/ivacaftor (NCT03768089) and in combination with tezacaftor, ivacaftor, and/or VX-561 (NCT03912233).

39Although certain CFTR mutants are functional and present at the PM, the protein may still display a significant reduction in half-life (Class VI, Figure 4), probably due to accelerated endocytosis and/or reduced recycling. Low temperature incubation rescues CFTR in F508del-expressing cells (r.F508del), but the protein still demonstrates reduced stability and is rapidly removed by peripheral quality control mechanisms. In fact, environmental stresses may also lead to destabilization and internalization of WT-CFTR. Stabilizers are agents that anchor CFTR at the PM, thus preventing its removal and degradation by lysosomes.

40Lumacaftor demonstrated to rescue functional expression of CFTR in F508del-expressing cells, but it does not confer long-term stability similar of that in WT-CFTR. Therefore, novel treatments have been investigated, alone or in combination, to rectify the intrinsic protein instability and thus prolong CFTR residence time at the PM. Hepatocyte growth factor (HGF) was demonstrated to promote CFTR stabilization at the PM in F508del-expressing cells by activating Rac1 GTPase signaling and thus inducing CFTR interaction with Na+/H+ exchanger regulatory factor 1 (NHERF1). Co-administration of HGF and lumacaftor has further enhanced CFTR maturation and anchoring at the PM. Prolonged HGF treatment also prevented ivacaftor-mediated destabilization of lumacaftor-rescued CFTR in F508del-expressing cells. Other strategies have demonstrated to enhance the PM stabilization of mutant CFTR protein, including administration of vasoactive intestine peptide (VIP), activation of exchange factor directly activated by cAMP 1 (EPAC1), and inhibition of S-nitrosoglutathione reductase or CFTR-associated ligand (CAL). More recently, keratin-19 was also demonstrated to stabilize WT-CFTR and lumacaftor-rescued F508del at the PM by decreasing Rab7-mediated lysosomal degradation.

41Cavosonstat (N91115; Nivalis) was the first CFTR stabilizer being tested in clinical trials. It was demonstrated to promote CFTR maturation and PM stability by inhibiting S-nitrosoglutathione reductase in vitro. In a phase I trial, cavosonstat demonstrated no safety concerns with the highest dose yet reducing sweat chloride concentration in F508del-homozygous patients. Nevertheless, cavosonstat failed to demonstrate any additional benefit in lung function and sweat chloride concentration when in combination with lumacaftor/ivacaftor or ivacaftor in phase II trials (NCT02589236 and NCT02724527). The clinical development of cavosonstat has been terminated for CF.

42A significant fraction of the CF-causing mutations are in-frame nonsense, frameshift, and splicing variants that introduce a premature termination codon (PTC) into the CFTR mRNA, thus abrogating CFTR protein synthesis or resulting in translation of shortened, truncated forms (Class I, Figure 4). PTCs are also subjected to nonsense-mediated mRNA decay (NMD), resulting in substantial decrease in the quantity of CFTR transcripts. Around 10% of CF patients worldwide carry a PTC mutation, with G542X and W1282X being the most common PTC variants found in CF alleles (2.5% and 1.2%, respectively). Read-through agents are compounds that induce a ribosomal “over-reading” of a PTC, enabling the incorporation of a foreign amino acid in that place and thus the continued translation to the normal end of the transcript.

43Read-through effects were first found in aminoglycoside antibiotics, such as gentamicin and geneticin. Gentamicin is also commonly used to eradicate P. aeruginosa infection in CF patients. In both cell lines and transgenic mice, these compounds demonstrated the ability to promote expression of full-length CFTR and partially restore CFTR-dependent chloride secretion. Furthermore, gentamicin improved nasal potential difference after administration either topically on the nasal mucosa or intravenously in patients carrying a CFTR PTC mutation. Despite such findings, gentamicin and geneticin cannot be used as read-through agents in the clinics, since high systemic levels or long-term use may result in severe nephrotoxicity and ototoxicity. Advances in the rational design of chemically modified aminoglycosides have enabled the identification of read-through agents with higher activity and less toxicity. In this line, ELX-02 (NB124; Eloxx Pharmaceuticals) has demonstrated to restore CFTR function in cells expressing any of the four most prevalent PTC mutations — G542X, R553X, R1162X, and W1282X. Moreover, ELX-02 was less cytotoxic than gentamicin in a model for ototoxicity. In phase I clinical trials with healthy volunteers, ELX-02 was well tolerated and exhibited a favorable safety profile, although mild side effects were also reported. Early-stage clinical trials are in progress to evaluate the effects of multiple dose escalation of ELX-02 in CF patients carrying the G542X mutation in at least one allele (NCT04126473, NCT04135495).

44Ataluren (PTC124; PTC Therapeutics) was identified by HTS, and demonstrated to restore CFTR expression and function in transgenic mice expressing human G542X. Despite therapeutic effects observed in phase II clinical trials, ataluren treatment only demonstrated a favorable trend, albeit not significant, to improve ppFEV1 of CF patients carrying a nonsense CFTR mutation in at least one allele in a phase III clinical trial. A new phase III trial was undertaken to exclude patients taking inhaled tobramycin, since it could interfere with ataluren actions on the ribosome; however, no improvements were observed in ppFEV1 and pulmonary exacerbations (NCT02139306). Although ataluren (Translarna®, PTC Therapeutics) is approved for the treatment of patients with Duchenne muscular dystrophy, its clinical development has been terminated for CF. Other studies have performed HTSs to identify potential read-through agents for the various PTC mutations.

45In addition to read-through agent-induced efficacy, the abundance of CFTR transcripts and the activity of the recoded protein are other factors that should be considered in order to efficiently rescue a PTC mutant. The number of transcripts may considerably differ depending on the identity (amber, ochre, or opal) and position of the termination codon as well as cell type and patient's genetic background, and association of an NMD inhibitor with a read-through agent may result in better therapeutic efficacy. In this line, amlexanox and escin are drugs already approved for unrelated diseases that demonstrated dual activity by concomitantly increasing the abundance of target transcripts and read-through efficacy for certain CFTR PTC mutations. Furthermore, incorporation of a foreign amino acid may result in full-length but misfolded and/or nonfunctional proteins. PTC suppression in combination with other modulators, such as lumacaftor and/or ivacaftor, has demonstrated to promote a further rescue of expression and function of CFTR PTC mutations. These approaches should be exploited in future clinical studies.

46A nucleic acid therapeutic has been developed for CF-causing nonsense mutations. RCT101 (ReCode Therapeutics) is a modified transfer RNA (tRNA) that is delivered into cells by nanoparticles to precisely recode the translating CFTR protein. In experimental studies, RCT101 demonstrated an increase in CFTR-dependent chloride secretion in primary bronchial epithelial cells carrying either G542X/G542X or G542X/F508del. Such effects were even greater when combined with lumacaftor/ivacaftor.

47Some CF-causing mutations lead to reduction in the synthesis or maturation of CFTR protein. The 3849+10kbC>T, 2789+5G>A, and A455E are common mutations that cause such abnormalities (Class V, Figure 4) and are found in 0.8%, 0.7%, and 0.4% of the CF alleles, respectively. Amplifiers are compounds that increase expression of CFTR mRNA and, consequently, biosynthesis of the CFTR protein.

48The PTI-428 (nesolicaftor; Proteostasis Therapeutics) is the first-in-class amplifier investigated in clinical trials. It was demonstrated to selectively increase the expression of immature CFTR protein carrying different mutations without eliciting alteration in the expression of cellular stress response genes. PTI-428 also enhanced the rescue of CFTR in F508del- and ΔI1234_R1239-expressing cells when co-administered with lumacaftor/ivacaftor. Furthermore, the triple combination PTI-428/PTI-808/PTI-801 enhanced the CFTR-dependent chloride secretion to almost normal levels in F508del-expressing cells. In phase I/II clinical trials (NCT03500263), this triple combination regimen resulted in significant reduction of sweat chloride concentration and improvement of lung function (8% in ppFEV1) compared to placebo in F508del-homozygous patients, being the greatest effects observed in those individuals with high disease burden. Based on these results, a phase III trial is planned to begin in early 2020. In F508del-heterozygous patients, the triple combination PTI-428/PTI-808/PTI-801 demonstrated a more variable change in sweat chloride concentration and lung function. These modulator drugs have also been tested in intestinal organoids of patients carrying rare CF genotypes in the HIT-CF project and the crossover clinical trial based on the individual responses is expected to initiate in the middle of 2020. Other early-stage clinical trials are in progress to evaluate the safety and efficacy of PTI-428 in CF patients on stable treatment with ivacaftor (NCT03258424), lumacaftor/ivacaftor (NCT02718495), or tezacaftor/ivacaftor (NCT03591094).

49Antisense oligonucleotides (ASOs) are chemically-modified synthetic RNA-like molecules that act by complementary base pairing to the target sequence. ASOs have been demonstrating promising results to correct nonsense and splicing mutations or even to replace missing bases caused by deletion mutations, such as the F508del.

50ASOs corrected the aberrant splicing in a cell line expressing the 2789+5G>A mutation minigene. Such correction resulted in recovery of CFTR protein levels at the PM. Recently, ASOs developed by SpliSense have also demonstrated to correct aberrant splicing and restore CFTR function in a 3849+10kbC>T-expressing cell line and in primary bronchial epithelial cells (3849+10kbC>T/F508del genotype).

51The mutant W1282X is subjected to NMD, thus resulting in significant reduction of CFTR mRNA abundance. Nevertheless, it still produces a certain amount of the truncated forms of both partially and fully glycosylated CFTR protein that may respond to CFTR modulators. ASOs designed to downregulate the serine/threonine-protein kinase SMG-1, a factor involved in the NMD pathway, led to upregulation of mRNA, protein maturation and traffic to the PM of the truncated CFTR products in W1282X-expressing cells. Furthermore, these ASOs increased CFTR-dependent chloride secretion in W1282X-homozygous cells.

52Eluforsen (QR-010; ProQR) is a modified RNA oligonucleotide that was demonstrated to restore CFTR function in a F508del-expressing cell line, patient-derived specimens and murine models. Furthermore, eluforsen was able to efficiently diffuse through CF-like mucus layer on air-liquid interface cell cultures. In an early-stage trial assessing single and multiple doses in F508del-homozygous patients, eluforsen was well tolerated and improved quality of life. Lung function also remained stable throughout the study. In a following clinical study, repeated intranasal administration of eluforsen resulted in improvement in the nasal potential difference in F508del-homozygous patients, but not in the F508del-heterozygous cohort. Although no severe safety concerns have been reported, the clinical development of eluforsen has been discontinued.

53CFTR modulators have become transformative therapeutic approaches for many CF patients, as mentioned above. Despite several breakthroughs, further research is needed to continue optimizing therapies and to identify novel modulators for patients carrying rare, ultra-rare, or even unique CFTR mutations, who still face an unmet need for efficient, corrective therapies. Furthermore, some barriers still pose substantial challenges in the equitable availability of these pharmacotherapies, including the excessive costs and regulatory national issues. The collaborative environment composed by academic researchers, healthcare professionals, pharmaceutical companies, and patient representatives has been crucial in developing better treatments for people with CF.

54The multifaceted nature of CF requires complex and time-consuming therapeutic regimens that should be periodically adapted according to disease progression. Furthermore, CF patients are subjected to substantial clinical, psychosocial, and economic burdens, which pose challenges to achieve optimal, lifelong treatment adherence. The adherence varies largely depending on treatment type, route of administration, duration, and number of distinct medications, as well as patient age and socioeconomic status. Moreover, CF patients usually spend twice and 20 times more time in daily treatment activities than diabetic and asthmatic patients, respectively, which may considerably affect adherence. Poor adherence has also been associated with higher healthcare costs, more frequent hospitalizations, and worse quality of life and clinical manifestations. Establishing a closer relationship among patient, families/caregivers and the multidisciplinary healthcare team may be a first step to overcome key barriers to treatment adherence.

55To date, only few publications have evaluated the adherence to ivacaftor treatment and adherence to modulator combinations remains yet to be demonstrated. From a clinical perspective, a life-transforming oral medication with a simple dosing schedule would supposedly be taken as prescribed. Adherence to ivacaftor has nevertheless varied from suboptimal to optimal. As these studies had a small sample size and applied distinct methods, it is still difficult to extrapolate the results to a broader CF population, and further studies are needed to better address this issue. Certain therapeutic benefits may also be more modest in a real-world setting compared to clinical trials, as patients should take these oral medications following specific recommendations, including dietary to ensure better drug absorption and availability in the body. Furthermore, recent studies have demonstrated that abrupt interruption of CFTR modulator therapy may cause severe clinical consequences. Ivacaftor withdrawal resulted in accelerated deterioration of lung function consistent with a pulmonary exacerbation episode in a case series. Patients who discontinued treatment with lumacaftor/ivacaftor, mainly due to early adverse effects, also demonstrated a higher risk of worsening clinical manifestations compared to patients who continued treatment or those who restarted it after temporary discontinuation in a real-world study. As patients have different lifestyles and socioeconomic conditions, the development of educational and motivational interventions at an individual level may help in ensuring optimal adherence to achieve the greatest clinical outcomes.

56CFTR modulators have been added to therapeutic regimens of eligible patients, rather than replacing some symptomatic therapies. This appears to be the optimal approach for most CF patients, although it also increases the burden of medications in use. Once the safety and efficacy of novel therapies are demonstrated in adults, extension clinical trials are pursued to evaluate the effects on younger patients, as adverse effects may vary across distinct age groups. Starting these transformative therapies in milder disease severity and earlier in life may offer more chances of significantly improving long-term outcomes or even preventing certain injury of affected organs, which may also result in a lower burden of medications in a long-term perspective. Some reports have also demonstrated that younger patients are more adherent to therapies than adolescents and adults, possibly due to higher parental supervision. Providing educational and supporting approaches to young children and their parents may result in optimal, lifelong treatment adherence. As patients should be transferred from pediatric to adult care at a certain age (generally between 18 and 21 years old), a planned transition is greatly helpful to maximize independence, minimize chances of interruption in the therapies and continue improving their quality of life.

57Most development programs of CFTR modulators has been initially focused on the correction of F508del mutation, since fully overcoming the defects in this mutation would result in an effective therapy for approximately 82% of the CF patient population worldwide. There are still nevertheless 10%–18% of patients without any CFTR-directed therapeutics. This percentage is even higher in countries where the prevalence of F508del is much lower, such as Brazil, Israel, Italy and Turkey (Figure 3).

58Identifying the putative binding sites of CFTR-directed modulators using the novel insights of CFTR structure may facilitate the rational design of novel compounds with enhanced pharmacological properties. The pipeline of CFTR modulators continues to expand and some recent drug development programs have also been pursuing the identification of modulators to less common CF-causing mutations. Identification of specific therapies for rare and ultra-rare mutations poses nevertheless several challenges due to the great variability of CF-causing mutations and the very small number of patients. In addition to CFTR-directed modulators, CFTR dysfunction might be compensated by targeting alternative ion channels, such as ENaC, the calcium-activated chloride channel transmembrane protein membrane 16A (TMEM16A), and the solute carrier 26A9 (SLC26A9). Strategies that modulate these alternative ion channels might be efficient therapies for all patients, regardless of their CF genotypes. These strategies might also be used alone or in combination with CFTR modulators to enhance clinical outcomes. Nevertheless, as CF patients are already subjected to a substantial burden of medications, drug-drug interaction profiles should be further exploited to avoid adverse effects or inhibitory effects of one therapy on another. In this line, itraconazole, an antifungal commonly used for the treatment of allergic bronchopulmonary aspergillosis, was demonstrated to significantly increase systemic exposures of tezacaftor and ivacaftor. Caution and appropriate monitoring are recommended when these therapies are used at the same period.

59Traditional trials with a placebo-controlled design have been providing evidence for the safety and efficacy of CFTR modulators (Table 1); however, alternatives will be needed in the near future, as more modulator options become available and the number of patients without any modulator therapy will certainly reduce. Furthermore, clinical trials in sicker or younger patients, and those carrying rarer CFTR mutations are more challenging due to small sample size, specific inclusion/exclusion criteria, or even for some hesitation on the part of the investigators. Strategies to adapt and optimize trial design and deliver for speed and efficacy have been discussed, including the use of patient-derived specimens, power calculations to compensate for group sampling, and N-of-1 and “basket” trials.

60The use of patient-derived specimens to comparatively evaluate drug efficacies may be a feasible starting point to identify the best candidate drug(s) in vitro and predict the magnitude of therapeutic responses for following clinical testing. In fact, a significant but variable clinical responsiveness was observed in clinical trials with CFTR modulators in patients carrying at least one G551D mutation or in F508del-homozygous patients, which suggests that patient responsiveness to a certain therapy is influenced not only by the CF genotype but also by the genetic background and/or epigenetic factors. In this line, some reports have demonstrated that single nucleotide polymorphisms in SLC26A9 gene contribute to heterogeneity in inter-individual responsiveness to CFTR modulator therapies. Such findings denote the relevance of assessing the drug effectiveness at an individual level in patient-derived specimens.

61A report pairing in vitro measurement of CFTR function in cell lines and clinical features demonstrated a strong correlation between CFTR function and sweat chloride concentration, and to a lesser extent but still significant with lung function and pancreatic status. Correlations between responses in patient-derived specimens and clinical parameters/biomarkers have been investigated to establish reliable prediction of drug effectiveness. A consistent correlation was found among forskolin-induced swelling of intestinal organoids, sweat chloride concentration and intestinal current measurements of infants with CF. Despite the clinical heterogeneity in adults with CF and homozygous for F508del mutation, forskolin-induced swelling of intestinal organoids positively correlated with FEV1 and BMI. Responses from intestinal organoids were also demonstrated to correlate with intestinal current measurements, reduction in sweat chloride concentration and improvement in lung function of patients after CFTR modulator therapies. In N-of-1 trial series, an increase in CFTR-dependent chloride transport in nasal epithelial cell cultures was only found in the three patients who also demonstrated a reduction in sweat chloride concentration after ivacaftor treatment. Furthermore, responses in F508del-homozygous patient-derived nasal epithelial cells were correlated to improvements in ppFEV1 and intestinal current measurements, but not with nasal potential difference after co-treatment with lumacaftor/ivacaftor. Nevertheless, no significant correlations were found among responses in intestinal current measurement, nasal potential difference and sweat chloride concentration, despite high concordance for all CFTR-dependent biomarkers in another study evaluating the co-treatment with lumacaftor/ivacaftor. Further studies are certainly needed to better correlate and validate drug effectiveness in patient-derived specimens with clinical features, and identification of novel biomarkers may also enrich strategies in efficacy trials.

62In an era of drugs targeting the underlying defects in CF-causing mutations, the development of symptomatic therapies might appear less attractive. Nevertheless, these therapies must continue to be developed as most (if not all) existing CF population will need them at some point, and CFTR modulators are very unlikely to reverse lung tissue remodeling already established. A recent study demonstrated that six months of ivacaftor treatment was unable to significantly change airway microbiome and several inflammation measurements in patients carrying at least one G551D mutation. Such findings indicate that antibiotics and anti-inflammatory drugs will still be required to control disease symptoms and prevent complications. As the disease progresses, patients may also develop comorbidities and thus require even more complex therapeutic regimens, adding further burdens. Ivacaftor treatment was demonstrated to improve exocrine pancreatic function as well as insulin secretion profile, which may alleviate or even reverse CF-related diabetes. Ivacaftor treatment was also demonstrated to improve bone health and vascular tone abnormalities. A recent review nicely summarizes the current understanding of CFTR modulators on extra-pulmonary complications in CF. Nevertheless, most studies are case reports or have a small sample size, and further studies are warranted to investigate the impact of CFTR modulator therapies on CF comorbidities.

63Treatment with more than one CFTR modulator appears to be the optimal approach for many CF-causing mutations. As heterozygous carriers are asymptomatic, fully overcoming CFTR dysfunction in one allele might be enough to halt disease progression, if treatment is started early in life and before severe lung injury occurs. Based on in vitro evidence, rescue of 25-50% of WT-CFTR function in both alleles might also be sufficient to restore normal rates of mucociliary clearance. It remains nevertheless unclear how many CFTR modulators would be needed to reach such threshold in patients. In addition to CFTR modulators, progress has been made in developing cell-based and gene-based therapies for CF lung disease.

64A major limitation of these novel pharmaceutical treatments for CF patients, such as the CFTR modulators, is the excessive costs when they reach the market (over US$250,000 per patient per year), which renders difficulties in their availability for many patients worldwide, especially for those living in low- and middle-income countries. In developed countries, certain health authorities have also been slow in approving reimbursement and the cost-effectiveness of these pharmacotherapies has yet been questioned. Even though the quality-adjusted life-year (QALY) analysis might not adequately address all concerns for rare diseases, such as CF, these therapies pose a substantial burden on national healthcare systems, as they are expensive and lifelong. It remains nevertheless unclear if such prices will persist over time, as several novel molecules are on the horizon and probably will reach the market over the next years, if they prove to be safe and to have efficacy in clinical studies. Further discussion should certainly be undertaken with patient representatives, healthcare providers, policymakers, government authorities and pharmaceutical companies to identify feasible and sustainable solutions that would enable equitable access to eligible patients for these “on-target” therapies. Hopefully, market competition will also reduce modulator prices with the approval of novel ones.

65Over the past three decades, the human disease target landscape considerably expanded, as approximately 40% of approved pharmaceuticals received an orphan designation. Drug discovery and development for a new molecule may be nevertheless far slower than expected, as it is a costly process with high attrition rates that also depends on several regulatory requirements. Drug repurposing (also known as drug repositioning or reprofiling) has become an increasingly attractive strategy that may save valuable time and funding investments in drug development for common and rare diseases. As approved drugs have already undergone extensive toxicological evaluations in both experimental and early-stage clinical studies, the time frame to obtain a new disease indication may be reduced, if safety and efficacy is demonstrated for the repurposed use in late-stage clinical studies. Furthermore, drug repurposing may unravel effective therapies for patients with common and rare CF-causing mutations in an expedited way and at a feasible cost for national healthcare systems. In experimental models, certain underlying defects in CFTR mutations have been rectified by administering clinically approved drugs, such as gentamicin, amlexanox, escin, ibuprofen, and genistein. These findings indicate that other existing and approved drugs for unrelated disease indications might have the potential to correct or circumvent CFTR dysfunction and should be exploited in the pre-clinical setting. Both cysteamine and thymosin α-1 were also claimed to restore functional expression of F508del-CFTR. Nevertheless, several independent CF research groups failed to demonstrate rescue of F508del-CFTR PM expression and function by either cysteamine or thymosin α-1. Although the immunomodulatory effect of these molecules in CF remains to be further exploited, they did not demonstrate F508del-CFTR correction. As the identification of highly efficient treatments often draws the attention of both scientific and lay audiences, a note of caution should be considered before such findings are diffused in the press to avoid creating premature expectations, especially in CF patients and their relatives.

66Thirty years have passed since the discovery of the CFTR gene, and numerous milestones in experimental and clinical research of CF have been achieved during this period (Figure 8). Understanding the cellular and molecular basis of the disease has paved the way for the development of therapeutic strategies targeting the underlying dysfunctions caused by CF mutations. CFTR modulator therapies are in clinics and they represent a landmark in patients' lives, demonstrating short- and long-term benefits in clinical outcomes. Nevertheless, strategies to achieve optimal, lifelong adherence to treatments should be optimized. Several barriers have still been preventing equitable access worldwide of the current CFTR modulators, including the costs and regulatory national issues, and as such further discussions are needed to identify feasible and sustainable solutions for these therapies to achieve all eligible patients. Furthermore, many rare and ultra-rare CF-causing mutations are still without any efficient, corrective therapy. Novel tools have been developed to accelerate and continue to expand the pipeline of CFTR modulators. From a translational perspective, the use of patient-derived specimens would ensure a comparative evaluation of drug efficacies in vitro to select the best candidate(s) and predict the therapeutic responses at an individual level. Every patient is unique, but everyone certainly wants the same: to have a longer and healthier life (ideally, with no symptoms or complications). Hopefully, precision medicine will enable “the highest attainable standard of health” for all patients with CF, and then we will see “all our brothers and sisters breathing free.”

67CF patients under care at accredited care centers in Argentina, Australia, Brazil, Canada, Europe, New Zealand, South Africa, United Kingdom, and United States of America. Figures 2 and 3 are data compiled from the last Patient Registry Report in Australia (Cystic Fibrosis Australia), Brazil (Brazilian Cystic Fibrosis Study Group), Canada (Cystic Fibrosis Canada), Europe (European Cystic Fibrosis Society), New Zealand (Cystic Fibrosis New Zealand), UK (Cystic Fibrosis Trust), and USA (Cystic Fibrosis Foundation). Data from Argentina (Associación Argentina de Lucha contra la Enfermedad Fibroquística del Páncreas) and South Africa (South Africa Cystic Fibrosis Association) have been obtained from Annals of scientific events. A citation with the link to access each Registry Report has been provided.

68The author confirms being the sole contributor of this work and has approved it for publication.

69The author is a recipient of the 2018 Gilead Sciences Research Scholars for Cystic Fibrosis.

70The author declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.