New treatments targeting the basic defects in cystic fibrosis

Presse Med. 2017; 46: e165–e175

CYSTIC FIBROSIS

on line on www.em-consulte.com/revue/lpm www.sciencedirect.com

Quarterly Medical Review

New treatments targeting the basic defects in cystic fibrosis Isabelle Fajac 1,2, Claire E. Wainwright 3,4

Available online: 26 May 2017

1. Université Paris Descartes, Sorbonne Paris Cité, site Cochin, 24, rue du FaubourgSaint-Jacques, 75014 Paris, France 2. AP–HP, hôpital Cochin, service de physiologie et explorations fonctionnelles,27, rue du Faubourg-Saint-Jacques, 75014 Paris, France 3. University of Queensland, St Lucia Queensland 4072,Brisbane, Australia 4. Lady Cilento Children's Hospital, 501 Stanley St, 4101 Brisbane, QLD, Australia

Correspondence: Isabelle Fajac, université Paris Descartes, Sorbonne Paris Cité, site Cochin, 24, rue du Faubourg-Saint-Jacques, 75014 Paris, France. [email protected]

Editorial. P.R. Burgel (France) The Changing Epidemiology and Demography of Cystic Fibrosis. A.L. Stephenson (Canada, France) et al. The diagnosis of cystic fibrosis. K.A. De Boeck (Belgium) et al. Common clinical features of cystic fibrosis (Respiratory Disease and Exocrine Pancreatic Insufficiency). R. Somayaji (Canada, United States) et al. Current and emerging comorbidities in cystic fibrosis. B. Plant (Ireland) et al. The treatment of the pulmonary and extrapulmonary manifestations of cystic fibrosis. M. Chin (Australia, Canada) et al. New treatments targeting the basic defects in cystic fibrosis. I. Fajac (France, Australia) et al.

tome 46 > n86 > June 2017 http://dx.doi.org/10.1016/j.lpm.2017.01.024 © 2017 Elsevier Masson SAS. All rights reserved.

Summary Cystic fibrosis (CF) is a monogenic autosomal recessive disorder affecting around 75,000 individuals worldwide. It is a multi-system disease but the main morbidity and mortality is caused by chronic lung disease. Due to newborn screening, a multidisciplinary approach to care and intensive symptomatic treatment, the prognosis has dramatically improved over the last decades and there are currently more adults than children in many countries. However, CF is still a very severe disease with a current median age of life expectancy in the fourth decade of life. The disease is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene which encodes the CFTR protein, a protein kinase A-activated ATP-gated anion channel that regulates the transport of electrolytes such as chloride and bicarbonate. More than 2000 mutations have been reported, although not all of these have functional consequences. An enormous research effort and progress has been made in understanding the consequences of these mutations on the CFTR protein structure and function, and this has led to the approval of two new drug therapies that are able to bind to defective CFTR proteins and partially restore their function. They are mutation-specific therapies and available at present for specific mutations only. They are the first personalized medicine for CF with a possible disease-modifying effect. A pipeline of other compounds is under development with different mechanisms of action. It is foreseeable that new combinations of compounds will further improve the correction of CFTR function. Other strategies including premature stop codon read-through drugs, antisense oligonucleotides that correct the basic defect at the mRNA level or gene editing to restore the defective gene as well as gene therapy approaches are all in the pipeline. All these strategies are needed to develop disease-modifying therapies for all patients with CF.

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In this issue

I. Fajac, C.E. Wainwright

Introduction Cystic fibrosis (CF) is an autosomal recessive multi-organ disease affecting approximately 75,000 individuals worldwide [1]. The main clinical features are exocrine pancreatic insufficiency and bronchiectasis with chronic airway infection leading to respiratory failure and premature death. The disease was described in 1938 [2] and although the biochemical basis of CF was not identified for 50 years, the disease was known to be associated with abnormalities of chloride and sodium transport in several epithelia [3,4]. In 1989, the cystic fibrosis transmembrane conductance regulator (CFTR) gene was cloned [5] and the CFTR protein identified as a chloride channel [6]. This led to a growing research output on the basic defects in CF: the CFTR gene and mutations, and the CFTR protein's maturation, structure, function and interactions with other ion channels. The mainstay of treatment in CF is symptomatic and focuses on compensating for exocrine pancreatic insufficiency with pancreatic enzymes, fatsoluble vitamins and high caloric intake; and slowing lung disease progression with inhaled and physical therapies that improve airway clearance, and antibiotic therapy [7]. The goals of new approaches to therapy include the development of drugs that correct the basic defect in CF that might delay the progression or prevent respiratory disease if given early enough in life. Since 2012, and twenty-three years after the cloning of the CFTR gene, two new drugs aimed at correcting defective CFTR protein have been made available to patients bearing specific CFTR mutations and they are the first personalized medicine in CF. Improvements on these approaches as well as many other corrective strategies are currently under clinical investigation. In this review, we will discuss the basic defects in CF that might be targeted to develop disease-modifying treatments, and provide an overview of the two currently available CFTR modulator therapies. Lastly, we will review other strategies currently in development to restore robust CFTR function along with some discussion around the remaining challenges that need to be overcome before such disease-modifying drugs are made available to all patients with CF.

Cystic fibrosis basic defects that are possible therapeutic targets CFTR gene and mRNA The CFTR gene was cloned in 1989 by using chromosome walking and jumping, and linkage disequilibrium analysis [5]. The gene comprises 27 coding exons, spanning over 250 kb on

Glossary

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CF CFTR ENaC ppFEV1

Cystic fibrosis Cystic Fibrosis transmembrane Conductance Regulator Epithelial sodium channel percent predicted forced expiratory volume in one second

the long arm of chromosome 7, and the transcript is 6.5 kb. To date, around 2000 CFTR mutations have been described. However, a molecular alteration in the DNA sequence does not necessarily lead to a defective CFTR protein and clinical disease. Around 250 variants have evidence supporting a disease-causing effect [8,9]. Only
20 mutations occur at a worldwide frequency above 0.1% in CF patients, and determining the disease liability of very rare mutations is often difficult [10]. As CF is a genetic disease due to mutations in a single gene, one approach to treat the basic defect would be to develop oligonucleotide-based drugs to bypass or correct the DNA or mRNA defects in order to produce a normal CFTR protein (figure 1). These strategies known as gene therapy, gene editing or RNA repair have been the subject of extensive research since the cloning of the CFTR gene. As oligonucleotides are poorly suited for oral or systemic delivery, these approaches are likely to remain restricted to local respiratory treatment via aerosol. The great advantage of the gene therapy approach is that it is not restricted to specific mutations and airway administration of normal cDNA could theoretically treat airway disease of all patients. Successes and pitfalls of targeting DNA or mRNA defects to treat CF are described later in this review.

CFTR protein The CFTR protein is an anion channel expressed at the apical membrane of many epithelial cells. It allows chloride and bicarbonate transport thereby creating an osmotic gradient for fluid secretion. The CFTR protein is a member of the ATP-binding cassette (ABC) family of transporter proteins. They are characterized by two membrane-spanning domains (MSD1 and MSD2) which form the channel pore, and two nucleotide-binding domains (NBD1 and NBD2) which bind and hydrolyse ATP. CFTR has an additional regulatory domain which regulates channel opening and closing (for review: [11]) (figure 2). When open or activated, the CFTR protein allows passive diffusion of chloride or bicarbonate ions down their electrochemical gradient. The CFTR protein also has many other roles such as inhibition of sodium transport through the epithelial sodium channel (ENaC) and regulation of other chloride channels. It is also thought to interact with cellular pathways related to inflammation [12]. Mucociliary clearance is a primary innate defense mechanism that helps to protect healthy airways from accumulation of inhaled particles including bacteria [13]. The airway epithelium is mainly composed of ciliated cells on which lies an airway surface liquid consisting of an overlying mucus layer and a periciliary liquid layer. The mucus layer is produced by submucosal glands and goblet cells and contains endogenous antimicrobial agents that kill bacteria. The periciliary liquid layer produced by the airway epithelium is crucial because it provides a low-viscosity solution in which cilia can beat rapidly [12,14]. Thus, in the healthy situation, the cilia beat within the periciliary liquid and sweep bacteria trapped in mucus out of the lung.

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Figure 1 Ion transports in healthy and CF airways, and therapeutic strategies targeting the basic defects in CF

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A. In healthy airways, effective mucociliary clearance requires proper hydration of the airway surface layer and this is achieved by movements of salts driving secretion of water. CFTR and other chloride channels such as TMEM16A and SLC26A9 allow chloride secretion. The ENaC channel is downregulated by CFTR and it allows passive sodium absorption. B. In the presence of CFTR mutations, the CFTR protein is defective and there is an imbalance between CFTR-dependent chloride secretion and ENaC-mediated sodium absorption. This leads to a low volume, dehydrated airway surface liquid and impaired airway mucociliary clearance. CFTR dysfunction also causes abnormal bicarbonate secretion resulting in reduced pH of the airway surface liquid and impaired bacterial killing. C. Gene therapy, gene editing and mRNA repair are strategies using oligonucleotides to target the DNA or the RNA in order to restore CFTR function. Read-through agents for class 1 mutations and CFTR modulators are small pharmacological molecules aiming at restoring CFTR function. ENaC inhibitors and activators of non-CFTR chloride channels could bypass the dysfunctional CFTR and restore normal sodium absorption and chloride secretion. Several of these approaches could be combined.

I. Fajac, C.E. Wainwright

Figure 2 Schematic diagram of the CFTR protein showing its domain organization The two transmembrane-spanning domains (MSD1 and MSD2) form the channel pore. Opening of the pore, and the subsequent anion flow through it, is powered by cycles of ATP binding and hydrolysis at the two ATP-binding sites located on the intracytoplasmic nucleotide-binding domains (NBD1 and NBD2). The intracellular regulatory domain (R domain) contains numerous sites of phosphorylation (P). Activation of CFTR requires phosphorylation of the R domain as it stimulates CFTR function by enhancing ATP-dependent channel gating at the NBDs.

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When the CFTR protein is defective, there is an imbalance between CFTR-dependent chloride secretion and ENaC-mediated sodium absorption. This leads to a low volume, dehydrated airway surface liquid, therefore impairing airway mucociliary clearance [14,15]. CFTR dysfunction also causes impaired bicarbonate secretion and this results in reduced pH of the airway surface liquid, thus interfering with the innate immune system's ability to kill bacteria [16]. These defects in the airways' innate defence trigger a chain of events including mucus stasis and plugging, airway obstruction, infection and inflammation [12,17] (figure 1). Due to mutations in the CFTR gene, the CFTR protein is defective in CF. One strategy to treat the basic defect is to use small molecules to modulate defective CFTR protein and restore functional ion transport (figure 1). However, the numerous CFTR mutations that have been identified lead to many different CFTR functional defects. Therefore, an approach to correct the CFTR protein would need to be mutation-specific or mutationclass-specific. Indeed, CFTR mutations have been grouped into

six classes according to their effects on the maturation and function of the CFTR protein [18,19] (table I):  class 1 mutations result in no protein production. They comprise mostly nonsense or premature stop codon mutations (e. g. G542X or Gly542X) and result in a truncated mRNA that is degraded by nonsense-mediated decay. These mutations are present in around 10% of patients worldwide. In class 1 mutations, CFTR production could be obtained by agents able to read through the premature stop codons and allow the cell to produce a full-length, functional protein;  class 2 mutations cause protein trafficking defects which result in premature degradation of CFTR. The most common mutation, Phe508del, belongs to this class. It is present on at least one allele in 70% of patients with CF. Agents allowing the CFTR protein resulting from class 2 mutations to reach the cell surface are called correctors. As the protein may still be poorly functional at the cell surface, other agents enhancing CFTR function are likely to be needed as well;  class 3 mutations are gating mutations, allowing trafficking of CFTR to the apical membrane but causing defective regulation of the chloride channel. They result in a very poorly functional CFTR protein. These class 3 mutations are present in 4–5% of patients worldwide. The most common class 3 mutation is G551D (Gly551Asp) and is observed in around 4% of patients worldwide (around 3000 patients). Agents increasing CFTR channel gating to enhance chloride transport are called potentiators;  class 4 mutations, such as R117H (Arg117His), lead to a CFTR protein present at the apical membrane but with decreased conductance. Potentiators and/or agents called stabilizers that stabilize the CFTR protein at the cell membrane might be able to correct the defective CFTR protein resulting from class 4 mutations;  class 5 mutations lead to the production of a normal CFTR protein but in a reduced quantity because of aberrant splicing (e.g. 3849 + 10kbC!T) or moderately decreased trafficking (A455E or Ala455Glu). Stabilizers or agents called amplifiers because they amplify the amount of CFTR in the cell could be used for class 5 mutations;  class 6 mutations (e.g. 120del23) lead to a high turnover of CFTR at the apical surface. Stabilizers and amplifiers might also be used for this class of mutations. Class 1, 2 and 3 mutations are commonly associated with pancreatic insufficiency and severe disease, whereas class 4, 5 and 6 mutations are frequently associated with pancreatic sufficiency and a milder phenotype [8]. This complex classification is useful but quite theoretical: some mutations occur rarely and their effect on CFTR function, and thus their class, is unknown. Many mutations induce several CFTR protein defects with overlap across different classes [20]. This is the case for the most frequent Phe508del mutation that belongs to class 2 but also has gating defects and higher cell turn over when expressed at the apical cell membrane.

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TABLE I Classes of CFTR mutations Mutation class

Class 1

Class 2

Class 3

Class 4

Class 5

Class 6

Consequences

No stable RNA

CFTR trafficking defects

Defective CFTR regulation

Decreased CFTR conductance

Reduced CFTR synthesis

Decreased CFTR stability

G542X

F508del

G551D

R117H

3849 + 10kbC!T

N287Y

W1282X

N1303K

G551S

R334W

2789 + 5G!A

4326delTC

10%

F508del allele: 70%

4%

3%

3%

Membrane location

No

Very low

Present

Present

Reduced

Reduced

Function

No

Poor

Poor

Partly

Good

Good

Read-through agents

Combination of correctors and potentiators

Potentiators

Correctors potentiators

Stabilizers amplifiers

Stabilizers amplifiers

Lumacaftor/ivacaftor for F508del/F508del

Ivacaftor

Ivacaftor for R117H

Examples of mutations

Frequency (% of patients) CFTR protein

Marketed drugs

The epithelial sodium channel (ENaC) and alternate chloride channels The CFTR channel interacts with other ion channels at the cell membrane (for review: [21]). CFTR inhibits sodium transport through the epithelial sodium channel and in CF, the overactivity of ENaC contributes to the airway surface liquid dehydration. CFTR also interacts with other chloride channels such as SLC26A9. Moreover, alternate chloride channels have been identified in the airways such as TMEM16A. Pharmacological correction of the ion transport defect observed in CF could be achieved by bypassing the abnormal CFTR protein and using ENaC blocking agents or activators of alternate chloride channels (figure 1).

Innovative therapies currently available for patients with CF The advent of CFTR modulator therapy in the last decade has revolutionized the approach to CF treatment by targeting the underlying CFTR defect. The rapidity that these novel agents have progressed from preclinical trials to clinical use, and the expanding research into more potent drugs targeting a broader range of mutation classes is at once exciting and challenging.

Ivacaftor, the first CFTR potentiator approved to treat CF patients with a gating mutation The first CFTR modulator approved for clinical use was ivacaftor, a CFTR potentiator developed by Vertex Pharmaceuticals, that increases the open probability (gating) of the CFTR channel. Class 3 CFTR gating mutations were the obvious target for this therapy although the benefit of potentiation of CFTR has also been exploited in improving CFTR function in patients carrying the residual function R117H (Arg117His) mutation which is predominently a Class 4 or conductance mutation but which also has some gating dysfunction.

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The most common gating mutation is the G551D (Gly551Asp) mutation, accounting for approximately 4% of CFTR alleles worldwide and associated with adequate CFTR protein production but no CFTR function. Two landmark phase 3 trials of orally-administered ivacaftor in patients carrying at least one copy of the G551D (Gly551Asp) mutation aged 6 to 11 years in one trial and in young people and adults aged 12 years and older in the other trial [22,23] demonstrated consistent and robust improvements in lung function, and nutritional status, and reported a marked reduction in sweat chloride measurement to below the diagnostic cut off for CF, confirming restoration of CFTR function. Important improvements in the frequency of pulmonary exacerbations was also reported in the phase 3 trial in children aged 12 and older and adults. These clinical benefits persisted in a 2 year open label extension study [24]. In 2012, the United States Food and Drug Administration (FDA) and the European Medicines Agency (EMA) approved the use of ivacaftor for patients aged 6 years and older with CF carrying at least one G551D (Gly551Asp) mutation. A cross over placebo-controlled trial of ivacaftor in patients aged 6 years and older carrying gating mutations other than the Gly551Asp mutation showed similar benefits to the phase 3 trials in patients with the Gly551Asp mutation [25]. This led to an extension of approval for the use of ivacaftor to patients carrying at least one copy of the eight other non-Gly551Asp gating mutations in July 2014. A study in patients aged 6 years and older carrying the residual function R117H (Arg117His) mutation was more challenging and did not meet the primary end point which was a change from baseline through 24 weeks in percent predicted forced expiratory volume in one second (ppFEV1) [26]. Sweat chloride and health-related quality-of-life improvements were shown and in patients 18 years and older who had more established disease, lung function improvement was observed. For younger patients despite the improvements in sweat chloride

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the lung function changes favored placebo. This study however led to extension of approval for patients with CF carrying Arg117His mutation. In 2015, the FDA and the EMA reduced the age of access for all of these patients to age 2 years and older based on the data from a small open-label study in 34 children aged 2–5 years with CF carrying a Gly551Asp mutation [27]. This study showed similar reduction in sweat chloride compared with older children confirming the effects on CFTR function. More surprisingly, there was a statistically significant improvement in faecal-elastase-1 concentrations as compared to baseline suggesting for the first time a window in early life wherein at least partial restoration of pancreatic exocrine function might be possible However, the study also reported more frequent adverse events related to raised transaminase levels in 15% of the cohort suggesting that care is needed in monitoring young patients [27]. Ivacaftor's development was a major breakthrough for CF, since it showed for the first time that improving the function of the defected CFTR protein by the use of pharmacological agents was feasible and associated with important clinical benefit. Moreover, the extent of the gain in respiratory function in both children and adults was unexpected [22]. Furthermore, the clinical improvement reported in clinical trials was also observed in real life after the drug was marketed and similar clinical benefits on respiratory function, pulmonary exacerbations and weight were reported in patients with severe lung disease that were not enrolled in the clinical trials [28]. Long-term disease-modifying effects are also becoming apparent with a slower rate of lung function decline in patients treated with ivacaftor [29]. However, despite all the novelty and the successes of ivacaftor, it was evaluated that it achieves a CFTR activity equivalent to approximately 35–40% of normal CFTR activity [30]. Since most patients are heterozygous for gating mutations, other approaches including targeting both mutations using combinations of therapies as well as the development of more potent potentiators are being investigated.

Lumacaftor associated with ivacaftor approved to treat CF patients homozygous for the Phe508del mutation

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Correcting the CFTR abnormalities associated with the Phe508del class 2 mutation is a much greater challenge. Therapies targeting the Phe508del mutation must modulate both the CFTR processing defect as well as the gating defect. In vitro studies of the Phe508del-CFTR corrector, lumacaftor (developed by Vertex Pharmaceuticals), showed increased CFTR production [31], and in combination with ivacaftor [32], greater chloride transport than with either drug alone [31]. As monotherapy with each of these agents was not found to have clinical benefit [33,34], combination therapy with lumacaftor and ivacaftor was studied in two large phase 3 trials in patients homozygous for Phe508del-CFTR aged 12 years and older. Significant but relatively modest improvements in lung

function and nutrition, and important reduction in the rate of pulmonary exacerbations were observed [35]. Whilst less impressive than the benefits of ivacaftor in patients with gating mutations, the findings are arguably no less clinically significant when the size of the target population is considered with the potential positive implications for long term morbidity at a population level. Combination therapy with lumacaftor and ivacaftor was approved by FDA and EMA in 2015 for patients aged 12 years and older homozygous for Phe508del-CFTR. An extension of approval for patients aged 6 years through 11 was further granted by the FDA on September 2016 following the results of an open-label study in this age range showing similar results than those published for adults [36]. While combination therapy with lumacafator and ivacaftor provides an enormous first step forward in improving health outcomes for patients homozygous for Phe508del-CFTR, it is recognised that many challenges remain. Lumacaftor is a strong inducer of CYP3A which has meant that larger doses of ivacaftor, which is a substrate for CYP3A, are required to achieve clinical effect when used in this combination therapy and the effective dose of ivacaftor is less than the one provided for patients with gating mutations taking ivacaftor monotherapy. This has led to the development of other corrector molecules such as tezacaftor (VX661 developed by Vertex Pharmaceuticals) that do not interact with ivacaftor. Two phase 3 studies have recently shown that tezacaftor in combination with ivacaftor treatment led to a significant improvement in respiratory function in patients homozygous for Phe508del-CFTR and in patients who have one Phe508delCFTRand one mutation that results in residual CFTR function (Vertex Press Release, 28 March 2017). Two in vitro studies have suggested that treatment ( 48 hours) with potentiators, including ivacaftor, may reduce the stability and expression of corrected Phe508del-CFTR [37,38]. A study examining the correction of CFTR folding mutations using a panel of correctors in a CFTR assay of CF intestinal organoids showed differences in correction efficacy for the different correctors supporting the development of mutationspecific correctors [39]. New potentiators as well as other corrector molecules or combinations of corrector molecules, and specific potentiator-corrector combinations are currently being investigated to both improve the current available CFTR therapies, as well as extend CFTR modulator therapies to a wider range of patients (http://ecysticfibrosisreview.org/newsletters/2016/ volume06_issue11.html).

Therapies in development Read-through agents Class 1 mutations comprise mostly premature stop codon mutations that result in a truncated mRNA degraded by nonsense-mediated decay. High concentrations of aminoglycoside antibiotics were first shown to act as read-through agents [40]. They interact with the ribosome and instead of the premature stop codon being read, they allow the erroneous addition of an amino

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CFTR amplifiers and stabilizers Novel compounds called amplifiers and stabilizers are being developed that could be combined with correctors and potentiators. Amplifiers such as PTI-428 from Proteostasis Therapeutics (NCT02718495) aim at increasing the amount of CFTR in the cell, so that more CFTR would be available for correctors and potentiators. Stabilizers such as Cavosonstat (N91115 from Nivalis) aim at stabilizing the modulated CFTR at the cell membrane. Cavosonstat is thought to inhibit S-nitrosoglutathione reductase that is increased in CF human bronchial epithelial cells [45]. Restoring intracellular S-nitrosoglutathione to normal levels would decrease CFTR degradation and improve CFTR stability at the cell surface. Unfortunately, two phase 2 studies of Cavosonstat combined with lumacaftor/ivacaftor or ivacaftor alone failed to show any benefit in respiratory function or sweat chloride at 12 weeks (NCT02589236 and NCT02724527) and the development of cavosonstat for CF has been terminated (Nivalis press releases, 28 November 2016 and 23 February 2017).

Oligonucleotide-based drugs to correct or bypass the DNA or RNA defects A very attractive approach to treat the basic defect is to develop oligonucleotide-based drugs to bypass or correct the DNA or mRNA defects in order to produce a normal CFTR protein. Oligonucleotides are poorly suited for oral or systemic delivery and these approaches usually aim at correcting airway disease by respiratory treatment administered via aerosol.

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Gene therapy consists of delivering the normal CFTR cDNA to cells in order to get a functional CFTR protein synthesis. The great advantage of the gene therapy approach is that it is not mutation-restricted and airway administration of normal CFTR cDNA could theoretically treat airway disease of all patients. The proofof-concept of gene therapy in CF epithelial cells was published very soon after the CFTR gene cloning and to date, more than 25 clinical trials have been completed (for review: [46]). But the hurdles of this approach have proven to be numerous: the CFTR cDNA is a large molecule that needs to be compacted by vectors to enter the cells and these vectors, either biochemical molecules or derived from viruses, allowed a low cDNA delivery into the very differentiated airway cells. Moreover, in studies to date, therapeutic gene expression was low, had a short duration and unwanted inflammatory responses were reported. It is noteworthy that most of the vectors permit the expression of the normal CFTR cDNA with the endogenous CFTR gene still being expressed, making this approach more gene bypass than gene therapy. A recent randomised, double-blind, placebo-controlled, multi-centre phase 2b trial included 140 patients with CF who received a monthly inhalation of a plasmid DNA encoding the CFTR cDNA complexed with a liposomal vector for 1 year. The results showed that the treatment was well tolerated and there was a stabilization of lung function as compared with some lung function loss in the placebo group [47]. Rather than moving forward with the liposomal vector used for the phase 2 trial, the authors have pseudotyped a lentiviral vector to achieve efficient gene transfer into airway epithelial cells in vitro and in vivo in mice. A single-dose, double-blinded, dose-escalating phase I/IIa safety and efficacy clinical trial is planned [48]. After more than 20 years of research in this area, the crucial challenge for CF gene therapy is still the need for more potent vectors to deliver the cDNA into the airway cells. Gene editing is a true gene therapy approach with permanent correction at the genome level by accurate genome engineering. It is still in early stages with no clinical data for CF so far but it is a promising approach. It relies on programmable nucleases allowing defined alterations in the genome with ease-of-use, efficiency, and specificity. Some in vitro data in intestinal organoids showed repair of the Phe508del mutation by gene editing [49]. CFTR mRNA repair is another therapeutic approach and partial transcriptional repair of Phe508del-CFTR mRNA has been shown as early as 2004 [50]. The drug QR-010 is developed by ProQR Therapeutics. It is a single-stranded antisense RNA-based oligonucleotide sequence containing the missing bases and acting as guide sequences to repair the targeted abnormal mRNA in patients with the Phe508del mutation. A phase 1 proof-ofconcept study is ongoing in CF patients homozygous for the Phe508del mutation (NCT02532764).

ENaC inhibitors In normal airways, the CFTR protein counteracts the absorptive function of ENaC [51]. In CF, the defective CFTR protein leads to

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acid to the polypeptide chain and the translation to continue to the end of the gene. A fourteen-day topical nasal administration of gentamicin in patients with CF bearing a premature stop codon mutation induced some correction of the chloride transport defect [41]. However, prolonged high doses of aminoglycoside antibiotics is prevented by serious renal toxicity and ototoxicity. Ataluren (formerly PTC124) is a read-through agent developed by PTC Therapeutics. It has no structural similarities to aminoglycosides. Phase 2 open-label studies with oral administration of ataluren in patients with CF bearing at least one CFTR nonsense mutation improved nasal epithelial chloride transport [42,43]. However, in a double-blind phase 3 clinical trial, improvement in respiratory function was only observed in patients not using chronic inhaled tobramycin [43,44]. It has been hypothesized that tobramycin interferes with the mechanism of action of ataluren.However, a new phase 3 trial on ataluren in patients with CF bearing a nonsense mutation and not receiving inhaled tobramycin has failed to show an improvement in respiratory function or in the rate of pulmonary exacerbations as compared with placebo. (NCT02139306). The development of ataluren for CF has thus been discontinued (PTC Therapeutics press release, 2 March 2017). Other genetic disorders are due to nonsense mutations and could benefit from these read-through agents. Indeed, conditional approval has been granted in Europe for ataluren (TranslarnaTM) in patients with Duchenne muscular dystrophy caused by a nonsense mutation.

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overactivity of ENaC and sodium hyperabsorption that contributes to airway surface liquid volume depletion. The effects of ENaC blocking agents on airway hydration were studied even before the cloning of the CFTR gene. There was no or little improvement in lung function upon a 6-month treatment of inhaled amiloride, the prototype of ENaC blocker [52,53]. It was attributed to limited potency and a short half-life in airways. More potent derivatives demonstrated poor duration of action or acute hyperkalemia due to airway absorption and inhibition of ENaC in the kidney [54]. Several other ENaC blockers chemically different from the classical amiloride are being developed and a new compound VX-371 (formerly P-1037) from Vertex Pharmaceuticals and Parion (NCT02709109) is currently evaluated. Indirect ENaC inhibitors are also being developed.

Activators of other chloride channels Chloride channels other than CFTR are expressed at the apical membrane of airway epithelial cells [21]. Stimulating them could compensate for the loss of CFTR function. TMEM16A or anoctamin-1 is a chloride channel activated by calcium. UTP binds to purinergic receptors called P2Y2 receptors and induces intracellular calcium elevation which activates TMEM16A. Native UTP is rapidly metabolized and one UTP analog named denufosol was developed. It was more resistant to enzymatic degradation than UTP but the phase 3 program failed to improve lung function or reduce pulmonary exacerbations in patients with CF [55]. Recently, significant preclinical progress has been made with identification of alternative chloride channels that should facilitate the development of specific activators [21].

Main challenges and future questions Developing disease-modifying therapies for all patients

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A major challenge for the future of CFTR modulation is to develop therapies that are effective in all patients, i.e., that are not mutation class-specific. As novel agents move from preclinical studies to clinical trials, there is an obligation to broaden the therapeutic repertoire to target the full range of basic defects caused by the different mutation classes, and ensure equitable access to all patients. Clinical trials provide evidence for the efficacy of new drugs, yet many patient groups are not included. Trials are more challenging in young children, in sicker patients, and in those with rarer CFTR mutations where patient numbers may be too small to power traditional clinical trials. New approaches will be needed to ensure equitable access to new therapies for all patients with CF. As more patients start to use CFTR modulator therapy, the pool of patients naïve to CFTR modulator therapy declines, making placebo-controlled trials less possible. For second-generation CFTR modulators, comparative efficacy studies with an approved modulator will likely be required. Alternatives to the traditional randomised placebo-controlled design will be needed in future

clinical trials, including one-person trials [56], protocols that accommodate small samples that use more sensitive outcome measures of effect, and with design approaches that can obtain sufficient meaningful data to enable extension of the findings to larger populations.

Optimizing the use of CFTR modulators in patients An important challenge for clinicians is how to optimize the use of CFTR modulators in their patients. Considerations include selection of patients on the basis of genotype, ability to adhere to therapy, and severity of disease. To date, the clinical trials of CFTR modulator therapy have been conducted in patient populations who are generally taking the full range of available preventative therapies and who are required to continue their usual therapies while participating in the trials. As new therapies become available, they are added on, rather than replacing some existing therapies, and the burden of treatment and health care costs continue to escalate. Once the trials are completed, and therapies are used in a 'real world setting', some of the efficacy seen in clinical trials may be more modest. This was even observed for ivacaftor in patients with a Gly551Asp mutation who took part in the GOAL study that examined the treatment effects in a cohort of patients starting therapy with ivacaftor once it was approved for clinical use and in whom the improvement in ppFEV1 was less than seen in the clinical trials [57]. The real world benefits may be more modest because some patients choose to stop some preventative therapies particularly if they feel healthier, although other reasons are also possible including the difficulty of generalisability of clinical trials across a larger population. We do need to understand the use of the usual repertoire of therapies as well as other new developing therapies in patients using CFTR modulator therapies. New investigative approaches such as adaptive design or platform trials may help to provide some much needed evidence in the future. Despite the promise of benefit with use of ivacaftor, adherence may not be much better than is seen for other CF therapies [58], and while no data are specifically available for combination therapy with lumacaftor and ivacaftor it is likely exactly the same. Correct use of currently available CFTR modulator therapies require oral administration with fatty food to ensure optimal drug availability along with the avoidance of foods and drugs that may interact. Ensuring ongoing education of patients and supporting optimal adherence will be hugely important in maximising the potential benefits for these drugs. Clinical decisions surrounding initiation of treatment in patients with mild or severe lung disease are difficult on the basis of current evidence. Clinical trials tend to exclude patients with more severe disease to avoid the potential adverse events that are frequently seen in this group of patients. Patients with milder disease may also be excluded as the outcome measures commonly used to determine efficacy may not be sensitive to

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improvement in this population as there are ceiling effects for many outcomes including standard spirometry measures such as ppFEV1. Indications for treatment with CFTR modulator therapies may also extend beyond improvement in lung function and nutrition and more evidence emerges regarding potential additional effects of CFTR modulators in CF, such as airway microbiology [57] and glucose homeostasis [59]. The ultimate goal of disease prevention has already driven clinicians to start therapies earlier in life and in more mild disease. While the potential benefit of this approach is clear, it raises the importance of long-term monitoring of therapies introduced in infancy and potentially continuing across the life span. Adverse event profiles may not be the same across all age groups as has been suggested by Davies et al. in the open label study of ivacaftor in children aged 2–5 years [27]. There are advantages to dropping the age of access rapidly based on efficacy in older patients as this provides the opportunity for earlier access to potentially beneficial therapies and takes advantage of the window of opportunity to effect the development and progression of disease substantially. Without appropriate clinical trials, however, in young infants and children, we will not have the evidence required to determine the risk/ benefit for this important and vulnerable patient population.

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processes internationally, encouraging extension of use of new medicines to other indications and providing a framework for funding mechanisms [62] may provide some opportunities. To address the challenges that we face with developing these exciting new therapies, patients, health-care providers, researchers, governments, regulatory authorities and pharmaceutical companies will need to work together closely to realize the potential for these new medicines for all people with CF.

Conclusion The cloning of the CFTR gene in 1989, the subsequent characterization of the CFTR protein and the huge work undertaken to understand the protein structure and how mutations translate into a defective CFTR protein have led to the recent approval of two drugs that bind to the defective CFTR protein and are able to partly restore CFTR function. Ivacaftor may target a narrow population and lumacaftor/ivacaftor may have a small impact on respiratory function. Nevertheless, these mutation-specific treatments are the landmark of a new era for CF where personalized medicine is now in the clinics and will have a diseasemodifying impact. Many other compounds are under development and combination of treatments is likely to lead to more effective CFTR correction.

Working to make CF health care cost sustainable New targeted or personalised medicines have high financial cost as we have seen not just for CF but also for indications in oncology, asthma, metabolic disease etc. The development of new medicines is a costly and risky undertaking and small markets will drive up the costs [60,61]. We will need as a society to find solutions to the high cost of personalised medicine if we are to enable equitable access to new therapies for a greater number of patients in the future. Harmonising regulatory

Disclosure of interest: IF: Principal or co-investigator for clinical trials sponsored by Bayer, Novartis and Vertex Pharmaceuticals. Consultancy for EPG Health Media, Galapagos and Vertex Pharmaceuticals. Conferences: invitation as speaker for Vertex Pharmaceuticals, as auditor by PTC Therapeutics. CW: Honoraria received by institution from Novartis Pharmaceuticals, Vertex Pharmaceuticals and Vertex Pharmaceuticals Australia, Principal or co-investigator for clinical trials sponsored by Novartis and Vertex Pharmaceuticals. Consultancy on clinical trials for Vertex Pharmaceuticals. Funding from Children's Hospital Foundation Brisbane, Australia.

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