Rapid Review

Progress in therapies for cystic fibrosis

Kris De Boeck, Margarida D Amaral

Lancet Respir Med 2016; Standard follow-up and symptomatic treatment have allowed most patients with cystic fibrosis to live to young
4: 662–74 adulthood. However, many patients still die prematurely from respiratory insufficiency. Hence, further investigations
Published Online to improve these therapies are important and might have relevance for other diseases—eg, exploring how to increase
April 1, 2016
airway hydration, how to safely downscale the increased inflammatory response in the lung, and how to better combat
http://dx.doi.org/10.1016/
S2213-2600(16)00023-0 lung infections associated with cystic fibrosis. In parallel, development of modulators that target the underlying
Pediatric Pulmonology,
dysfunction in the cystic fibrosis transmembrane conductance regulator (CFTR) is fast moving forward. Existing
Department of Pediatrics, treatments are specific to certain mutations, or mutation class, in CFTR. An effective, although not yet entirely
University of Leuven, Leuven, corrective, treatment is available for patients with class III mutations, and a treatment with modest effectiveness is
Belgium (Prof K De Boeck PhD);
available for patients who are homozygous for Phe508del, albeit at a very high cost. Corrective treatments that are
and Faculty of Sciences,
Biosystems and Integrative non-specific to mutation class and thus applicable to all patients—eg, gene therapy, cell-based therapies, and activation
Sciences Institute (BioISI), of alternative ion channels that bypass CFTR—are being explored, but they are still in early stages of development.
University of Lisbon, Lisbon, In view of the large number of patients with very rare mutations, a plan to advance personalised biomarkers to predict
Portugal (M D Amaral PhD)
treatment effect is also being investigated and validated.
Correspondence to:
Prof Kris De Boeck, Pediatric
Pulmonology, Department of
Introduction (CFTR)—which encodes an epithelial chloride and
Pediatrics, University of Leuven, Cystic fibrosis is the most common life-shortening rare bicarbonate ion channel.4,5 Most patients ultimately
Leuven 3000, Belgium disease, affecting around 32 000 individuals in Europe develop progressive lung disease with airway mucus
christiane.deboeck@
and about 85 000 individuals worldwide. In most obstruction, bacterial infection, and inflammation
uzleuven.be
European Union (EU) countries, adult patients now (figure 1) despite intense symptomatic (ie, standard)
outnumber paediatric patients, but the median age at treatments, which do not treat the molecular cause of the
death remains low at roughly 28 years.1 However, for disease (ie, defective CFTR protein). These symptomatic
patients born in the past 15 years, median predicted treatments include mucolytics to dissolve thick mucus,
survival in the UK is now greater than 50 years.2 The antibiotics to treat or prevent infections, and anti-
number of patients reported to disease registries inflammatory agents to dampen chronic inflammation.
worldwide rises steadily because of the widespread However, treatments that act at the level of the CFTR
implementation of newborn screening, increased molecular defect are necessary to block the series of
For the Cystic Fibrosis Mutation
Database see http://www.genet.
diagnosis in low-income and middle-income countries, events that lead to progressive lung disease. Patients
sickkids.on.ca and improved survival.3 have a large range of clinical phenotypes, which can be
For more on CFTR2 see Cystic fibrosis is caused by mutations in one gene— partly explained by the roughly 2000 CFTR gene
http://www.cftr2.org/ cystic fibrosis transmembrane conductance regulator mutations so far identified (see Cystic Fibrosis Mutation
Database), of which only around 200 have been
characterised in terms of disease liability (see CFTR2).7
Key messages Other genetic, cellular, and environmental factors, which
• 80 000 people worldwide have cystic fibrosis and will die prematurely from respiratory remain largely unknown, also modify the clinical course
insufficiency of the disease and each individual’s response to therapy.8–11
• Standard follow-up and symptomatic treatments have allowed most patients to live Cystic fibrosis is often regarded as a model disease,
to young adulthood since many pioneering studies in genetics, molecular
• Improvement of conventional drugs continues to reduce disease complications by and cellular pathogenesis, and drug discovery that have
increasing airway hydration and ameliorate chronic lung inflammation and infection, been done in cystic fibrosis paved the way for other rare
thereby raising the expected age of survival genetics disorders. Furthermore, evidence shows that the
• To further increase the lifespan and quality of life of patients, more effective CFTR protein has a key role in several major respiratory
treatments are needed and therapies correcting the underlying defect hold promise of conditions of high public health relevance that are rapidly
achieving this goal becoming more prevalent in the EU, such as asthma and
• Modulators that target the underlying dysfunction in cystic fibrosis transmembrane chronic obstructive pulmonary disease (COPD; estimated
conductance regulator (CFTR) in a mutation-specific or mutation-class-specific way as the fourth leading cause of death worldwide), in which
are being developed at a steady pace lack of functional CFTR at the cell surface has been
• An effective treatment is available for patients with class III mutations (around 5% of demonstrated.12–14 The CFTR protein is even thought to
all patients) and is becoming available for patients who are homozygous for have a role in smoking-related respiratory disease, since
Phe508del (roughly 40–45% of all patients) loss of CFTR at the plasma membrane is a major and
• So-called mutation agnostic corrective treatments—such as gene therapy, cell-based early event in cells exposed to cigarette smoke and
therapies, and activation of alternative ion channels to bypass CFTR—are in early pollutants.15–18 These findings led some researchers to
stages of development consider COPD and heavy smoking as so-called acquired
CFTR deficiency, by contrast with genetic cystic fibrosis.

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 Rapid Review

Molecular-defect treatments Symptomatic treatments

6 Destructive cycle 7 Progressive loss of lung function

4 Decreased water content

in airway surface liquid;
3 Abnormal CI– permeability thick mucus
altered ionic transport Bacterial infection
Inflammation and scarring
CI–
2 Defective 5 Mucus obstruction
CFTR protein and bronchiectasis
Na+

Na+ ENaC
CFTR
Na+
Na+

1 Two defective CFTR genes

Figure 1: Pathogenetic cascade that causes cystic fibrosis lung disease

Cystic fibrosis is caused by mutations in the CFTR gene, which trigger a series of events that ultimately lead to severe lung deficiency. Most standard therapies treat the
symptoms (eg, mucolytics to dissolve the thick mucus). However, treatments that target molecular defects acting earlier in this cascade of events (eg, CFTR modulators) can
prevent progression to end-stage lung disease.6 CFTR=cystic fibrosis transmembrane conductance regulator. ENaC=epithelial sodium channel. Modified from Amaral.6

In this Review, we highlight progress in both standard prevented by eradicating the root cause and entirely
symptomatic treatments and new therapies targeting the blocking the pathophysiological cascade,23 the
molecular defect in CFTR. In the fast-moving field of conventional symptomatic therapies, which enable most
cystic fibrosis research, our discussion of these new patients to live to adulthood, remain important.24 Further
therapies can be seen as an update to the 2013 Review in research to optimise these treatment modalities
The Lancet Respiratory Medicine.19 Our focus here is to continues to be highly relevant for patients with cystic
highlight recent and ongoing trials rather than past trials fibrosis and might also benefit patients with other
or preclinical studies. chronic lung diseases, such as non-cystic fibrosis
bronchiectasis and COPD.
Symptomatic therapies In this section, we discuss developments in the
Understandably, most attention and resources are now strategies to restore the airway surface liquid layer and
focused on correction of the molecular defect with CFTR improve mucociliary clearance, to dampen the excessive
modulators—ie, correctors and potentiators. However, inflammatory response in the lung, and to control
we should stress that none of these therapies are chronic lung infection.
sufficiently effective to be used as stand-alone treatments
at present. Even the most successful of these therapies Restoration of airway surface liquid and mucociliary
(ie, ivacaftor in patients with a class III mutation) is used clearance
in addition to existing standard therapies.20 During Central in the pathophysiology of cystic fibrosis lung
treatment with ivacaftor, sweat chloride value, a marker disease are abnormally viscid secretions and deficient
of CFTR function, comes close to, but does not reach, the mucociliary clearance, leading to airway obstruction. The
normal range.20,21 Additionally, although the clinical absence of chloride and bicarbonate secretion via the
benefit of the drug is impressive (a 10% predicted CFTR channel, coupled to excess sodium ion absorption
improvement in forced expiratory volume in 1 s [FEV1]), via the epithelial sodium channel (ENaC, which is not
disease progression is not stopped: the treatment is downregulated by CFTR), leads to insufficient water
estimated to only halve the FEV1 rate of decline.22 secretion to the airway surface liquid layer. Whether this
Furthermore, during ivacaftor treatment, patients still insufficiency results directly in decreased height of the
have pulmonary exacerbations and other lung periciliary layer, or whether the increased oncotic
complications. Thus, until disease manifestations can be pressure in the overlying mucus layer pushes the

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 Rapid Review

periciliary layer down (the new so-called gel-on-brush discontinued prematurely because of severe safety
model),25 is under investigation. At present, inhalation of concerns. The low-dose treatment (1 mg prednisone per
hypertonic saline and mannitol are used to improve kg bodyweight) on alternating days did show a small
airway hydration by altering osmolarity of the airway benefit on repeated measurements of FEV1 (% predicted)
surface liquid.26,27 but was again associated with serious side-effects, such
Dornase alfa (ie, recombinant human DNase-1) as growth retardation, cataract, and more frequent
increases mucociliary clearance by decreasing sputum pseudomonas infection.38 Inhaled steroids have no
viscosity and is part of standard care for cystic fibrosis.24 proven benefit.39 Leukotriene B4 (LTB4) is a potent
As shown in the Epidemiologic Study of Cystic Fibrosis,28 activator of inflammatory responses mediated by
the use of dornase alfa is associated with a reduction in neutrophils, macrophages, and monocytes, and its
the rate of FEV1 decline. However, around 30% of patients concentration is increased in the lung of patients with
might be non-responders,29,30 probably because of cystic fibrosis. In a clinical trial with the LTB4 receptor
insufficient sputum concentration of magnesium needed antagonist BIIL 284,37 active treatment was associated
for DNase-1 activity or excessive sputum actin content, with increased pulmonary exacerbations in adult
the naturally occurring inhibitor of DNase-1.30 When patients. In an animal model, BIIL 284 increased the
tested in vitro, PRX-110, a plant-cell-expressed incidence of bacterial infections in the mouse lung.40
recombinant form of human DNase-1, was more Therefore, existing anti-inflammatory strategies are
resistant than dornase alfa to the inhibitory effect of mainly restricted to non-steroidal drugs. Oral high-dose
actin.31 Another strategy is to use actin depolymerising ibuprofen decreases the rate of decline of lung function,
agents such as gelsolin or polyanions (eg, polyaspartate),32 but fear of serious side-effects and the need for frequent
which await proof-of-concept trials. drug level monitoring severely limit its use.41,42 In 2015,
To further restore mucociliary clearance, ENaC ibuprofen was identified to have some CFTR corrector
inhibitors are being considered as stand-alone therapy, in activity in a human bronchial epithelial cell assay and in
combination with existing hydrators such as hypertonic a mouse model.43
saline, or in combination with CFTR modulators. Indeed, With treatments aiming to decrease inflammation, a
ENaC inhibitors sit in between symptomatic therapy to more realistic expectation is to decrease pulmonary
improve mucociliary clearance and therapies aiming to exacerbations and stabilise lung function (ie, less decline)
improve the molecular defect by targeting non-CFTR rather than acute improvement in lung function.44
channels (see below). Obviously, a longer treatment period is needed to
measure these outcomes.44 Several new compounds are
Safe reduction of excessive lung inflammation being studied in clinical trials (table 1). Acebilustat is a
The complex association between inflammation and cystic small molecule that blocks the enzyme leukotriene A4
fibrosis lung disease has been reviewed elsewhere.33,34 The hydrolase, thereby decreasing the production of LTB4.
hallmark of cystic fibrosis lung disease is infection by The aim of acebilustat is to reduce airway obstruction by
bacteria and other pathogens. However, compared with blocking excessive neutrophil influx and activation. In a
lung infection in other disorders, in cystic fibrosis the phase 1 clinical trial with oral acebilustat (50 mg and
associated inflammation is increased but is still 100 mg once daily) for 15 days in 17 adult patients with
ineffective to clear pathogens from the lung. Lung cystic fibrosis and mild to moderate lung disease, positive
inflammation in cystic fibrosis is driven by neutrophils, trends were seen in blood and sputum biomarkers,
which contribute to structural lung damage, for example including LTB4, without changes in sputum
via release of neutrophil elastase. Although the microbiology.45 The planned phase 2 trial, EMPIRE-CF
mechanism is not fully understood, another hallmark is (ClinicalTrials.gov identifier NCT02443688), will aim to
reduced invasiveness of pathogens despite a high local show a reduction in pulmonary exacerbations and in
bacterial burden.35,36 Therefore, the balance between FEV1 decline. The oral anti-inflammatory compound
For more on Resunab see http:// excessive inflammation (which contributes to lung ajulemic acid (Resunab) is designed to enhance the
www.corbuspharma.com/ damage) and insufficient inflammation (which possibly resolution of chronic inflammation by binding and
product-pipeline/resunab
allows progression to more invasive disease) needs to be activating cannabinoid receptor type 2, which is present
maintained, and potent anti-inflammatory therapies on immune cells such as monocytes, T cells, and B cells.
have been shown to worsen cystic fibrosis lung disease.37 Ajulemic acid induces apoptosis of T cells, inhibits
Therapeutic strategies that only modestly decrease the leucocyte migration, reduces cytokine release (eg,
inflammatory response, promote resolution of interleukin-6 and interleukin-8), decreases production of
inflammation, and increase local antiprotease or LTB4, and increases production of the anti-inflammatory
antioxidant activity might prove safer and are now being lipoxin A4. The drug had a favourable safety profile in a
explored. phase 1 trial46 and will progress to phase 2 trials in cystic
In a 4 year clinical trial in patients aged 6–14 years,38 fibrosis (NCT02465450) and dermatomyositis.
the administration of high-dose systemic steroids (2 mg Research into strategies that increase local antiprotease
prednisone per kg bodyweight) on alternating days was and antioxidant activity to combat damage by neutrophils

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Trial description Intervention Sponsor

Improving airway hydration
NCT01619657 Phase 2 pilot study of safety and efficacy in Preventive inhalation of 6% hypertonic saline Heidelberg University,
newborns and infants (4 mL) versus 0·9% isotonic saline (4 mL), twice Heidelberg, Germany
daily for 52 weeks
NCT02378467 Phase 3 study of safety and efficacy (improvement Inhalation of 7% hypertonic saline versus 0·9% University of Washington,
in lung clearance index) in children aged 3–5 years isotonic saline, twice daily for 48 weeks Seattle, WA, USA
NCT02343445 Phase 2 placebo-controlled trial of safety and Inhalation of ENaC blocker (P-1037) with saline or Parion Sciences
efficacy in patients aged 12 years or older hypertonic saline versus saline or hypertonic
saline
Safely combatting inflammation
NCT01270074 Phase 3 placebo-controlled trial of safety and Azithromycin (10 mg/kg bodyweight) three times Queensland Children’s Medical
efficacy in infants to prevent development of per week, from age 3 months until 3 years Research Institute, Brisbane,
bronchiectasis at age 3 years QLD, Australia
NCT02443688 Phase 2 placebo-controlled trial of safety, Oral acebilustat, once daily for 48 weeks Celtaxsys
tolerability, and efficacy in adult patients
NCT02465450 Phase 2 placebo-controlled trial of safety, tolerability, Ajulemic acid (JBT-101) Corbus Pharmaceuticals
pharmacokinetics, and efficacy in adult patients
Improving control of lung infection
NCT01455675 Phase 3 trial of safety and efficacy for prevention of Avian polyclonal anti-P aeruginosa antibodies Mukoviszidose Institut, Bonn,
re-infection with Pseudomonas aeruginosa versus placebo Germany
NCT02526004 Phase 3 trial of microbiome-determined antibiotic Microbiome-guided therapy versus standard University College Cork, Cork,
therapy in cystic fibrosis exacerbations therapy Ireland

ENaC=epithelial sodium channel.

Table 1: Key ongoing clinical trials of standard therapies

has been active for more than a decade, but progress is exacerbations. However, in view of the small sample size,
slow. The continued interest is explained by the safety of the possibility of this being a chance finding cannot be
these approaches; unfortunately, efficacy has been more excluded. The authors hypothesised other potential
difficult to prove. In an open-label multicentre study in mechanisms of action, such as a positive effect of
Germany in 52 patients with cystic fibrosis, pseudomonas N-acetylcysteine on other pathways of inflammation or on
lung infection, and raised free elastase levels in induced CFTR trafficking.
sputum,47 daily inhalation of α1 antitrypsin (25 mg) for
4 weeks resulted in raised sputum α1 antitrypsin levels and Efficient control of chronic lung infection: treatments
reductions in free elastase levels and concentrations of beyond antibiotics
interleukin-8, TNF-α, interleukin-1β, and LTB4; decreased Intensive use of oral, inhaled, and systemic antibiotics
proportion of neutrophils; and a reduction in Pseudomonas has undoubtedly improved survival of patients with
aeruginosa colony count. However, these changes were not cystic fibrosis and is still one of the mainstay therapies.24
associated with any change in lung function.46 In a double- Ultimately, however, Pseudomonas species will develop
blind phase 2 study (NCT01684410),48 higher doses (ie, widespread antibiotic resistance, and pathogens that are
100 mg and 200 mg) of aerosolised α1 antitrypsin (a liquid increasingly more difficult to treat—such as Burkholderia
preparation of purified α1 proteinase inhibitor from pooled cepacia complex, Achromobacter species, atypical
human plasma) inhaled daily via a nebuliser for 3 weeks mycobacteria, and fungi—might also emerge.
were safe and well tolerated. The development of new inhaled antibiotics and better
Increasing the amount of the antioxidant glutathione in or faster inhalation devices has been the topic of several
the lungs has been attempted, either by inhalation of recent reviews.55–57 Therefore, we focus on strategies
glutathione (which is deficiently transported by CFTR) or other than antibiotics to combat chronic lung infection
by oral administration of the prodrug N-acetylcysteine, in cystic fibrosis.
which is then converted to glutathione.49–54 Although some Biofilm production is a common adaptation mechanism
changes in inflammatory parameters were noted with during chronic infection, and is typical for chronic
inhaled glutathione,49–54 no sustained change in lung pseudomonas infection in the cystic fibrosis lung.
function or exacerbations was seen in a subsequent Disruption of the biofilm could thus help to eradicate
6-month trial.52 Conversely, in a 24-week double-blind trial Pseudomonas species, and several strategies that are being
with oral N-acetylcysteine (n=70),54 no change in explored include saccharides such as liposomal β glycan58
inflammatory markers (eg, sputum neutrophil elastase or and OligoG,59,60 an oligosaccharide derived from brown
plasma interleukin-8) was seen, but lung function was algae. Safety and tolerability of nebulised OligoG have been
stabilised without a significant change in pulmonary proven in healthy volunteers and in patients,61 and patterns

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 Rapid Review

of lung deposition after inhalation are now being studied. of antibiotics during exacerbations. An EU-funded
Another compound that has been reported to disrupt the project and clinical trial have started to address these
For more on CF Matters see biofilm (by a yet-unknown mechanism), oral cysteamine, is issues and fill this knowledge gap (see CF Matters).
http://www.cfmatters.eu/ being tested in a phase 1 trial for tolerability and
pharmacokinetics.62 Interestingly, this compound, in Therapies to correct molecular defects
combination with a tea flavonoid, has been reported to CFTR modification therapies: classes of CFTR mutations
rescue traffic of Phe508del-CFTR by restoring autophagy.63,64 The roughly 2000 CFTR gene alterations that have so far
Nitric oxide concentrations are decreased in exhaled air been described consist of missense (39·6%), frameshift
from patients with cystic fibrosis compared with healthy (15·6%), splicing (11·4%), and nonsense (8·3%)
individuals.65 The importance of this finding is not entirely mutations; large (2·6%) and in-frame (2·0%) deletions
clear but has been linked to the increased susceptibility to or insertions; promoter mutations (0·7%); and presumed
infection.66 Attempts to increase nitric oxide production by non-pathological variants (15·0%).6,74 However, the
inhalation of interferon-γ to stimulate nitric oxide 3 base-pair deletion of phenylalanine 508 (Phe508del) is
synthase proved unsafe, since more exacerbations were present in around 85% of patients worldwide, with a
seen in patients given the high dose; neither high-dose higher frequency reported in northern Europeans than
treatment nor low-dose treatment demonstrated benefit in southern Europeans.75
on lung function, sputum imflammatory markers, or All mutations that cause cystic fibrosis ultimately lead to
bacterial density.67 Strategies to increase nitric a defect in cAMP-regulated chloride and bicarbonate
oxide concentration without interfering with complex secretion by epithelial cells, but many reasons exist for the
inflammatory cell signalling seem safer, but the different causative mechanisms.76 Not only is elucidation
effectiveness of these approaches is still to be proven. of the molecular and cellular effects brought about by
Direct inhalation of nitric oxide effectively decreased CFTR mutations informative for structure–function
bacterial load in a rat model of P aeruginosa pneumonia.68 studies, but it can also provide the scientific basis for
The safety and tolerability of nitric oxide inhalation mutation-specific corrective therapies.23 Therefore, these
(160 ppm for 30 min three times per day) have also been mutations can be grouped according to their functional
assessed in patients with cystic fibrosis, and at least defect into seven classes (figure 2).6,77,78
transient reductions in bacterial load were noted.69 Nitric Class I mutations affect protein production and include
oxide concentrations in the airways can also be increased mostly nonsense mutations (ie, those with premature
by repeated inhalations of L-arginine, the substrate for stop codons), thus often causing degradation of mRNA
nitric oxide synthase. In a pilot trial,70 inhalation of 500 mg by nonsense-mediated decay. Class II mutations include
L-arginine twice daily for 2 weeks was well tolerated, and Phe508del and affect CFTR protein traffic as a result of
FEV1 increased by 56 mL on average, although this protein misfolding and retention at the endoplasmic
increase was not significant; no change in inflammatory reticulum (ER) by the ER quality control mechanism.
markers in sputum was reported. Such retention is followed by premature degradation,
Interest in treatment with bacteriophages—ie, viruses which prevents the protein from trafficking to the cell
that infect and replicate in specific strains of bacteria— surface, thus severely reducing CFTR function.79,80
is mounting.71 Especially for patients colonised with Class III mutations impair gating of the CFTR channel.
multiresistant organisms, controlling the infection with Class IV mutations cause a substantial decrease in CFTR
bacteriophage treatment could be a last resort. At channel conductance (ie, flow) of chloride and
present, no clinical trial has started, but preparatory bicarbonate ions. Class V mutations lead to a major
work specific for cystic fibrosis is advancing. Anti- reduction in the levels of normal CFTR protein, often
Pseudomonas bacteriophages have been shown to retain because of alternative splicing that generates both
their activity when nebulised.72 Strict safety regulations aberrant and normal mRNA species, the proportion
imposed by health authorities are one of the major between which might vary among patients and in
hurdles to assess the efficacy of this therapy in the different organs of each patient. Class VI mutations
clinic. destabilise CFTR at the cell surface, either by increasing
Finally, a new and rapidly expanding research area is CFTR endocytosis or by decreasing its recycling back to
the lung microbiota, but a full discussion of this topic is the cell surface. Finally, class VII mutations are so-called
beyond the scope of this Review. Knowledge of lung unrescuable mutations, because they cannot be
microbiota has considerably changed the way lung pharmacologically rescued per se—eg, large deletions
infection in cystic fibrosis is viewed. The healthy airways such as the dele2,3(21kb) mutation.81 However, promising
are colonised by a wide spectrum of bacteria. By contrast, so-called one-size-fits-all therapeutic strategies (also
in the airways of patients with cystic fibrosis, bacterial known as mutation agnostic; ie, independent of the
diversity and species richness decrease as the disease mutation class) will also be suitable for this class of
progresses and lung function decreases.73 However, this mutations (see below).
improved knowledge of the microbial community has Novel emerging drugs targeting the underlying
not yet translated into more judicious or efficacious use defect—ie, mutant CFTR—open up new opportunities

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CI– CI– CI– CI– CI–

CI–
–
CI CI– CI
–

CFTR

Wild-type CFTR

Class I Class II Class III Class IV Class V Class VI Class VII

CFTR defect No protein No traffic Impaired Decreased Less protein Less stable No mRNA
gating conductance

Mutation GLy542X, Phe508del, Gly551Asp, Arg117His, Ala455Glu, c. 120del23, dele2,3(21 kb),

examples Trp1282X Asn1303Lys, Ser549Arg, Arg334Trp, 3272-26A→G, rPhe508del 1717-1G→A
Ala561Glu Gly1349Asp Ala455Glu 3849+10 kg C→T

Corrective Rescue synthesis Rescue traffic Restore channel Restore channel Correct Promote Unrescuable
therapy activity activity splicing stability

Drug Read-through Correctors (yes) Potentiators Potentiators Antisense Stabilisers (no) Bypass
(approved) compounds (yes) (no) oligonucleotides, therapies (no)
(no) correctors,
potentiators?
(no)

Figure 2: Classes of gene mutations and respective therapeutic strategies

CFTR mutations are grouped into seven functional classes, with the expectation that the same modulators will be applicable to all the defects in one class. However,
class VII mutations are not expected to be rescuable by any modulator. A therapeutic strategy for class VII mutations could involve stimulation of alternative chloride
channels (ie, bypass therapies). CFTR=cystic fibrosis transmembrane conductance regulator. rPhe508del=rescued Phe508del. Modified from Amaral.6

for so-called curative treatment. At present, two of these mutants but also the normal CFTR channel and some
drugs have been approved for clinical use: ivacaftor mutant proteins of classes IV and V.91 In a phase 3 trial,82 a
benefits only patients with gating mutations (around 5% improvement in lung function was reported in adult
5% of all patients) and, to some extent, patients with patients with the class IV mutation Arg117His; however,
the Arg117His mutation; lumacaftor is beneficial for this improvement was not seen in children with the same
patients with the homozygous Phe508del genotype (ie, mutation. In the entire cohort with this mutation,
roughly 40–45% of all patients with cystic fibrosis; improvements in sweat chloride (−24 mmol/L) and
table 2).20,21,82–84 quality-of-life score were seen.82 However, clinical benefit
Ivacaftor was shown to decrease sweat chloride in patients with other class IV or V mutations with
concentration by a mean of 50 mmol/L, augment residual function still needs to be firmly proven. New
predicted FEV1 by a mean of 10%, reduce the number of strategies are emerging for class V mutations that affect
pulmonary exacerbations, and improve body-mass index splicing, and a particular area of interest is the correction
(BMI), Z scores, and quality of life, not only in patients of alternative splicing with antisense oligonucleotides.92
with the Gly551Asp mutation but also in patients with The introduction of ivacaftor led to great optimism,
other class III mutations.20,21 Ivacaftor has now been used even though this drug is indicated in only up to 10% of
in patients with the Gly551Asp mutation for more than patients with cystic fibrosis. Ivacaftor provides proof of
5 years. Across age and disease severity groups, a robust concept that small-molecule therapy that interferes with
benefit was seen after marketing authorisation by both the molecular defect is possible, which encouraged
the US Food and Drug Administration (FDA) and the patients to participate in clinical trials with CFTR
European Medicines Agency (EMA).85–87 Long-term modulators. Yet, many challenges remain to ensure that
benefits are starting to become apparent87—ie, less patients have access to new treatments. Ivacaftor is very
frequent infections with pseudomonas,88 reduced rate of expensive (around US$311 000 per patient per year), and
lung function decline,21 improved glucose tolerance,89 some health authorities are slow to approve re-
sustained weight gain, and better growth in children.90 In imbursement, since the need for lifelong treatment
vitro, ivacaftor potentiates not only class III CFTR poses a substantial burden on health-care budgets.93,94

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Several companies are now developing other drugs that 30–40% reduction in pulmonary exacerbations—
potentiate CFTR channel function, and clinical trials are defined as treatments with additional antibiotics
ongoing (table 3). because of an increase in pulmonary symptoms—over
Results from phase 3 trials in patients homozygous the 24 week treatment period.83 Nonetheless, this
for Phe508del showed that combination therapy combination therapy has already been approved by the
of lumacaftor (a corrector that partly rescues FDA and EMA for patients homozygous for Phe508del
Phe508del-CFTR traffic) and ivacaftor (which potentiates aged 12 years or older (table 2). Again, cost (around
channel function) induced a significant but modest US$250 000 per patient per year) is a major issue, since
(2·6–4·0%) improvement in lung function and a 40–50% of patients qualify for this treatment.

Frequency Corrective therapy Limitations Status

Gly551Asp and eight other gating 4–5% Ivacaftor Very high cost (around US$311 000 per Approved in the EU and USA for
mutations (class III) patient per year) patients aged 2 years or above
Arg117His (class IV) 1–2% Ivacaftor Very high cost; variable response Approved in the EU for patients aged
18 years or above, and in the USA for
patients aged 6 years or above
Homozygous Phe508del 40–45% Lumacaftor plus Very high cost (around US$250 000 per Approved in the EU and USA for
ivacaftor patient per year); low efficacy; variable patients aged 12 years or above
response
Heterozygous Phe508del 40% None ¨ ¨
Nonsense mutations 10% Ataluren Benefit uncertain Phase 3 trial (NCT02139306) ongoing
in patients who are not using inhaled
aminoglycosides
Other mutations (including 10–15% None ¨ ¨
class VII mutations)

EU=European Union.

Table 2: Corrective therapies targeting the molecular defect in cystic fibrosis, by mutation type

Mechanism of action Stage of development Sponsor

Ataluren Rescue CFTR protein synthesis by Phase 3 confirmatory study (NCT02139306) of efficacy and safety is ongoing in patients PTC Therapeutics
read-through of nonsense mutations with nonsense CFTR mutations who are not taking inhaled aminoglycosides
VX-661 (corrector) plus Rescue Phe508del-CFTR protein to the cell Phase 3 studies (NCT02347657, NCT02392234, and NCT02412111) of efficacy and safety Vertex Pharmaceuticals
ivacaftor (potentiator) surface with a corrector and stimulate are ongoing in patients aged 12 years or above with homozygous and heterozygous
channel function with a potentiator Phe508del-CFTR mutation
N91115 (proteostasis Stabilise Phe508del-CFTR protein after Phase 2 study (NCT02589236) is ongoing in adults who are homozygous for Phe508del Nivalis Therapeutics
regulator) being rescued to the cell surface and receiving lumacaftor–ivacaftor combination treatment
Riociguat Improve CFTR expression Phase 2 trial (NCT02170025) of safety, tolerability, and efficacy is underway in adults Bayer
homozygous for Phe508del
QR-010 (antisense In-vivo correction of the defect in Phase 1b trial (NCT02532764) is ongoing to assess the safety of single and multiple ProQR Therapeutics
oligonucleotide) Phe508del-CFTR mRNA ascending doses of inhaled QR-010 in adult patients with homozygous Phe508del; phase 1
open-label exploratory study (NCT02564354) is underway to assess the efficacy of
intranasal administration on nasal potential difference (a measure of CFTR function) in
patients with homozygous and heterozygous Phe508del
GLPG1837 (potentiator) Rescue CFTR function by stimulating Phase 2a trial of safety and tolerability is ongoing in adults with at least one class III Galapagos NV
channel function mutation
QBW251 (potentiator) Rescue CFTR function by stimulating Phase 1 (NCT02190604) and phase 2a (NCT02190604) trials are underway to assess Novartis Pharmaceuticals
channel function safety, tolerability, and pharmacokinetics in healthy volunteers and adults with at least one
class III–VI mutations
FDL169 (potentiator) Rescue CFTR function by stimulating Phase 1 trial (NCT02359357) of safety, tolerability, and pharmacokinetics of single and Flatley Discovery
channel function repeat oral doses in healthy male volunteers is underway Laboratory LLC
C-10355 and C-10358 Rescue CFTR function by stimulating Phase 1 trial (NCT02392702) of safety, tolerability, and pharmacokinetics in healthy Concert Pharmaceuticals
(potentiators) channel function volunteers, with a pharmacokinetic comparison with ivacaftor, completed.
Other correctors and Rescue CFTR function at the cell surface by In the pipeline of several companies, pending results from preclinical and phase 1 studies of Vertex Pharmaceuticals,
potentiators two correctors and stimulate channel correctors with complementary action Galapagos NV, and Flatley
function with a potentiator in patients Discovery Laboratory LLC
homozygous or heterozygous for Phe508del

CFTR=cystic fibrosis transmembrane conductance regulator.

Table 3: Treatments targeting molecular defects in CFTR and relevant clinical trials

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At present, combination therapy has not shown conductance but also for other cellular defects, including
efficacy in patients heterozygous for Phe508del. Few ENaC hyperactivity and the reduced height and pH of
treatment options are available for patients with the airway surface liquid, which in turn impair bacterial
predominant Phe508del mutation (present in roughly clearance and killing,101 enhance sodium absorption,102
85% of all patients),19,78,95 since the rescue of Phe508del and reduce mucus fluidity.103–105
by one corrector is indeed inefficient. An improved Many alternative ion channels and transporters are
understanding of how the complex three-dimensional promising drug targets. Anoctamin 1 (ANO1; also known
folding of the Phe508del-CFTR protein is disturbed as TMEM16A) is a calcium-activated chloride channel
points to the need for a combination of correctors with present in many epithelial cells,106–110 and ANO6
different mechanisms of action, together with a (also known as TMEM16F) is an essential component of
potentiator to allow more efficient rescue of the mutant the outwardly rectifying chloride channel.111 Genetic
protein.96,97 Ivacaftor potentiates normal CFTR, class III variants of SLC9A3, which encodes a sodium–proton
mutants, and several mutated CFTR proteins exchanger, have been associated with pseudomonas
associated with residual function.91 However, different infections and decline in pulmonary function.110,112 The
corrector–potentiator combinations are probably chloride–bicarbonate transporter SLC26A9 is associated
needed to rescue different misfolded class II mutant with susceptibility to meconium ileus113 and diabetes,114
proteins.98 which are frequent complications in cystic fibrosis.
As has been shown for class V splicing defects,92 However, the logical strategy of stimulating alternative
antisense oligonucleotides can also be used to correct chloride channels has been unsuccessful so far.
the Phe508del mutation. Single-stranded antisense Inhalation of denufosol, which stimulates calcium-
RNA-based oligonucleotides can act as guide sequences activated chloride channels through the P2Y2 subtype of
to repair the targeted abnormal mRNA. The repaired purinergic receptors, was promising in phase 2
mRNA is then translated into a wild-type CFTR protein.99 investigation, but robust clinical benefit was lacking.115
For one such therapy (QR-010), clinical trials are underway Nevertheless, innovative approaches to find new
to assess the safety, tolerability, and pharmacokinetics of pathways to stimulate these channels are underway (eg,
single and multiple increasing doses of the inhaled drug INOVCF project116 and Cystic Fibrosis Trust in the UK). For more on the Cystic Fibrosis
in patients homozygous for Phe508del (NCT02532764 Another approach is to target ENaC, a major regulator of Trust see http://www.
cysticfibrosis.org.uk/
and NCT02564354). These studies will also investigate salt and water re-absorption. Since ENaC is downregulated
changes in sweat chloride, weight, Cystic Fibrosis by CFTR, the absence of functional CFTR leads to ENaC
Questionnaire Revised Respiratory Symptom Score, hyperactivity in cystic fibrosis,117,118 which in turn results in
and FEV1. water depletion from the airway surface liquid and
The so-called read-through strategy for patients with reduction in its height and fluidity.119 Mice that
premature termination codons (ie, class I mutations) overexpressed β ENaC mimicked various aspects of human
remains an important concept because it has potential in respiratory disease in cystic fibrosis,120 whereas mice with
all diseases caused by a nonsense mutation. The EMA has CFTR knockout or mutations did not develop lung
granted conditional approval for ataluren in patients with disease.121 In view of the major role of ENaC in the airway,
Duchenne’s muscular dystrophy caused by a premature ENaC inhibitors have been developed to reduce ENaC-
stop codon. For patients with cystic fibrosis, the 48 week mediated sodium hyperabsorption and increase hydration
double-blind phase 3 trial of ataluren100 did not lead to a of airway surface liquid. These include specific inhibitors
firm conclusion; indeed, the primary endpoint of such as amiloride (the short-lived prototype ENaC blocker)
improvement in FEV1 was not met. However, a pre- and its derivatives benzamil or PS552, and activators of
specified subgroup analysis showed that patients who purinergic receptors (ATP, UTP, or denufosol) that inhibit
were not using inhaled aminoglycosides had less decline ENaC through stimulation of calcium-activated chloride
in lung function than had those given placebo. In view of channels.23 However, repeated inhalations of amiloride did
this finding, a confirmatory phase 3 trial in patients who not show benefits in clinical trials.122 Development of long-
are not using inhaled aminoglycosides (NCT02139306) is acting blockers was halted because of hyperkalaemia or
ongoing. Moreover, the number of new read-through other side-effects. Newer ENaC blockers, such as P-1037
drugs in preclinical development is growing. and GS-9411 (NCT00999531), are being developed. The
safety and tolerability of P-1037 (NCT02343445) are being
Targeting non-CFTR channels: the bypass approach studied as monotherapy and in combination with
For patients with so-called unrescuable mutations hypertonic saline. The inhaled combination had a transitory
(class VII), the most straightforward approach is to target effect on mucus hydration and mucociliary clearance in
alternative non-CFTR anion channels, such that the loss healthy individuals and could lead to a greater or more
of CFTR-mediated chloride transport in airway epithelia prolonged benefit than that with inhaled hypertonic saline
can be bypassed to restore ion transport.21 These therapies, alone.123
which are sometimes called mutation agnostic, might Along these lines, a systems biology screen of more
compensate not only for the loss of CFTR-mediated anion than 6000 genes identified a key ENaC regulator, DGK1,

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 Rapid Review

which normalises (but does not totally block) ENaC benefits another 40–45% of patients (ie, those who are
function and restores fluid absorption to normal levels in homozygous for Phe508del). Thus, effective treatment for
primary lung cells from patients with cystic fibrosis.124 the remaining 50% of patients remains an unmet need.
However, excessive blocking of ENaC might cause severe Indeed, the wide range of CFTR mutations consists of
harm due to hyperkalaemia or undesirable accumulation many uncommon variants (so-called orphan mutations),
of fluid in the lungs (ie, pulmonary oedema).125 for which prediction of disease outcome is difficult, since
the respective functional defect has not been defined.
Gene, cell-based, and other mutation agnostic therapies Although phenotypes vary widely, many of these mutations
After cloning the CFTR gene, gene therapy was originally in classes IV–VI result in partial CFTR function and
the priority for therapy, but gene transfer into lung cells milder, so-called atypical, forms of disease.131,132 Since each
in vivo soon proved more challenging than anticipated. of these mutations affect very few patients worldwide, they
Nevertheless, research into gene therapy led to a phase 2b pose considerable challenges not only in establishing the
clinical trial (NCT01621867)126 of DNA plasmid containing diagnosis but also in assessing the potential of the new
the CFTR cDNA (ie, a copy of the CFTR mRNA) mutation-based therapies (so-called theranostics—ie, the
complexed with cationic liposomes (ie, a non-viral vector), use of molecular or functional diagnostic tests and targeted
which was delivered once a month to the lungs of patients therapeutics in an interdependent, collaborative manner,
with cystic fibrosis. Results from this double-blind which aims to individualise treatment by targeting therapy
randomised trial showed a significant, albeit modest, to an individual’s disease subtype and genetic or functional
effect in the treatment group versus placebo at 12-month profile).131–133 Some of these patients with orphan mutations
follow-up. The 3·7% difference in FEV1 resulted from are likely to respond to existing CFTR modulators.
stabilisation of lung function in treated patients and a Therefore, there is a need to functionally characterise these
decline in lung function in the placebo group.126 Despite orphan mutations and to establish validated biomarkers
the fact that the placebo was only 0·9% saline (ie, no for theranostics. Without any preselection of suitable
empty vector, CFTR DNA, or liposomes), efficacy data are candidates, the N-of-1 clinical trial that considers an
at least encouraging, and improved versions of this individual patient as the sole unit of observation is an
therapy might become a treatment option in the future. inefficient, costly, and time-consuming approach.
Cell-based therapy is another mutation agnostic
therapeutic approach with high potential. Indeed, cellular Using ex-vivo response to predict in-vivo response
reprogramming and genome editing technologies For many CFTR mutations, how they disturb the CFTR
(based on targeting of nucleases) have made possible the pathway is unknown, and most are very rare. For these
correction of patient-specific pluripotent stem cells and rare mutations, use of ex-vivo models is a promising
induction of their differentiation into a specific cell type. approach to predict in-vivo response. Bioelectric
This approach has been shown to be feasible for cystic measurements of CFTR dysfunction in native or
fibrosis in intestinal organoids and induced pluripotent cultured tissues from patients that are already in use for
stem cells.127,128 Therefore, the disease-target tissues could cystic fibrosis diagnosis and prognosis134,135 have also
potentially be repopulated with these corrected cells. been proposed for personalised therapies.6,136 The most
However, safety issues still need to be resolved regarding promising models for treatment response are intestinal
the clinical use of stem cells before cell-based approaches organoids and primary cultures of epithelial cells
can become a therapeutic option. derived from bronchi or nasal scrapings.98,136,137 Primary
Another class of emerging therapies focus on systems intestinal organoids are grown from rectum crypts
approaches to target both mechanisms of disease and obtained via suction biopsy and contain stem cells;
their therapeutic correction.11 One such strategy targets therefore, they can be expanded indefinitely to obtain a
proteostasis (ie, maintenance of the cellular proteome in continuous source of patient-specific material. Forskolin
a folded, functionally competent state) and can be induces swelling of organoids from healthy individuals
applied not only for cystic fibrosis but also for a group of via stimulation of the CFTR channel, but not in
so-called protein misfolding diseases.129 A successful organoids from patients with cystic fibrosis, and the
example is suberolyanilide hydroxamic acid (SAHA), a magnitude of swelling correlates with CFTR activity.137
histone deacetylase inhibitor and regulator of the Organoids from patients can be incubated with drug
chaperone protein Hsp90, which has been shown to candidates to assess their effect on CFTR function. If
restore the cell surface expression and channel activity CFTR function and organoid swelling are restored by a
of Phe508del-CFTR to 28% of wild-type levels in human specific modulator in a patient’s tissue ex vivo, then
primary airway epithelial cells.130 testing the clinical benefit in vivo is the next logical step.
There is therefore an unmet need to test compounds
Repurposing existing drugs for so-called orphan mutations that improve organoid function ex vivo in patients via
Despite the great breakthrough in drug development, the N-of-1 trials, so as to prove that a positive ex-vivo
first CFTR modulator, ivacaftor, targets only 5–10% of organoid response indeed correlates with a good in-vivo
patients at best, and its combination with lumacaftor clinical response. Therefore, the research community

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