Review
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doi: 10.1111/joim.12314
Novel personalized therapies for cystic fibrosis: treating the
basic defect in all patients
M. D. Amaral
From the BioFIG-Center for Biodiversity, Functional and Integrative Genomics, Faculty of Sciences, University of Lisboa, Lisboa, Portugal
Abstract. Amaral MD (University of Lisboa, Lisboa,
Portugal). Novel personalized therapy for cystic
fibrosis: treating the basic defect in all patients
(Review). J Intern Med 2015; 277: 155–166.
Cystic fibrosis (CF) is the most common genetic
life-shortening condition in Caucasians. Despite
being a multi-organ disease, CF is classically
diagnosed by symptoms of acute/chronic respiratory disease, with persistent pulmonary infections
and mucus plugging of the airways and failure to
thrive. These multiple symptoms originate from
dysfunction of the CF transmembrane conductance regulator (CFTR) protein, a channel that
mediates anion transport across epithelia. Indeed,
establishment of a definite CF diagnosis requires
proof of CFTR dysfunction, commonly through the
so-called sweat Cl test. Many drug therapies,
including mucolytics and antibiotics, aim to alleviate the symptoms of CF lung disease. However,
new therapies to modulate defective CFTR, the
Introduction
Although classified as a rare disease, cystic fibrosis
(CF) is the most common life-threatening monogenic condition in Caucasians. The estimated incidence of CF is 1 in 2500–4000 newborns, with a
recognized heterogeneity in the geographic distribution [1, 2]. CF affects >70 000 individuals worldwide, including more than 30 000 in Europe [3].
Despite being a multi-organ disease, CF predominantly affects the lungs. There is a wide clinical
variability in organ involvement; the dominant
cause of morbidity and mortality is lung disease,
but other CF symptoms include pancreatic insufficiency, intestinal obstruction, elevated electrolyte
levels in sweat (the basis of the most common
diagnostic test) and male infertility [4–6].
This inherited condition is caused by mutations in
the CF transmembrane conductance regulator
(CFTR) gene, which encodes a cAMP-regulated Cl
and HCO3 channel expressed at the apical
basic defect underlying CF, have started to reach
the clinic, and several others are in development or
in clinical trials. The novelty of these therapies is
that, besides targeting the basic defect underlying
CF, they are mutation specific. Indeed, even this
monogenic disease is influenced by a large number
of different genes and biological pathways as well
as by environmental factors that are difficult to
assess. Accordingly, every person with CF is
unique and so functional assessment of patients’
tissues ex vivo is key for diagnosing and predicting
the severity of this disease. Of note, such assessment will also be crucial to assess drug responses,
in order to effectively treat all CF patients. It is not
because it is a monogenic disorder that personalized treatment for CF is much easier than for
complex disorders.
Keywords: monogenic disorder, mutation-specific
therapies, personalized therapy, rare diseases.
membrane of epithelial cells [7]. The primary
absence of or reduction in anion permeability due
to CFTR gene defects triggers the so-called CF
pathogenesis cascade that characterizes CF respiratory disease [8]: (i) Lack of CFTR, the major
epithelial ion regulator, leads to deficient transport
of other ion conductances, for example excess Na+
mediated by the epithelial Na+ channel (ENaC;
which is negatively regulated by CFTR) [9]; (ii) This
ionic dysregulation in turn leads to a reduction in
the water content of the airway surface liquid and
excessively thick mucus that is resistant to
removal; (iii) Then a cycle of destruction is initiated
[10], involving airway mucus obstruction and disseminated bronchiectasis, bacterial infections,
chronic inflammation and lung damage/scarring;
(v) Finally, this cascade leads to end-stage lung
disease which can only be resolved by lung transplantation (Fig. 1).
Classical CF is diagnosed early in infancy and
suggested by one or more characteristic clinical
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M. D. Amaral
Review: Novel personalized therapies for CF
Defective
CFTR protein
Decreased water in
ASL thick mucus
Two defective
CF genes
Abnormal Cl– permeability
Altered ionic transport
Cl–
Cl–
CFTR
Cl–
Bronchiectasis
Scarring
Progressive loss of
lung function
Mucus obstruction
Inflammation
Bacterial infection
Na+
ENaC
Cl–
Na+
Fig. 1 The cystic fibrosis (CF) pathogenesis cascade in the lung. The mechanism of CF dysfunction starts with the primary
CFTR gene defect and ultimate leads to severe lung deficiency. CFTR, cystic fibrosis transmembrane conductance regulator;
ASL, airway surface liquid; ENaC, epithelial Na+ channel.
features, a history of CF in a sibling or, more
recently, a positive newborn screening result [11,
12]. The recently widely implemented neonatal
screening programmes identify increasing numbers of still asymptomatic CF patients, posing new
challenges to the CF diagnosis paradigm and
requiring new diagnosis assays [13] (see below).
Major clinical advances in treating the symptoms
and delaying disease progression have significantly
improved survival from 5 years in the early 1960s
to beyond the third decade at present [14]. Much of
the progress in extending life expectancy has been
due to standardized multisystem treatments comprising antibiotics to eradicate major bacterial lung
infections (especially by Pseudomonas aeruginosa,
a hallmark of CF) and mucolytics to loosen and
clear the thick mucus characteristic of CF as well
as high-calorie nutrition and chest physiotherapy
[2, 15]. As a result of these approaches, together
with widespread lung transplantation programmes, approximately 50% of CF patients are
now adults in several countries [16, 17].
Nevertheless, despite great advances in supportive
care and in our understanding of the pathophysiology of CF, there is still no cure for this disease.
To further increase the life expectancy of CF
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patients and significantly reduce the current therapeutic burden, the disease must be treated
beyond its symptoms (i.e. through treatments
addressing the basic defect associated with CFTR
gene mutations) in order to effectively halt the
cascade of effects downstream of CFTR dysfunction
[18, 19]. However, this may not be an easy task; as
to date, almost 2000 mostly disease-causing CFTR
mutations have been reported [20]. Notwithstanding, a single mutation – F508del, associated with
severe CF – remains the most common mutation,
occurring in ~85% of CF patients in at least one
allele [21].
Cystic fibrosis diagnosis
Traditionally, the diagnosis of CF is based on
clinical symptoms suggestive of the disease and/
or a positive family history. Such symptoms
include mostly those affecting the airways and
the gastrointestinal tract, but also those affecting
other systems (see Table 1).
Airway disease dominates the clinical phenotype
mostly due to the production of very thick mucus
and impaired mucociliary clearance which lead to
accumulation of purulent secretions. As mucociliary clearance is an important defence mechanism
M. D. Amaral
Review: Novel personalized therapies for CF
Table 1 Phenotypic characteristics of cystic fibrosis (CF)
Lower airways
Acute or persistent respiratory
symptoms
Chronic cough and sputum
production
Hyperviscous mucus
Obstructive lung disease
Recurrent pneumonia or lung
infections
Persistent colonization with CF
pathogens
Low performance in lung function
tests
Chronic chest radiograph
abnormalities (i.e. bronchiectasis)
Upper airways
Nasal polyps/sinus disease
aeruginosa (a hallmark of CF) is found in 80% of
patients by the age of 18 years, and Staphylococcus
aureus and Haemophilus are the main pathogens
in younger patients [22].
Nevertheless, to confirm a diagnosis of CF, it is
necessary to obtain evidence of CFTR dysfunction
through the identification of two CFTR gene mutations previously assigned as CF disease causing,
two tests showing a high Cl concentration in
sweat (>60 mEq L 1), distinctive transepithelial
nasal potential difference measurements and/or
assessment of CFTR (dys) function in native colonic
epithelia ex vivo [4, 12, 13]. For individuals with
symptoms suggestive of CF but intermediate sweat
Cl values (30–60 mEq L 1), the need for additional proof of CFTR function (through NPD measurements or CFTR functional assessment in rectal
biopsies) is particularly important.
Chronic suppurative sinopulmonary
disease
Chronic pansinusitis
Gastrointestinal
tract/nutrition
Pancreatic insufficiency with
malabsorption Failure to thrive/
malnutrition
Steatorrhoea/abnormal stools
Meconium ileus/intestinal
obstruction
Rectal prolapse
Hepatobiliary disease
Recurrent pancreatitis
Distal intestinal obstruction
syndrome
Sweat glands
High Cl concentration in sweat
Absence of ß-adrenergic sweat
Male reproductive
system
Congenital bilateral/unilateral
absence of the vas deferens
More recently, the diagnostic paradigm has changed with the widespread introduction of neonatal
screening programmes which often identify CF
patients before they develop any classical CF
symptoms. For these programmes, it is essential
to (i) confirm/exclude the CF diagnosis in a timely
manner, (ii) achieve this with a high degree of
accuracy to avoid excessive testing (and the associated costs), (iii) provide accurate prognostic
information and genetic counselling and, most
importantly, (iv) deliver appropriate treatment
and ensure early access to specialized CF reference
centres with multidisciplinary specialized services
[12, 23].
The search for new and reliable CF biomarkers
continues and is likely to provide results in the
short term. This is important not only to establish a
diagnosis of CF but also to monitor clinical trials of
new CFTR modulating drugs [24, 25].
Obstructive azoospermia
Metabolism
Hypoproteinaemia
The CF gene and CF disease: a paradigm for rare disorders
Fat-soluble vitamin deficiencies
Despite the apparent ‘simplicity’ of being monogenic (and ‘simplification’ of the name to ‘65 roses’
by children with CF [26]), CF still poses many
diagnostic challenges and its clinical management
is not straightforward. CF is in fact a complex
disorder for several reasons. First, as mentioned
above, almost 2000 CFTR gene mutations have so
far been reported [20], and this is further complicated by the presence of ‘complex alleles’, that is
those containing more than one CFTR mutation
[27]. Secondly, the CFTR genotype often poorly
Salt-loss syndrome with salt
depletion, with or without
metabolic alkalosis
Data from [5, 11, 12, 23, 46].
against pathogens and dust particles, its reduction
in CF patients leads to chronic infections by a
restricted group of pathogens: Pseudomonas
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M. D. Amaral
predicts the full spectrum of the clinical phenotypes and consequences in multiple organs [28];
for example, chronic lung inflammation and infection, which play a major role in the course of the
respiratory disease, are highly influenced by environmental factors such as tobacco smoke and
outdoor pollution and the bacterial microbiome of
the lung [29, 30]. Thirdly, an increasing number
of CFTR mutations are associated with isolated
disease characteristics such as chronic pancreatitis, chronic sinusitis, disseminated bronchiectasis or male infertility; the distinction between
these CFTR-related disorders (CF-RD) [31, 32] (or
‘CFTR-Opathies’ [33]) and CF (especially in its
milder forms) is not always straightforward.
Fourthly, several modifier genes have been identified but how these genes influence disease is not
always clear [34–36]. Finally, it has been suggested
that CFTR plays several other roles in the cell,
regulating (directly or indirectly) other cellular
proteins and functions [37, 38]. It is still unclear
whether such ‘secondary’ functions are regulated
by CFTR itself or by other channels which in turn
are regulated by CFTR [39]. Of note, it remains to be
determined whether correction of the primary function of CFTR as an anion channel will also restore
these additional ‘secondary’ functions.
Such complexity makes CF a devastating disease
still hard to manage at its root. Nevertheless,
despite being a ‘simple’ monogenic disease, CF
has given many lessons of complexity and biomedical science has gained much from CF research. In
many respects, CF is indeed a paradigmatic monogenic rare disease, greatly contributing to the
advancement of both biomedical science and clinical practice. As Jack Riordan, who together with
Lap-Chee Tsui and Francis Collins made the original
CFTR gene discovery, stated in a recent publication
for the 20th anniversary of the discovery, ‘The
disease has contributed much more to science than
science has contributed to the disease’ [40].
The knowledge gained during the long research
path to the discovery of the basic defect in CF may
accelerate the pace of translational medicine for
future gene discovery, as hard work and mistakes
made in this field may contribute to advances in
other areas. For new genes recently found to be
causal for other diseases, those 20 years of
research can be largely reduced because of work
so far done on CF [40]. Some of these highlights are
described below.
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Review: Novel personalized therapies for CF
Geneticists have been interested in the gene
responsible for CF as the disease was first identified in the 1930s [41]. Its final discovery in 1989
was a major breakthrough, establishing the
ground-breaking human gene cloning techniques
of chromosome jumping and chromosome walking
[42]. The major goal of identifying the CF gene was
to be able to move quickly to gene therapy. The
concept of giving patients a correct copy of the CF
gene, so that they could produce the functional
protein, was simple and elegant. Thus, gene therapy for a monogenic disorder was pioneered for CF
[43]. However, gene therapy trials soon demonstrated how difficult it is to express foreign genes in
the lung; this was a ‘lesson’ for other disorders [44].
Although gene therapy trials for CF have come to a
halt at present, knowledge gained from the preclinical and clinical gene therapy studies has
informed protocols and efficacy end-points for
further novel therapeutic approaches [45, 46].
The large number and variety of CFTR gene mutations led to the creation of a widely used global
repository of mutations by an international consortium [20]. Nevertheless, determining the genotype–phenotype correlations proved to be difficult
[47–49]. Moreover, such widespread genetic testing
is of limited value, given the substantial number of
DNA variants of uncertain pathogenic significance.
Therefore, the next challenge in the CF field was to
identify the molecular and cellular dysfunction
caused by these gene mutations (see below).
Although still far from accomplished, this has been
achieved for the most common mutations [50] and
the CFTR2 (Clinical and Functional TRanslation of
CFTR website) database has been established to
disseminate these findings [51].
Along the path to finding a cure for CF, many
advances in basic biology have provided a better
understanding of the pathophysiology of the disease [8], including, notably, the mechanisms of
protein folding, pathways of secretory traffic and
the physiology of epithelial channels (reviewed in
[39]). Therefore, CF has become a paradigm for
trafficking disorders and other ‘channelopathies’.
Knowledge in the field of CF may thus translate
into understanding other rare/orphan diseases, in
particular those sharing a similar basic defect [52].
Similarly, findings may also apply to other major
respiratory diseases in which CFTR has been found
to play a role such as in chronic obstructive
pulmonary disease (COPD) [53–56] or asthma
[57–60], both of which are currently increasing in
M. D. Amaral
prevalence worldwide. Therefore, it is expected that
CFTR-modifying drugs (see below) may also benefit
patients with these common disorders.
The generation of animal models for CF also led the
field of animal disease models through the application of the gene targeting principle. This endeavour, that is, generation of CF mouse models,
involved the three 2007 Nobel Prize awardees in
this field [61–63].
Finally, the development of protein conformation
change-inducing drugs, which has been a model
for drug discovery in rare diseases, has been led by
research in the field of CF. This programme was the
result of the innovative association between a
highly committed CF patients association (CFFCystic Fibrosis Foundation, USA) and the pharmaceutical industry (Vertex Pharmaceuticals) [46,
64]. The Foundation started work several decades
ago by (i) funding research that led to the discovery
of the gene in 1989 [42, 65, 66], (ii) building an
extensive patient registry and a clinical trial network, both of which were required for investigating
CF genetics and (iii) efficiently recruiting participants for trials of investigational drugs [64]. The
Foundation also funded a focused multidisciplinary scientific effort to understand the molecular
basis of this disorder [40, 67]. The ambitious goal
was to systematically investigate the basic CF
defect(s) using small molecules (see below). The
drug discovery programme that followed has been
exemplary in many respects [46]. By 2009,
20 years after the discovery of the CF gene, it was
anticipated that innovative life-changing treatment
for CF would soon be available [67]; this in fact
became a reality in 2013. This experience has
paved the way for personalized medicine in other
genetic diseases [46].
Functional classification of mutations
In the years after the discovery of the CFTR gene,
there were considerable advances in the understanding of the structure and function of the CFTR
protein: a complex multidomain 1480-amino acid
membrane protein that is the only member of the
ATP-binding cassette (ABC) transporters family
that functions as an ion channel [39]. However,
the major difficulty is tackling the almost 2000
CFTR mutations so far described [20].
To correct for such a variety of gene and protein
defects effectively, CFTR mutants are grouped into
Review: Novel personalized therapies for CF
functional classes in which the same restorative
strategy may be effective; this approach has been
termed ‘mutation-repairing therapy’ [18]. The first
step is thus to identify the basic molecular and
cellular defect underlying each individual mutation. This was the goal of the CFTR2 project [51]
which evaluated, in terms of both clinical severity
and functional consequences, the most common
CFTR gene mutations (i.e. those with an allele
frequency of ≥0.01%) altogether accounting for
96.4% of CF alleles, in a multicentre international
cohort of almost 40 000 patients (i.e. 57% of the
estimated 70 000 individuals with CF worldwide)
[50]. The complete list of mutations studied so far
(177 in July 2013) is available online [51]. To
achieve the aim of the CFTR2 project, molecular
and functional characterization of many CFTR
mutations was carried out thus enabling classification of each of these mutations into one of the
established six functional classes [8, 18, 19, 68,
69] (Fig. 2).
Class I (no protein) comprises mutations that
produce premature termination signals because
of splice site abnormalities, frameshifts due to
insertions or deletions, or nonsense mutations [i.e.
mutations generating premature stop codons
(PTCs)], for example G542X or R1162X. Class II
(no traffic) includes mutants that fail to traffic to
the cell surface (i.e. to the correct CFTR cellular
location), due to misfolding and premature degradation by the endoplasmic reticulum (ER) quality
control (ERQC) (reviewed in [70]); this class
includes the most common mutation, F508del.
Class III (no function) comprises CFTR mutants
that, although reaching the plasma membrane,
exhibit defective channel gating (i.e. the channel
pore does not open) due to impaired response to
channel agonists; an example of this class is
G551D. When F508del-CFTR is promoted to reach
the plasma membrane by corrector compounds, it
still has a partial gating defect, and thus, it can
also be in Class III. Class IV (less function) mutants
display substantially reduced conductance (i.e.
flow of ions through the CFTR channel pore), with
a resulting decrease in net Cl channel activity;
R334W, a mutation in the CFTR channel pore, is
an example. Class V (less protein) includes mainly
alternative splicing mutants (e.g. 3272-26A>G [71,
72]) that allow synthesis of some normal CFTR
mRNA (and protein), albeit at very low levels [73];
this class also includes promoter mutations that
reduce transcription (e.g. -94G>T [74]) and amino
acid substitutions that cause inefficient protein
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M. D. Amaral
Review: Novel personalized therapies for CF
CFTR defect type:
WT-CFTR
Cl–
Cl–
Cl–
Cl–
Cl–
Cl–
I
II
III
IV
V
VI
No protein
No traffic
No function
Less function
Less protein
Less stable
Cl–
Cl–
Cl–
Cl–
Cl–
Cl–
Cl–
Cl–
Cl–
Cl–
Cl–
CFTR
Cl–
Cl–
Cl–
Mutation
examples
G542X (a)
W1282X (a)
1717-1G (b)
F508del
N1303K
A561E
G551D
S549R
G1349D
R117H
R334W
A455E
A455E
3272-26A>G
3849+10 kb C>T
c.120del23
rF508del
Corrective
therapy
Rescue
synthesis
Rescue
traffic
Restore
channel
activity
Restore
channel
activity
Correct
splicing
Promote
stability
Drug
Read-through
compounds
Correctors
Potentiators
Potentiators
AONs
Correctors
Potentiators
Stabilizers
Drug approved
(Yes/No)
No
No
Yes
No
No
No
Fig. 2 Classes of CFTR gene mutations. The aim of stratification of CFTR mutations into functional classes is to apply the
same therapeutic correction for the basic defect in each class. The strategies, the types of molecules required to achieve such
strategies and their current status of clinical approval are shown. CFTR, cystic fibrosis transmembrane conductance
regulator; WT, wild-type; Antisense oligonucleotide, (AON).
maturation (e.g. A455E [75]). Finally, Class VI (less
stability) mutants impair the CFTR plasma membrane stability [76]; an example of this class is
c.120del23 which lacks the cytoskeleton-anchoring N-terminus of CFTR [77]. When rescued to the
cell surface, F508del-CFTR also behaves as a Class
VI mutant due to its intrinsic instability [78, 79].
F508del has multiple defects and is thus included
in Classes II, III and VI; this illustrates a limitation
of this CFTR mutation classification. Applying
rescuing strategies to achieve full correction of
such mutants (see below) is therefore complex, as
more than one type of CFTR-modulator drug will
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Journal of Internal Medicine, 2015, 277; 155–166
probably have to be used [19]. Another limitation of
this classification arises from the fact that the
majority of mutations have not been assigned to a
mutation class due to the absence of functional
studies. Although the most common mutations
have been assigned into a functional class [50],
much work regarding functional characterization
of CFTR mutations remains to be done if CFTR
modulating therapies are to reach 100% of CF
patients (see below). This becomes even more
relevant given the increasing number of novel
variants identified in full gene screening protocols
by next-generation sequencing as part of several
neonatal CF screening programmes [80, 81].
M. D. Amaral
Correcting the basic CFTR defects: personalized therapies
The major advantage of the above classification of
mutations is the possibility of adopting the same
therapeutic strategy for each class [82] (see Fig. 2).
Such strategies may even apply to the correction of
similar defects causing other diseases that share
the same basic molecular defect [8]. The design and
outcomes of the clinical trials discussed here are
reviewed in detail elsewhere [82].
Class I agents
As many Class I mutations (Class Ia) lead to the
complete lack of CFTR protein due to the presence
of a PTC, compounds promoting the read-through
of stop codons by the ribosome have been investigated for these mutants. Compounds that achieve
this goal are the aminoglycoside antibiotics (gentamicin and tobramycin) [83], which have also
been proposed to correct PTCs in other genetic
disorders such as Duchenne muscular dystrophy
(DMD) [84] and cancer [85].
Another compound, ataluren (formerly PTC124),
was also shown to lead to some degree of ‘overreading’ of PTCs and thus entered clinical trials;
however, no improvement in the primary endpoint, lung function, was observed [86]. Clinical
trials have so far also shown limited efficacy of
ataluren in patients with DMD with nonsense
mutations (nmDMD) [87]. Accordingly, this drug
is not approved by the US Food and Drug Administration (FDA), although in the EU, it has received
conditional marketing authorization for patients
with nmDMD aged 5 years and above [82]. Nonetheless, further optimization of read-through compounds is required to achieve significant clinical
efficacy.
For other Class I mutants (Class Ib), involving
small deletions or insertions that cause frameshift
mutations during protein production (Fig. 2), there
is at present no therapeutic strategy except perhaps the so-called bypass therapies which target
other (non-CFTR) channels (see below).
‘Correctors’ for Class II mutants
Mutations in Class II fail to traffic to the cell surface
and are mainly retained at the ER [88]. As the most
common CF-causing mutation (F508del) is
included in this class, it has been the focus of
major efforts to elucidate the molecular mechanism
Review: Novel personalized therapies for CF
of F508del-CFTR dysfunction. Due to inefficient
folding, this mutant acquires an abnormal conformation which is recognized and retained by the ER
quality control (ERQC) that targets it for premature
degradation [19]. Proof of concept that F508delCFTR could be rescued to the cell surface came
from initial studies showing that this could be
achieved by incubation at low temperatures [89].
Several compounds, chemical chaperones such as
glycerol or TMAO that promote protein folding, also
showed similar but nonspecific effects [70].
The identification of compounds that rescue
mutant CFTR in a specific way resulted from
high-throughput screening (HTS) for drug discovery [90, 91]. These screening studies led to the
identification of CFTR modulators: ‘correctors’ that
rescue the trafficking defect of F508del-CFTR and
‘potentiators’ that stimulate channel gating. To
date, the most successful corrector is the investigational drug lumacaftor (VX-809) [92] which,
despite very promising results in primary cells
[92], only promoted a significant decrease in sweat
Cl levels and no effect on lung function in F508del
homozygous patients during a Phase II clinical trial
[93]. Currently, lumacaftor and VX-661 (a secondgeneration corrector) are being tested in a clinical
trial in combination with the potentiator ivacaftor
(Kalydeco; previously VX-770) (see below). Interim
results from a Phase II trial of VX-661 and ivacaftor
showed a modest but statistically significant
change in sweat Cl levels as well as a small and
variable but also significant improvement in lung
function at day 28 in F508del-homozygous
patients [94].
Potentiators for Class III and IV mutants
After major success in a Phase III clinical trial [95],
the clinical approval in 2013 by both the FDA and
the European Medicines Agency of the first drug to
target mutant CFTR was met with great enthusiasm and optimism in the CF community. The CFTR
potentiator ivacaftor was approved for clinical use
in individuals with G551D, although this mutation
is only present in ~4% of CF patients. More
recently, following demonstration of in vitro effectiveness [96], ivacaftor was approved by the FDA
for another eight gating (Class III) mutations
which, together with G551D, account for ~5% of
all CF patients. Potentiators such as genistein and
related flavonoids can also activate Cl conductance and thus overcome the gating defects of
Class III mutants [97–99].
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M. D. Amaral
For Class IV mutants, compensation for reduced
ionic flow may be achieved by potentiators. Similarly, correctors could increase the overall cell
surface density of these mutants. However, this
assumption requires in vitro demonstration of
efficacy.
Agents that rescue Class V mutants
As many Class V defects are the result of alternative splicing, increasing the levels of splicing
factors that correct such missplicing may constitute an effective therapeutic strategy for CF
patients with these mutations (i.e. ~10% of all
patients with this disease). Although which factors
require manipulation to correct mRNA splicing is
still unclear, recent advances in the delivery of
‘splice switching’ oligomers to cells appear to be
promising [100]. Meanwhile, the approved potentiator ivacaftor, with proven efficacy on wild-typeCFTR, is also likely to provide benefit for patients
with Class V mutations; however, this requires
confirmation.
Restoring mutations in Class VI
Compounds that enhance CFTR retention/
anchoring at the cell surface will benefit Class VI
mutants that have intrinsic plasma membrane
instability. This is the case for F508del-CFTR
when rescued to the plasma membrane by novel
small-molecule correctors [101], which can partially account for the limited success of clinical
trials with correctors. F508del-CFTR stabilizers
include activators of Rac1 signalling, such as
hepatocyte growth factor (HGF), which promote
anchoring to the actin cytoskeleton via NHERF1
[102].
Correcting the basic CF defect in all CF patients: still an unmet
need
Despite the great breakthrough in CF drug development with the licensing of the first CFTR modulator drug, ivacaftor still only targets ~5% of CF
patients. Thus, there is still an unmet need to
effectively treat the remaining ~95% of CF patients.
Treatment for Class II mutations should be available in the 2–3 years, whereas Class I mutation
treatments might take longer to develop [103].
Importantly, however, only ~40% of patients are
F508del-homozygous, and the efficacy of correctors in patients with only one F508del allele is
expected to be even lower than in homozygotes.
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Review: Novel personalized therapies for CF
Moreover, at least 15% of all CF patients lack
F508del in both alleles and thus will not benefit
from F508del correctors.
The aim of bypass therapies is to manipulate other
(non-CFTR) ionic conductances to restore the ionic
homeostasis of the epithelia and correct the fluid
and pH imbalance in CF. The great advantage of
this therapeutic approach is that it is equally
suitable for all patients with CF, irrespective of
their mutations. This approach can be achieved by
normalizing the hyperabsorption of Na+ that occurs
in CF epithelia [104] through ENaC directly or
indirectly [105] or through activation of alternative
Cl channels, particularly the anoctamins, a family
of Ca2+-activated Cl channels (CaCCs) [106].
Stimulation of basolateral K+ channels could also
increase the driving force for Cl secretion and
hence favour CFTR-mediated secretion in patients
with residual CFTR function [107].
These
innovative
therapeutic
approaches,
although very appealing, may take years to reach
the clinic. Thus, it is important to find effective
means of testing the response of rarer mutations to
the approved drug ivacaftor (and new CFTR modulators), in order to treat a wider population of CF
patients.
Ivacaftor may indeed be one of many therapeutic
agents that point to the emergence of a new era of
personalized medicine. Rare mutations could be
tested in modified single-patient (‘n-of-1’) trials
[108], in which the subject is exposed to treatment
over variable blinded time periods, and outcome
parameters are measured repeatedly to compare
outcomes during ‘on’ versus ‘off’ drug periods
[108]. However, treatment effect and drug efficacy
are hard to prove. There is thus an urgent unmet
need to test the efficacy of emerging CFTR
modulators directly on patients’ tissues ex vivo to
identify responders who will benefit from these
innovative therapies. Not unsurprisingly, CF
patients associations support such approaches to
accelerate the process of bringing these new drugs
to a greater number of patients [25]. Bioelectric
measurements of CFTR (dys)function in native (or
cultured) tissues from CF patients have been
proposed as the basis of personalizing therapies
[25], as well as for CF diagnosis and prognosis [13,
109]. These are original paradigmatic technological
developments in science that offer new promise for
developing targeted therapeutics and tools for
predicting those patients who will respond to a
M. D. Amaral
medical therapy and those who will experience no
effects at all, without actually taking the drug.
Conclusions
The aims of personalized medicine are to assess
the medical risks and monitor, diagnose and treat
patients according to their specific genetic composition and molecular phenotype. It has been
shown for CF as a paradigmatic disease that using
the genetic variations to predict clinical phenotype
is not easy. Indeed, even this ‘simple’ monogenic
disease is influenced by a large number of different genes and biological pathways as well as by
environmental factors that are difficult to assess.
Furthermore, every person with CF is unique and
requires personalized diagnosis. It is not because
it is a monogenic disorder that personalized
treatment for CF is much easier than for diabetes,
neurological disorders, cancer or other diseases
involving a large number of different genes and
biological pathways [110]. Consequently, the
combined knowledge of gene variants along with
a functional assessment of responses ex vivo will
be crucial for predictive personalized treatment of
CF.
Conflict of interest statement
MDA has served as a consultant to Vertex and
Galapagos, has been supported to attend and
speak at symposia by Novartis, Gilead and Vertex
and participated in an educational grant programme by Facilitate Ltd.
Acknowledgements
Work in the author’s laboratory has been supported by strategic grant PEst-OE/BIA/UI4046/
2011 (to BioFIG) and research grants PTDC/SAUGMG/122299/2010 (to MDA) from FCT/MCTES,
Portugal; CFF-Cystic Fibrosis Foundation, USA,
(Ref. 7207534), Gilead GENESE-Portugal
Programme (Ref 002/2013); ‘INOVCF’ from CF Trust,
UK (Strategic Research Centre Award No. SRC 003)
(to MDA).
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Correspondence: Margarida D. Amaral, BioFIG-Center for
Biodiversity, Functional and Integrative Genomics, Faculty of
Sciences, University of Lisboa, Campo Grande, C8 bdg, 1749-016
Lisboa, Portugal.
(fax:+351-21-750 0088; e-mail: [email protected]).