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Novel personalized therapies for cystic fibrosis: treating thebasic defect in all patientsM. 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 cysticfibrosis: treating the basic defect in all patients(Review). J Intern Med 2015; 277: 155–166.

Cystic fibrosis (CF) is the most common geneticlife-shortening condition in Caucasians. Despitebeing a multi-organ disease, CF is classicallydiagnosed by symptoms of acute/chronic respira-tory disease, with persistent pulmonary infectionsand mucus plugging of the airways and failure tothrive. These multiple symptoms originate fromdysfunction of the CF transmembrane conduc-tance regulator (CFTR) protein, a channel thatmediates anion transport across epithelia. Indeed,establishment of a definite CF diagnosis requiresproof of CFTR dysfunction, commonly through theso-called sweat Cl� test. Many drug therapies,including mucolytics and antibiotics, aim to allevi-ate the symptoms of CF lung disease. However,new therapies to modulate defective CFTR, the

basic defect underlying CF, have started to reachthe clinic, and several others are in development orin clinical trials. The novelty of these therapies isthat, besides targeting the basic defect underlyingCF, they are mutation specific. Indeed, even thismonogenic disease is influenced by a large numberof different genes and biological pathways as wellas by environmental factors that are difficult toassess. Accordingly, every person with CF isunique and so functional assessment of patients’tissues ex vivo is key for diagnosing and predictingthe severity of this disease. Of note, such assess-ment will also be crucial to assess drug responses,in order to effectively treat all CF patients. It is notbecause it is a monogenic disorder that personal-ized treatment for CF is much easier than forcomplex disorders.

Keywords: monogenic disorder, mutation-specifictherapies, personalized therapy, rare diseases.

Introduction

Although classified as a rare disease, cystic fibrosis(CF) is the most common life-threatening mono-genic condition in Caucasians. The estimated inci-dence of CF is 1 in 2500–4000 newborns, with arecognized heterogeneity in the geographic distri-bution [1, 2]. CF affects >70 000 individuals world-wide, including more than 30 000 in Europe [3].Despite being a multi-organ disease, CF predomi-nantly affects the lungs. There is a wide clinicalvariability in organ involvement; the dominantcause of morbidity and mortality is lung disease,but other CF symptoms include pancreatic insuf-ficiency, intestinal obstruction, elevated electrolytelevels in sweat (the basis of the most commondiagnostic test) and male infertility [4–6].

This inherited condition is caused by mutations inthe CF transmembrane conductance regulator(CFTR) gene, which encodes a cAMP-regulated Cl�

and HCO3� channel expressed at the apical

membrane of epithelial cells [7]. The primaryabsence of or reduction in anion permeability dueto CFTR gene defects triggers the so-called CFpathogenesis cascade that characterizes CF respi-ratory disease [8]: (i) Lack of CFTR, the majorepithelial ion regulator, leads to deficient transportof other ion conductances, for example excess Na+

mediated by the epithelial Na+ channel (ENaC;which is negatively regulated by CFTR) [9]; (ii) Thisionic dysregulation in turn leads to a reduction inthe water content of the airway surface liquid andexcessively thick mucus that is resistant toremoval; (iii) Then a cycle of destruction is initiated[10], involving airway mucus obstruction and dis-seminated bronchiectasis, bacterial infections,chronic inflammation and lung damage/scarring;(v) Finally, this cascade leads to end-stage lungdisease which can only be resolved by lung trans-plantation (Fig. 1).

Classical CF is diagnosed early in infancy andsuggested by one or more characteristic clinical

ª 2014 The Association for the Publication of the Journal of Internal Medicine 155

Review

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features, a history of CF in a sibling or, morerecently, a positive newborn screening result [11,12]. The recently widely implemented neonatalscreening programmes identify increasing num-bers of still asymptomatic CF patients, posing newchallenges to the CF diagnosis paradigm andrequiring new diagnosis assays [13] (see below).

Major clinical advances in treating the symptomsand delaying disease progression have significantlyimproved survival from 5 years in the early 1960sto beyond the third decade at present [14]. Much ofthe progress in extending life expectancy has beendue to standardized multisystem treatments com-prising antibiotics to eradicate major bacterial lunginfections (especially by Pseudomonas aeruginosa,a hallmark of CF) and mucolytics to loosen andclear the thick mucus characteristic of CF as wellas high-calorie nutrition and chest physiotherapy[2, 15]. As a result of these approaches, togetherwith widespread lung transplantation pro-grammes, approximately 50% of CF patients arenow adults in several countries [16, 17].

Nevertheless, despite great advances in supportivecare and in our understanding of the pathophys-iology of CF, there is still no cure for this disease.To further increase the life expectancy of CF

patients and significantly reduce the current ther-apeutic burden, the disease must be treatedbeyond its symptoms (i.e. through treatmentsaddressing the basic defect associated with CFTRgene mutations) in order to effectively halt thecascade of effects downstream of CFTR dysfunction[18, 19]. However, this may not be an easy task; asto date, almost 2000 mostly disease-causing CFTRmutations have been reported [20]. Notwithstand-ing, a single mutation – F508del, associated withsevere CF – remains the most common mutation,occurring in ~85% of CF patients in at least oneallele [21].

Cystic fibrosis diagnosis

Traditionally, the diagnosis of CF is based onclinical symptoms suggestive of the disease and/or a positive family history. Such symptomsinclude mostly those affecting the airways andthe gastrointestinal tract, but also those affectingother systems (see Table 1).

Airway disease dominates the clinical phenotypemostly due to the production of very thick mucusand impaired mucociliary clearance which lead toaccumulation of purulent secretions. As mucocil-iary clearance is an important defence mechanism

Cl–

CFTR

Cl– Na+

Cl– Cl– Na+

ENaC

Two defective CF genes

DefectiveCFTR protein

Abnormal Cl– permeabilityAltered ionic transport

Decreased water in ASL thick mucus

Mucus obstruction

Bronchiectasis

Inflammation

Scarring

Bacterial infection

Progressive loss oflung function

Fig. 1 The cystic fibrosis (CF) pathogenesis cascade in the lung. The mechanism of CF dysfunction starts with the primaryCFTR gene defect and ultimate leads to severe lung deficiency. CFTR, cystic fibrosis transmembrane conductance regulator;ASL, airway surface liquid; ENaC, epithelial Na+ channel.

M. D. Amaral Review: Novel personalized therapies for CF

156 ª 2014 The Association for the Publication of the Journal of Internal MedicineJournal of Internal Medicine, 2015, 277; 155–166

against pathogens and dust particles, its reductionin CF patients leads to chronic infections by arestricted group of pathogens: Pseudomonas

aeruginosa (a hallmark of CF) is found in 80% ofpatients by the age of 18 years, and Staphylococcusaureus and Haemophilus are the main pathogensin younger patients [22].

Nevertheless, to confirm a diagnosis of CF, it isnecessary to obtain evidence of CFTR dysfunctionthrough the identification of two CFTR gene muta-tions previously assigned as CF disease causing,two tests showing a high Cl� concentration insweat (>60 mEq L�1), distinctive transepithelialnasal potential difference measurements and/orassessment of CFTR (dys) function in native colonicepithelia ex vivo [4, 12, 13]. For individuals withsymptoms suggestive of CF but intermediate sweatCl� values (30–60 mEq L�1), the need for addi-tional proof of CFTR function (through NPD mea-surements or CFTR functional assessment in rectalbiopsies) is particularly important.

More recently, the diagnostic paradigm has chan-ged with the widespread introduction of neonatalscreening programmes which often identify CFpatients before they develop any classical CFsymptoms. For these programmes, it is essentialto (i) confirm/exclude the CF diagnosis in a timelymanner, (ii) achieve this with a high degree ofaccuracy to avoid excessive testing (and the asso-ciated costs), (iii) provide accurate prognosticinformation and genetic counselling and, mostimportantly, (iv) deliver appropriate treatmentand ensure early access to specialized CF referencecentres with multidisciplinary specialized services[12, 23].

The search for new and reliable CF biomarkerscontinues and is likely to provide results in theshort term. This is important not only to establish adiagnosis of CF but also to monitor clinical trials ofnew CFTR modulating drugs [24, 25].

The CF gene and CF disease: a paradigm for rare disorders

Despite the apparent ‘simplicity’ of being mono-genic (and ‘simplification’ of the name to ‘65 roses’by children with CF [26]), CF still poses manydiagnostic challenges and its clinical managementis not straightforward. CF is in fact a complexdisorder for several reasons. First, as mentionedabove, almost 2000 CFTR gene mutations have sofar been reported [20], and this is further compli-cated by the presence of ‘complex alleles’, that isthose containing more than one CFTR mutation[27]. Secondly, the CFTR genotype often poorly

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

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

Obstructive azoospermia

Metabolism Hypoproteinaemia

Fat-soluble vitamin deficiencies

Salt-loss syndrome with salt

depletion, with or without

metabolic alkalosis

Data from [5, 11, 12, 23, 46].


ª 2014 The Association for the Publication of the Journal of Internal Medicine 157Journal of Internal Medicine, 2015, 277; 155–166

predicts the full spectrum of the clinical pheno-types and consequences in multiple organs [28];for example, chronic lung inflammation and infec-tion, which play a major role in the course of therespiratory disease, are highly influenced by envi-ronmental factors such as tobacco smoke andoutdoor pollution and the bacterial microbiome ofthe lung [29, 30]. Thirdly, an increasing numberof CFTR mutations are associated with isolateddisease characteristics such as chronic pancrea-titis, chronic sinusitis, disseminated bronchiecta-sis or male infertility; the distinction betweenthese CFTR-related disorders (CF-RD) [31, 32] (or‘CFTR-Opathies’ [33]) and CF (especially in itsmilder forms) is not always straightforward.Fourthly, several modifier genes have been identi-fied but how these genes influence disease is notalways clear [34–36]. Finally, it has been suggestedthat CFTR plays several other roles in the cell,regulating (directly or indirectly) other cellularproteins and functions [37, 38]. It is still unclearwhether such ‘secondary’ functions are regulatedby CFTR itself or by other channels which in turnare regulated by CFTR [39]. Of note, it remains to bedetermined whether correction of the primary func-tion of CFTR as an anion channel will also restorethese additional ‘secondary’ functions.

Such complexity makes CF a devastating diseasestill hard to manage at its root. Nevertheless,despite being a ‘simple’ monogenic disease, CFhas given many lessons of complexity and biomed-ical science has gained much from CF research. Inmany respects, CF is indeed a paradigmatic mono-genic rare disease, greatly contributing to theadvancement of both biomedical science and clin-ical practice. As Jack Riordan, who together withLap-CheeTsuiandFrancisCollinsmade theoriginalCFTR gene discovery, stated in a recent publicationfor the 20th anniversary of the discovery, ‘Thedisease has contributed muchmore to science thanscience has contributed to the disease’ [40].

The knowledge gained during the long researchpath to the discovery of the basic defect in CF mayaccelerate the pace of translational medicine forfuture gene discovery, as hard work and mistakesmade in this field may contribute to advances inother areas. For new genes recently found to becausal for other diseases, those 20 years ofresearch can be largely reduced because of workso far done on CF [40]. Some of these highlights aredescribed below.

Geneticists have been interested in the generesponsible for CF as the disease was first identi-fied in the 1930s [41]. Its final discovery in 1989was a major breakthrough, establishing theground-breaking human gene cloning techniquesof chromosome jumping and chromosome walking[42]. The major goal of identifying the CF gene wasto be able to move quickly to gene therapy. Theconcept of giving patients a correct copy of the CFgene, so that they could produce the functionalprotein, was simple and elegant. Thus, gene ther-apy for a monogenic disorder was pioneered for CF[43]. However, gene therapy trials soon demon-strated how difficult it is to express foreign genes inthe lung; this was a ‘lesson’ for other disorders [44].Although gene therapy trials for CF have come to ahalt at present, knowledge gained from the pre-clinical and clinical gene therapy studies hasinformed protocols and efficacy end-points forfurther novel therapeutic approaches [45, 46].

The large number and variety of CFTR gene muta-tions led to the creation of a widely used globalrepository of mutations by an international con-sortium [20]. Nevertheless, determining the geno-type–phenotype correlations proved to be difficult[47–49]. Moreover, such widespread genetic testingis of limited value, given the substantial number ofDNA variants of uncertain pathogenic significance.Therefore, the next challenge in the CF field was toidentify the molecular and cellular dysfunctioncaused by these gene mutations (see below).Although still far from accomplished, this has beenachieved for the most common mutations [50] andthe CFTR2 (Clinical and Functional TRanslation ofCFTR website) database has been established todisseminate these findings [51].

Along the path to finding a cure for CF, manyadvances in basic biology have provided a betterunderstanding of the pathophysiology of the dis-ease [8], including, notably, the mechanisms ofprotein folding, pathways of secretory traffic andthe physiology of epithelial channels (reviewed in[39]). Therefore, CF has become a paradigm fortrafficking disorders and other ‘channelopathies’.Knowledge in the field of CF may thus translateinto understanding other rare/orphan diseases, inparticular those sharing a similar basic defect [52].Similarly, findings may also apply to other majorrespiratory diseases in which CFTR has been foundto play a role such as in chronic obstructivepulmonary disease (COPD) [53–56] or asthma[57–60], both of which are currently increasing in



prevalence worldwide. Therefore, it is expected thatCFTR-modifying drugs (see below) may also benefitpatients with these common disorders.

The generation of animal models for CF also led thefield of animal disease models through the appli-cation of the gene targeting principle. This endeav-our, that is, generation of CF mouse models,involved the three 2007 Nobel Prize awardees inthis field [61–63].

Finally, the development of protein conformationchange-inducing drugs, which has been a modelfor drug discovery in rare diseases, has been led byresearch in the field of CF. This programme was theresult of the innovative association between ahighly committed CF patients association (CFF-Cystic Fibrosis Foundation, USA) and the pharma-ceutical industry (Vertex Pharmaceuticals) [46,64]. The Foundation started work several decadesago by (i) funding research that led to the discoveryof the gene in 1989 [42, 65, 66], (ii) building anextensive patient registry and a clinical trial net-work, both of which were required for investigatingCF genetics and (iii) efficiently recruiting partici-pants for trials of investigational drugs [64]. TheFoundation also funded a focused multidisciplin-ary scientific effort to understand the molecularbasis of this disorder [40, 67]. The ambitious goalwas to systematically investigate the basic CFdefect(s) using small molecules (see below). Thedrug discovery programme that followed has beenexemplary in many respects [46]. By 2009,20 years after the discovery of the CF gene, it wasanticipated that innovative life-changing treatmentfor CF would soon be available [67]; this in factbecame a reality in 2013. This experience haspaved the way for personalized medicine in othergenetic diseases [46].

Functional classification of mutations

In the years after the discovery of the CFTR gene,there were considerable advances in the under-standing of the structure and function of the CFTRprotein: a complex multidomain 1480-amino acidmembrane protein that is the only member of theATP-binding cassette (ABC) transporters familythat functions as an ion channel [39]. However,the major difficulty is tackling the almost 2000CFTR mutations so far described [20].

To correct for such a variety of gene and proteindefects effectively, CFTR mutants are grouped into

functional classes in which the same restorativestrategy may be effective; this approach has beentermed ‘mutation-repairing therapy’ [18]. The firststep is thus to identify the basic molecular andcellular defect underlying each individual muta-tion. This was the goal of the CFTR2 project [51]which evaluated, in terms of both clinical severityand functional consequences, the most commonCFTR gene mutations (i.e. those with an allelefrequency of ≥0.01%) altogether accounting for96.4% of CF alleles, in a multicentre internationalcohort of almost 40 000 patients (i.e. 57% of theestimated 70 000 individuals with CF worldwide)[50]. The complete list of mutations studied so far(177 in July 2013) is available online [51]. Toachieve the aim of the CFTR2 project, molecularand functional characterization of many CFTRmutations was carried out thus enabling classifi-cation of each of these mutations into one of theestablished six functional classes [8, 18, 19, 68,69] (Fig. 2).

Class I (no protein) comprises mutations thatproduce premature termination signals becauseof splice site abnormalities, frameshifts due toinsertions 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 tothe cell surface (i.e. to the correct CFTR cellularlocation), due to misfolding and premature degra-dation by the endoplasmic reticulum (ER) qualitycontrol (ERQC) (reviewed in [70]); this classincludes the most common mutation, F508del.Class III (no function) comprises CFTR mutantsthat, although reaching the plasma membrane,exhibit defective channel gating (i.e. the channelpore does not open) due to impaired response tochannel agonists; an example of this class isG551D. When F508del-CFTR is promoted to reachthe plasma membrane by corrector compounds, itstill has a partial gating defect, and thus, it canalso be in Class III. Class IV (less function) mutantsdisplay substantially reduced conductance (i.e.flow of ions through the CFTR channel pore), witha resulting decrease in net Cl� channel activity;R334W, a mutation in the CFTR channel pore, isan example. Class V (less protein) includes mainlyalternative splicing mutants (e.g. 3272-26A>G [71,72]) that allow synthesis of some normal CFTRmRNA (and protein), albeit at very low levels [73];this class also includes promoter mutations thatreduce transcription (e.g. -94G>T [74]) and aminoacid substitutions that cause inefficient protein



maturation (e.g. A455E [75]). Finally, Class VI (lessstability) mutants impair the CFTR plasma mem-brane stability [76]; an example of this class isc.120del23 which lacks the cytoskeleton-anchor-ing N-terminus of CFTR [77]. When rescued to thecell surface, F508del-CFTR also behaves as a ClassVI mutant due to its intrinsic instability [78, 79].

F508del has multiple defects and is thus includedin Classes II, III and VI; this illustrates a limitationof this CFTR mutation classification. Applyingrescuing strategies to achieve full correction ofsuch mutants (see below) is therefore complex, asmore than one type of CFTR-modulator drug will

probably have to be used [19]. Another limitation ofthis classification arises from the fact that themajority of mutations have not been assigned to amutation class due to the absence of functionalstudies. Although the most common mutationshave been assigned into a functional class [50],much work regarding functional characterizationof CFTR mutations remains to be done if CFTRmodulating therapies are to reach 100% of CFpatients (see below). This becomes even morerelevant given the increasing number of novelvariants identified in full gene screening protocolsby next-generation sequencing as part of severalneonatal CF screening programmes [80, 81].

Cl–Cl–Cl– Cl– Cl–

Cl–

Cl–

Cl–

Cl–Cl–

Cl–

Cl–

Cl–

Cl–

Cl– Cl–

Cl–

Cl–

Cl–

Cl–

WT-CFTR

CFTR

I II III IV V VI

CFTR defect type:

No protein No traffic No function Less function Less protein Less stable

Mutationexamples

Corrective therapy

Drug

G542X (a)W1282X (a)1717-1G (b)

F508delN1303KA561E

G551DS549R

G1349D

R117HR334WA455E

A455E3272-26A>G

3849+10 kb C>T

c.120del23rF508del

Rescuesynthesis

Rescuetraffic

Restorechannelactivity

Restorechannelactivity

Correctsplicing

Promotestability

Read-throughcompounds

Drug approved (Yes/No) No No No No NoYes

Correctors Potentiators PotentiatorsAONs

CorrectorsPotentiators

Stabilizers

Fig. 2 Classes of CFTR gene mutations. The aim of stratification of CFTR mutations into functional classes is to apply thesame therapeutic correction for the basic defect in each class. The strategies, the types of molecules required to achieve suchstrategies and their current status of clinical approval are shown. CFTR, cystic fibrosis transmembrane conductanceregulator; WT, wild-type; Antisense oligonucleotide, (AON).



Correcting the basic CFTR defects: personalized therapies

The major advantage of the above classification ofmutations is the possibility of adopting the sametherapeutic strategy for each class [82] (see Fig. 2).Such strategies may even apply to the correction ofsimilar defects causing other diseases that sharethe same basic molecular defect [8]. The design andoutcomes of the clinical trials discussed here arereviewed in detail elsewhere [82].

Class I agents

As many Class I mutations (Class Ia) lead to thecomplete lack of CFTR protein due to the presenceof a PTC, compounds promoting the read-throughof stop codons by the ribosome have been investi-gated for these mutants. Compounds that achievethis goal are the aminoglycoside antibiotics (gen-tamicin and tobramycin) [83], which have alsobeen proposed to correct PTCs in other geneticdisorders such as Duchenne muscular dystrophy(DMD) [84] and cancer [85].

Another compound, ataluren (formerly PTC124),was also shown to lead to some degree of ‘over-reading’ of PTCs and thus entered clinical trials;however, no improvement in the primary end-point, lung function, was observed [86]. Clinicaltrials have so far also shown limited efficacy ofataluren in patients with DMD with nonsensemutations (nmDMD) [87]. Accordingly, this drugis not approved by the US Food and Drug Admin-istration (FDA), although in the EU, it has receivedconditional marketing authorization for patientswith nmDMD aged 5 years and above [82]. None-theless, further optimization of read-through com-pounds is required to achieve significant clinicalefficacy.

For other Class I mutants (Class Ib), involvingsmall deletions or insertions that cause frameshiftmutations during protein production (Fig. 2), thereis at present no therapeutic strategy except per-haps the so-called bypass therapies which targetother (non-CFTR) channels (see below).

‘Correctors’ for Class II mutants

Mutations in Class II fail to traffic to the cell surfaceand are mainly retained at the ER [88]. As the mostcommon CF-causing mutation (F508del) isincluded in this class, it has been the focus ofmajor efforts to elucidate the molecular mechanism

of F508del-CFTR dysfunction. Due to inefficientfolding, this mutant acquires an abnormal confor-mation which is recognized and retained by the ERquality control (ERQC) that targets it for prematuredegradation [19]. Proof of concept that F508del-CFTR could be rescued to the cell surface camefrom initial studies showing that this could beachieved by incubation at low temperatures [89].Several compounds, chemical chaperones such asglycerol or TMAO that promote protein folding, alsoshowed similar but nonspecific effects [70].

The identification of compounds that rescuemutant CFTR in a specific way resulted fromhigh-throughput screening (HTS) for drug discov-ery [90, 91]. These screening studies led to theidentification of CFTR modulators: ‘correctors’ thatrescue the trafficking defect of F508del-CFTR and‘potentiators’ that stimulate channel gating. Todate, the most successful corrector is the investi-gational drug lumacaftor (VX-809) [92] which,despite very promising results in primary cells[92], only promoted a significant decrease in sweatCl� levels and no effect on lung function in F508delhomozygous patients during a Phase II clinical trial[93]. Currently, lumacaftor and VX-661 (a second-generation corrector) are being tested in a clinicaltrial in combination with the potentiator ivacaftor(Kalydeco; previously VX-770) (see below). Interimresults from a Phase II trial of VX-661 and ivacaftorshowed a modest but statistically significantchange in sweat Cl� levels as well as a small andvariable but also significant improvement in lungfunction at day 28 in F508del-homozygouspatients [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 andthe European Medicines Agency of the first drug totarget mutant CFTR was met with great enthusi-asm and optimism in the CF community. The CFTRpotentiator ivacaftor was approved for clinical usein individuals with G551D, although this mutationis only present in ~4% of CF patients. Morerecently, following demonstration of in vitro effec-tiveness [96], ivacaftor was approved by the FDAfor another eight gating (Class III) mutationswhich, together with G551D, account for ~5% ofall CF patients. Potentiators such as genistein andrelated flavonoids can also activate Cl� conduc-tance and thus overcome the gating defects ofClass III mutants [97–99].



For Class IV mutants, compensation for reducedionic flow may be achieved by potentiators. Simi-larly, correctors could increase the overall cellsurface density of these mutants. However, thisassumption requires in vitro demonstration ofefficacy.

Agents that rescue Class V mutants

As many Class V defects are the result of alterna-tive splicing, increasing the levels of splicingfactors that correct such missplicing may consti-tute an effective therapeutic strategy for CFpatients with these mutations (i.e. ~10% of allpatients with this disease). Although which factorsrequire manipulation to correct mRNA splicing isstill unclear, recent advances in the delivery of‘splice switching’ oligomers to cells appear to bepromising [100]. Meanwhile, the approved potenti-ator ivacaftor, with proven efficacy on wild-type-CFTR, is also likely to provide benefit for patientswith Class V mutations; however, this requiresconfirmation.

Restoring mutations in Class VI

Compounds that enhance CFTR retention/anchoring at the cell surface will benefit Class VImutants that have intrinsic plasma membraneinstability. This is the case for F508del-CFTRwhen rescued to the plasma membrane by novelsmall-molecule correctors [101], which can par-tially account for the limited success of clinicaltrials with correctors. F508del-CFTR stabilizersinclude activators of Rac1 signalling, such ashepatocyte growth factor (HGF), which promoteanchoring to the actin cytoskeleton via NHERF1[102].

Correcting the basic CF defect in all CF patients: still an unmetneed

Despite the great breakthrough in CF drug devel-opment with the licensing of the first CFTR mod-ulator drug, ivacaftor still only targets ~5% of CFpatients. Thus, there is still an unmet need toeffectively treat the remaining ~95% of CF patients.Treatment for Class II mutations should be avail-able in the 2–3 years, whereas Class I mutationtreatments might take longer to develop [103].Importantly, however, only ~40% of patients areF508del-homozygous, and the efficacy of correc-tors in patients with only one F508del allele isexpected to be even lower than in homozygotes.

Moreover, at least 15% of all CF patients lackF508del in both alleles and thus will not benefitfrom F508del correctors.

The aim of bypass therapies is to manipulate other(non-CFTR) ionic conductances to restore the ionichomeostasis of the epithelia and correct the fluidand pH imbalance in CF. The great advantage ofthis therapeutic approach is that it is equallysuitable for all patients with CF, irrespective oftheir mutations. This approach can be achieved bynormalizing the hyperabsorption of Na+ that occursin CF epithelia [104] through ENaC directly orindirectly [105] or through activation of alternativeCl� channels, particularly the anoctamins, a familyof Ca2+-activated Cl� channels (CaCCs) [106].Stimulation of basolateral K+ channels could alsoincrease the driving force for Cl� secretion andhence favour CFTR-mediated secretion in patientswith residual CFTR function [107].

These innovative therapeutic approaches,although very appealing, may take years to reachthe clinic. Thus, it is important to find effectivemeans of testing the response of rarer mutations tothe approved drug ivacaftor (and new CFTR mod-ulators), in order to treat a wider population of CFpatients.

Ivacaftor may indeed be one of many therapeuticagents that point to the emergence of a new era ofpersonalized medicine. Rare mutations could betested in modified single-patient (‘n-of-1’) trials[108], in which the subject is exposed to treatmentover variable blinded time periods, and outcomeparameters are measured repeatedly to compareoutcomes during ‘on’ versus ‘off’ drug periods[108]. However, treatment effect and drug efficacyare hard to prove. There is thus an urgent unmetneed to test the efficacy of emerging CFTRmodulators directly on patients’ tissues ex vivo toidentify responders who will benefit from theseinnovative therapies. Not unsurprisingly, CFpatients associations support such approaches toaccelerate the process of bringing these new drugsto a greater number of patients [25]. Bioelectricmeasurements of CFTR (dys)function in native (orcultured) tissues from CF patients have beenproposed as the basis of personalizing therapies[25], as well as for CF diagnosis and prognosis [13,109]. These are original paradigmatic technologicaldevelopments in science that offer new promise fordeveloping targeted therapeutics and tools forpredicting those patients who will respond to a



medical therapy and those who will experience noeffects at all, without actually taking the drug.

Conclusions

The aims of personalized medicine are to assessthe medical risks and monitor, diagnose and treatpatients according to their specific genetic com-position and molecular phenotype. It has beenshown for CF as a paradigmatic disease that usingthe genetic variations to predict clinical phenotypeis not easy. Indeed, even this ‘simple’ monogenicdisease is influenced by a large number of differ-ent genes and biological pathways as well as byenvironmental factors that are difficult to assess.Furthermore, every person with CF is unique andrequires personalized diagnosis. It is not becauseit is a monogenic disorder that personalizedtreatment for CF is much easier than for diabetes,neurological disorders, cancer or other diseasesinvolving a large number of different genes andbiological pathways [110]. Consequently, thecombined knowledge of gene variants along witha functional assessment of responses ex vivo willbe crucial for predictive personalized treatment ofCF.

Conflict of interest statement

MDA has served as a consultant to Vertex andGalapagos, has been supported to attend andspeak at symposia by Novartis, Gilead and Vertexand participated in an educational grant pro-gramme by Facilitate Ltd.

Acknowledgements

Work in the author’s laboratory has been sup-ported by strategic grant PEst-OE/BIA/UI4046/2011 (to BioFIG) and research grants PTDC/SAU-GMG/122299/2010 (to MDA) from FCT/MCTES,Portugal; CFF-Cystic Fibrosis Foundation, USA,(Ref. 7207534), Gilead G�ENESE-Portugal Pro-gramme (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]).



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