REVIEW URRENT C OPINION

Recent advances in cystic fibrosis Carlos E. Milla a and Richard B. Moss b

Purpose of review The field of cystic fibrosis (CF) continues to evolve at a fast pace thanks to novel observations that have enabled deeper understanding of the disease pathophysiology. Parallel groundbreaking developments in innovative therapies permit, for the first time, distinct disease modification. Recent findings This review highlights important discoveries in fluid homeostasis and mucus secretion in CF that further informs the pathophysiology of the airway disease that characterizes CF. In addition, current concepts and novel paradigms, such as ‘theratypes’ and ‘CF transmembrane conductance regulator chaperome’, which will be important for the continued development of disease modifying therapies, are reviewed. Summary The rate of progress in the field continues to accelerate with new knowledge informing the development of innovative therapies. This has already led to tangible substantial and unprecedented clinical benefit for selected subsets of the CF patient population. In the years ahead, further knowledge acquisition may motivate the extension of these benefits to the larger population of people with CF. Keywords cystic fibrosis, disease modification, theratypes

INTRODUCTION Cystic fibrosis (CF) is a life-shortening autosomal recessive disease caused by mutations in the gene encoding the CF transmembrane conductance regulator (CFTR), a protein with anion channeling properties that participates in electrolyte homeostasis and fluid movement across mucosal surfaces [1]. At the surface epithelium of the respiratory tract, gastrointestinal tract and exocrine gland ducts, the presence of functional CFTR is critical. Although a multisystemic defect, the morbidity and mortality associated with CF primarily centers on the pulmonary manifestations. Although great progress has been made since CF was initially identified as a distinct clinical entity [2,3], progress was considerably accelerated when the CFTR gene was cloned and localization of the normal CF protein to the surface of mucosal epithelium occurred 25 years ago [4]. This allowed an explosion of knowledge that identified important aspects of the basic mechanisms of the disease, as well as pinpointing potential targets for intervention [5]. In parallel, this also permitted the development of the necessary tools for drug development aimed at the basic defect. In this review, we will elaborate on the recent advances in the understanding of the disease pathophysiology and the therapeutic developments that are

leading us into an era of disease modification for patients with CF.

CURRENT STATUS: WHERE ARE WE NOW Without question from the time of the CFTR gene cloning, we have witnessed an incremental and progressive increase in the fundamental understanding of the disease pathophysiology in CF. This has been aided greatly by the development of transgenic murine [6] and recently large mammal CF animal models [7–9]. A striking defect in mucociliary clearance is a hallmark of CF that results in an airway environment prone to chronic bacterial infection and severe inflammation [10]. Although there has long been strong focus on defective Cl
and fluid movement at the airway surface as a key a The Stanford Cystic Fibrosis Center, Palo Alto and bCenter for Excellence in Pulmonary Biology, Stanford University School of Medicine, Stanford, California, USA

Correspondence to Carlos E. Milla, MD, Center for Excellence in Pulmonary Biology, Stanford University School of Medicine, 770 Welch Road, Suite 350, Palo Alto, CA 94304, USA. Tel: +1 650 723 8325; fax: +1 650 723 5201; e-mail: [email protected] Curr Opin Pediatr 2015, 27:317–324 DOI:10.1097/MOP.0000000000000226

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KEY POINTS  The role of bicarbonate and mucus abnormalities in the pathophysiology of cystic fibrosis is now well established.  The understanding of the complexity of microbial infections continues to evolve with the use of molecular tools.  The disease modifying effects of the first drug approved as a CFTR modulator are now well demonstrated.  The ‘theratype’ paradigm, that is the concept of targeting specific CFTR functional defects with the application defect-specific CFTR modulators, should lead to further advances in CF therapeutics.  Therapeutics for CF in the near future is likely to include combination drug regimens to achieve additive and even synergistic effects in the restoration of CFTR function to maximize clinical benefit.

secretion: a cyclic adenosine monophosphatedependent current proven to be CFTR mediated and a purinergic-dependent current mediated by a calcium-activated chloride channel. This finding provides further support for the notion that CFTRmediated HCO3
secretion plays a crucial role in the proper unfolding and release of mucins [24,25,26 ]. Additional evidence for a primary defect in the clearance of mucus in the airway independent of airway surface fluid volume has been provided by two recent CF pig studies. In CF pig tracheas, submucosal glands secreted mucus strands that remained tethered to gland ducts, and this defect could be replicated in airways from healthy pigs by blocking anion secretion [27 ]. In the second study, direct measurements of airway surface fluid depth and mucus clearance demonstrated that in CF mucus viscosity was increased in situ as a result of compromised bicarbonate transport, suggesting that the defect in mucus clearance in CF airways occurs even in the presence of adequate hydration [28 ]. This cumulative evidence implicates defective release and transport of mucus as a primary defect in CF due to defective HCO3
secretion in the small airways as a consequence of CFTR dysfunction. This compromises the patency of the small airway lumen, and prompts small airways collapse and accumulation of viscous secretions to set the stage for chronic infection and inflammation. Moreover, acidification of the airway surface fluid (absent bicarbonate secretion) may be involved in the failure to clear infection. In newborn pigs, the airway surface fluid rapidly kills bacteria in vivo, and this is defective in CF pigs. Simply reducing the pH of the surface fluid impairs its antimicrobial activity, and increasing the pH rescues the antibacterial activity in CF airways [29]. Although direct evidence for this mechanism is lacking in human airways, studies in infants with CF have noted lower pH in exhaled breath condensate (a surrogate for lower airway fluid composition) compared with healthy controls [30]. Further evidence for a role of airway surface pH in disease pathogenesis derives from the link between epithelial sodium channel (ENaC) hyperactivity and CFTR dysfunction wherein the pH sensitive secreted peptide Short Palate Lung and Nasal Epithelial Clone 1 (SPLUNC1) modulates ENaC activity [11]. Thus, in the acidic CF airway environment, SPLUNC1 is deactivated, which increases ENaC activity to cause airway surface fluid hyperabsorption [31 ]. As a result of compromised CFTR function, the airway lumen becomes overburdened with bacterial and fungal microbes, as well as by large concentrations of inflammatory mediators. Animal model and human epidemiologic studies provide evidence &

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pathogenic event [11], a growing body of evidence suggests that defects in bicarbonate (HCO3
) and mucus secretion [12] play an important pathophysiologic role. The earliest systematic descriptions of the lung disease in young children with CF pointed to focal changes at the small airway level [13,14], and this has had a well-recognized functional correlate in abnormalities noted in pulmonary function studies of young children with CF [15]. The first detailed pathologic studies of the lungs of deceased infants noted the presence of bronchial gland hypertrophy, and mucus plugging and dilatation of the small airways’ lumen by the inspissated secretions [16]. Work on bronchial glands pioneered by Wine et al. [17] at Stanford has provided evidence for fundamental defects in glandular secretions that result in impaired clearance and disruption of an important airway innate defense mechanism. Work with bronchial glands obtained from explanted CF lungs has provided direct evidence of defective secretory function and abnormalities in the viscosity and elasticity of the mucus produced in CF bronchial glands [18–21]. However, how this relates to the small airway disease identified in patients was difficult to establish given technical limitations in the handling of such small airways (1–2 mm luminal diameter) for in-vitro studies. Taking advantage of a novel system developed to study ion transport directly in small airway preparations [22], Shamsuddin and Quinton [23 ] were recently able to demonstrate HCO3
transport in small airways in response to agonists. This work identified two independent pathways that result in HCO3
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that this process likely starts early after birth, one best defined as an inability to clear microorganisms instead of a propensity to acquire infection. A chronic, progressive process slowly destroys the airways and often results in severe lung dysfunction and death from respiratory failure. Infection with Pseudomonas aeruginosa, the prototypical pathogen associated with CF morbidity, elicits and drives a persistent exuberant inflammatory response that damages the airways. A plethora of other microbial pathogens, such as Staphylococcus aureus, Haemophilus influenzae, Burkholderia cepacia, Stenotrophomonas maltophilia, Achromobacter xylosoxidans, Aspergillus spp. and nontuberculous mycobacteria, can also be involved in this complex process. Informed by the knowledge of this vicious cycle of events, therapeutic developments over the last 3 decades endeavored to curtail the progression of the lung disease. Therapies proven of benefit to CF patients have been primarily focused on facilitating clearance of secretions (dornase alfa [32]), controlling chronic Pseudomonas infections (inhaled tobramycin [33] and inhaled aztreonam [34]), and rehydrating the airway surface (hypertonic saline [35] and mannitol [36]). Despite these advances, morbidity and mortality associated with CF continues to be a result of its pulmonary manifestations and its progressive nature, so the therapeutic armamentarium developed has produced at best modest disease modifying effects, likely due to the fact that the vicious cycle of airway obstruction by secretions, chronic infection and severe inflammation occurs downstream from the basic genetic defect and the resultant pathophysiologic mechanisms discussed above. Additionally, the clinical response to the seemingly most appropriate antibiotic regimens based on laboratory sensitivity tests cannot be fully predicted, and tremendous variability exists from patient to patient [37]. Conversely, it is a common clinical observation that antibiotic therapy may confer benefit even in the context of continuing infection with antibioticresistant organisms. This apparent paradox is likely the result of a complex multifactorial process in which the conditions of the airway environment, including the host inflammatory response, and the characteristics of the offending microorganisms, including phenotypic changes with reduced expression of virulence genes and adaptive mechanisms, determine disease manifestations [38]. However, this paradigm is still based on the presumption of a sole causative (or at least dominant) microorganism and ignores the possibility of a more complex microbial composition in the airway. The availability of nonculture-based microbial identification methodologies has opened the possibility of performing a more comprehensive assessment of the

microbial ecology in different niches of the human body, referred to as the microbiota. The application of microbiologic identification tools based on 16s ribosomal RNA gene analysis to better characterize the CF airway microbiota has revealed a highly complex ecology. In parallel, evidence for the presence in the lower airway of non-CF healthy individuals of low levels of many bacterial taxa challenged the commonly held notion of a sterile lower airway [39]. This suggests that bacteria are normally present in the lower airway but are also likely effectively cleared, a condition that is clearly defective in CF patients; this parallels the observations made in transgenic CF pigs. However, it remains to be demonstrated what relationship the development and composition of microbial communities in the CF airway have to the progression of lung disease. Given the large variability seen in lung disease manifestations among CF patients, this also opens the door for the possibility of certain bacterial communities offering a protective role from disease progression.

WHERE WE ARE GOING Deeper understanding of CFTR structure and function at the molecular level has led to the development of a revolutionary pipeline of small molecules aimed at restoring CFTR function [40 ]. This has been grounded in the realization 2 decades ago that the application of molecular tools at the cellular level was required to craft groundbreaking therapies. As a consequence, perhaps one of the most remarkable advances in CF pertains to the application of highthroughput technology to screen cell cultures for activity of exogenous small molecules on defective CF chloride transport. The ability to rapidly evaluate thousands of chemical compounds permitted testing the concept, proposed more than 2 decades ago, that restoring function could be accomplished by rescuing defective CFTR protein from degradation and by enhancing its activity in the cell membrane. Broadly speaking such molecules can be termed CFTR modulators. Some modulators, termed correctors, work by improving the production of CFTR protein and others, termed potentiators, by improving CFTR’s anion transport function. Figure 1 shows the general structure of CFTR predicted from its primary amino acid sequence and largely confirmed by subsequent empirical study [41,42]. With now close to 2000 mutations identified in patients affected by CF, a mechanism-based classification of CFTR mutations into specific functional defects [43] has been a crucial construct in understanding CF pathophysiology and CFTR modulator treatment goals (Fig. 2) [44]. Class I mutations result in premature stop codons or affect

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Extracellular

MSD1

MSD2

N-terminus

R domain

Intracellular

ATP

ICL1 F508del ICL4 NBD1

PKA ICL2 ICL3 NBD2 C-terminus

FIGURE 1. Theoretical domain organization of CFTR at the apical membrane based on analysis of crystal structures and structural homology models. PKA, by hydrolyzing ATP, phosphorylates the R domain to facilitate channel opening. Also shown is the location of the phenylalanine residue at position 508 in NBD1, where the most prevalent mutation F508del occurs, which places it in proximity to ICL4 and destabilizes the interface between this ICL and NBD1. MSD1 and MSD2 (dark and light green, respectively); NBD1 and NBD2 (light and dark pink, respectively); R (black); ICL1, ICL2, ICL3 and ICL4 (red, yellow, bright green and blue, respectively). ATP, adenosine triphosphate; CFTR, cystic fibrosis transmembrane conductance regulator; ICL, intracellular loop; MSD, membrane spanning domain; NBD, nucleotide-binding domain; PKA, phosphokinase-A; R, regulatory domain. Adapted with permission from [41].

canonical splice sites, leading to a lack of CFTR protein. Class II mutations affect CFTR production through protein folding defects, with nascent mutant protein retained in the endoplasmic reticulum (ER) and degraded by cellular ER quality control mechanisms instead of trafficking to the apical cell membrane. Class III mutants produce a regulatory defect that reduces channel open probability (gating defect). Class IV mutants reduce ion flow through the channel (conduction defect). Class V mutants reduce quantity of mRNA or protein, usually via alternative splicing. Class VI mutants result in instability of CFTR at the plasma membrane. A shortcoming of this classification scheme is the occurrence of multiple functional (class) defects caused by a single mutation. For example, F508del is known to produce a folding and trafficking defect, but if rescued it also demonstrates membrane gating and stability defects; R117H causes both conductance and gating defects and, depending on upstream polythymidine and TG repeat tracts, a variable splice defect influencing protein abundance. 320

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It is now clear that functional assessment of individual CFTR mutations is necessary to evaluate the response achievable by treatment with a single modulator or a combination of modulators (which might involve evaluating permutations of individual correctors and potentiators) [45]. The template for this approach has been demonstrated and validated in the extension of FDA approval for ivacaftor (Kalydeco; Vertex Pharmaceuticals, Boston, Massachusetts, USA), a CFTR potentiator, from its initial approval for treatment of G551D-CFTR (a gating defect) to 8 additional gating and R117H mutations; initial in-vitro analysis of the mutationspecific drug effect in a mutant CFTR cell expression system was followed by a phase 3 clinical trial guided by those results [46,47] (Moss RB, Flume PA, Elborn JS, et al., unpublished observation). In addition to ivacaftor, novel CFTR potentiators have been identified, including one that has entered clinical trials [48–50]. Groups of mutations that display similar CFTR functional defects and respond similarly to defectspecific CFTR modulators can be considered in principle as ‘theratypes’, forming distinct genotypebased target populations for treatment with specific individual or combination CFTR modulators [51 ]. In the theratype approach, measures of CFTR function can be made using a variety of in-vitro, ex-vivo cell culture and in-vivo measures. Some of these (e.g., sweat chloride, nasal potential difference) have been employed for decades and standardized with uniform operating procedures for multicenter clinical trials [52,53], whereas others (e.g., Ussing chamber intestinal current measures of anion efflux in rectal biopsies, or CFTR protein expression in epithelial cells obtained by nasal brushing) require further validation and standardization for widespread use [54,55]. In addition to cell culture-based CFTR functional assays, new measures directly assessing CFTR function in vivo are being developed. The sweat gland is an easily accessible target organ, and quantitative measures of sweat rate under pharmacologic stimulation yield an estimate of CFTR function that could be utilized to assess response to CFTR modulators [56,57]. Additional, and perhaps more indirect, in-vivo measures of CFTR function include mucociliary clearance, intestinal pH, microbiology and structural airway changes [58 ,59,60]. The theratype paradigm offers an opportunity for true personalized medicine for people with CF, but it is not absolute. It is very well appreciated that large phenotypic and treatment differences exist between individuals who share the same CFTR genotype. At the individual level, novel predictive assays based on ex-vivo assessment of CFTR function &&

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CFTR defect type: WT-CFTR –

– – 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

CFTR –

Cl

–

Cl

–

–

Cl

Cl

–

Cl

–

Cl

–

Cl

–

Cl

Mutation examples

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

F508del N1303K A561E

G551D S549R G1329D

R117H R334W A455E

A455E 3272-26A>G 3849+10kb C>T

c. 120del23 rF508del

Corrective therapy

Rescue synthesis

Rescue traffic

Restore channel activity

Restore channel activity

Correct splicing

Promote stability

Read-through compounds

Drug

Drug approved (yes/no)

No

Correctors

Potentiators

No

Yes

AONs Potentiators correctors potentiators No

No

Stabilizers

No

FIGURE 2. Functional classification of CFTR mutations with examples of more prevalent mutations of each class, theratype approach to small molecule pharmacotherapy and regulatory status of drugs in each category. AON, antisense oligonucleotide; CFTR, cystic fibrosis transmembrane conductance regulator; WT, wild type. Reproduced with permission from [44].

have great potential in this regard. Epithelial cell samples from the gastrointestinal or respiratory tracts (rectal biopsies or nasal scrapings) can be relatively easily obtained and expanded to provide ongoing ex-vivo testing of CFTR functional impairment and drug response that is specific to the individual patient [61,62,63 ]. Such ex-vivo assessments have revealed differences in responses to a CFTR modulator between CF patients with identical CFTR genotypes, demonstrating a proof of concept for the ability to distinguish both CFTR-dependent and independent interindividual differences, as well as responsiveness to CFTR modulation [64]. In approaching the treatment of CF from a theratype perspective, the major target is the F508del mutation, as it is by far the most prevalent mutation causing CF (i.e., homozygous in 50% of CF patients with another 40% heterozygous for a different CFTR mutation in trans). The F508del mutation occurs in the cytosolic first nucleotidebinding domain (NBD1) (Fig. 1). Although described in 1990 as a protein folding defect that results in a lack of proper trafficking to the normal epithelial apical cell membrane site of function &

[65], the molecular mechanisms underlying the F508del folding defect are complex and only recently understood. In the ER, CFTR folding requires organizing two transmembrane domains and three cytosolic domains before export to the Golgi for glycosylation and on to the cell surface. In this complex process, several distinct intramolecular interactions have been identified, as well as intermolecular interactions with nearly 200 cytoplasmic proteins (the ‘CFTR interactome’) including several dozen proteins comprising a cytosolic and ER lumenal folding assembly machinery (the ‘CFTR chaperome’) [66,67]. The intramolecular CFTR folding process involves separate assembly steps in primary NBD-1 folding that promote thermodynamic stabilization and further assembly interactions between NBD-1 and several intracellular loops of the membrane spanning domains (MSDs) [41,68–71,72 ]. One type of small molecule corrector has been found to stabilize the NB1– MSD2 interface (these have been termed class I correctors, e.g., lumacaftor), a second type (class II correctors) stabilizes the NBD1–MSD2 interface, and a third type (class III correctors) stabilizes

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NBD1; class III correctors target proteins in the CF chaperome rather than directly binding to CFTR [72 ]. In-vitro experiments indicate that combining different classes of correctors markedly improves CFTR function in F508del cells [73,74,51 ]. Many new insights into the CFTR chaperome indicate that multiple class III correctors exist as potential therapeutics [75–82]. One such class III corrector, an inhibitor of S-nitrosoglutathione reductase, has entered clinical trials [83]. A recent surprise has been the demonstration that the popular antiinflammatory agent ibuprofen is also a class III CFTR corrector via COX-1 inhibition [84]. Another breakthrough technology applicable to CF-personalized drug testing has come from the ability to induced pluripotent stem cells from skin fibroblasts or other tissue sources and differentiate them along desired lines with defined culture factors in an air–liquid interface system [85,86]. Marker proteins introduced into these cells can provide readouts of CFTR function and drug response. Finally, these stable culture systems of patientderived cells allow work on direct genetic correction of CFTR mutations using recently developed geneediting technologies [87]; here, proof of principle has been established for F508del-CFTR correction at the DNA level by direct gene-edit repair in CF patient-derived intestinal organoids or fibroblastderived induced pluripotent stem cells [88,89]. An additional exciting possibility of CFTR modulation at the mRNA rather than protein level is mRNA repair with an antisense oligonucleotide specific for the F508del region, and one such agent, QR-010, targeted directly to lung epithelial cells by inhalation will soon enter clinical trials in CF patients [90]. Although F508del remains a major focus for drug development, other classes of CFTR mutations are also being probed for response to specific modulators. Class I mutations can be addressed with drugs that provide ribosomal read through of premature stop codons. These include aminoglycoside derivatives and ataluren, which failed a phase 3 trial for efficacy, but which may be effective in a subgroup of CF patients who are not chronically treated with aminoglycosides (these compete with ataluren at the ribosomal target site) [91]. Class I mutations responsive to aminoglycoside derivatives may also be potentiated by ivacaftor [92]. Class IV and V mutations, which are usually phenotypically milder, may also be treatable with potentiators or, perhaps, novel agents that override disease-causing mRNA splicing such as splice siteannealing antisense oligonucleotides [93]. Are there possibilities for the treatment of CF agnostic to the mutation-specific functional defect? &

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CF gene therapy represents such an approach that has frustrated attempts at success for nearly 2 decades. However, a current CF gene therapy trial utilizing an inhaled liposomal vector has been completed in the United Kingdom with results pending [94 ]. A second approach, also awaiting success, lies in bypassing defective CFTR chloride channel function via stimulating alternative respiratory epithelial chloride channels or suppressing the ENaC activity. The CF community was disappointed in the failure of the purinergic agonist denufosol to demonstrate clinical efficacy as an activator of a respiratory epithelial calcium-dependent alternative chloride channel [95]. The discovery of TMEM16A (a.k.a. anoctamin-1) as the calciumdependent, CFTR-independent chloride channel in the lung epithelium has renewed interest in this potential therapeutic mechanism [96]. &

CONCLUSION Progress in the understanding of the pathophysiology and the development of novel therapies continues to accelerate in CF. What lies ahead for those involved in the field is a new era in which, although a true cure is not in sight, novel therapeutics targeting the basic defect effect true disease modification. In addition, it is now apparent that for this new paradigm in therapy the specific genotype of the patient will be a key determinant of the prescribed regimen. Thus, CF is becoming a clear example of the emerging concepts of genomically based precision medicine. Acknowledgements None. Financial support and sponsorship None. Conflicts of interest C.E.M. is currently receiving research funding in the form of grants from the US National Institutes of Health (1P01HL108797 and 9U54HL096458-06) and the US Cystic Fibrosis Foundation (MILLA08A0, 09Y0, 14PE0, and C011-09-166), as well as clinical trials funding from Vertex Pharmaceuticals and KaloBios Inc, and consultative and advisory fees from Gilead Sciences and KaloBios Inc. R.B.M. has received research funding from Vertex Pharmaceuticals, PTC Therapeutics, N30 Pharma, and ProQR Inc, and consultative and advisory fees from Novartis Pharmaceuticals, Gilead Sciences, Genentech, Vertex Pharmaceuticals, Celtaxsys, Asubio, and Proteostasis Therapeutics Inc. Volume 27  Number 3  June 2015

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Volume 27  Number 3  June 2015

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