Cystic fibrosis - European Journal of Internal Medicine

European Journal of Internal Medicine 25 (2014) 803–807

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European Journal of Internal Medicine journal homepage: www.elsevier.com/locate/ejim

Review Article

Cystic fibrosis — What are the prospects for a cure? Shankar Kumar a,⁎, Anand Tana a, Anu Shankar b a b

St. George's, University of London, United Kingdom St. George's Hospital, London, United Kingdom

a r t i c l e

i n f o

Article history: Received 20 June 2014 Received in revised form 2 September 2014 Accepted 26 September 2014 Available online 18 October 2014 Keywords: Cystic fibrosis Cystic fibrosis transmembrane regulator Ivacaftor Lumacaftor Ataluren Gene therapy

a b s t r a c t Significant improvements in the treatment of cystic fibrosis over the last few decades have altered this lethal disease in children to a multisystem disorder with survival into adult life now common. In most developed countries the numbers of adult cystic fibrosis patients outnumber children. This is mainly due to improvements in care during early life. The principal cause of morbidity and mortality is pulmonary disease, and so the focus of new treatments has targeted the lungs. Identification of the underlying gene defect in the cystic fibrosis transmembrane conductance regulator has ushered in a new era in cystic fibrosis research, with prospects of a cure. In this article, we review the most exciting recent advances that correct defects in cellular processing, chloride channel function and gene therapy. © 2014 European Federation of Internal Medicine. Published by Elsevier B.V. All rights reserved.

1. Background Cystic fibrosis (CF) is the commonest life-limiting autosomal recessive condition in Europe affecting 1 in 2000 to 3000 newborns, with a carrier rate of 4% in the United Kingdom, rising to as much as 70% in northern, western, and north-eastern Europe [1–3]. It is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, found on the surface of cells in a variety of tissues including the lungs, gut and pancreas, where it functions as a regulated chloride ion channel. Loss or defective function of the gene product gives rise to disease. Viscous secretions predispose to pulmonary and pancreatic disorders [4]. In the lungs, tenacious secretions predispose to repeated infections, leading to bronchiectasis, respiratory failure and cor pulmonale. Exocrine pancreatic insufficiency develops with occlusion of the pancreatic duct by viscous secretions, and manifest clinically as steatorrhoea. In the latter stages of disease, pancreatic endocrine dysfunction gives rise to diabetes mellitus. Median predicted survival is now over 40 years and in the United Kingdom, 57.6% of CF patients are classified as adults with 9.1% aged living beyond 40 years [5]. 2. Molecular basis of cystic fibrosis In health, respiratory epithelium is lined by a thin layer of fluid referred to as the airway surface liquid (ASL) (Fig. 1). It comprises periciliary liquid over which lies the mucus layer, secreted by goblet ⁎ Corresponding author at: St George's, University of London, Cranmer Terrace, London, SW17 0RE. Tel.: +44 7949628454; fax: +44 1534 485628. E-mail address: [email protected] (S. Kumar).

and Clara cells. Mucus traps microorganisms before being cleared by the muco-ciliary escalator. Periciliary fluid secretion is tightly regulated by absorption of sodium by the apical membrane epithelial sodium channel (ENaC), together with active chloride secretion by the CFTR to maintain the ASL in which the cilia bathe [6]. In CF, the gene mutation resides on chromosome 7 in the region of 7q31.2 which codes for the CFTR protein. Almost 2000 mutations have been identified, divided into 5 classes (Fig. 2) [7]. The most prevalent abnormality is a deletion in delta F508 (dF508). The mutation alters the secondary and tertiary structure of the protein, so that chloride channels fail to open in response to elevated cAMP in epithelial cells. Defective expression, trafficking or function of CFTR leads to impaired secretion of chloride and an increase in sodium absorption. This causes depletion of the airway surface liquid and, in turn, to defective mucociliary action and reduced mucus clearance. This encourages bacterial colonisation, recurrent infections, chronic inflammation and irreversible damage to the airway epithelium. In the 1980s, children with CF rarely lived beyond adolescence. Innovative management strategies based on daily chest physiotherapy to facilitate mucus clearance, correct nutrition, and early intervention with antimicrobials to stem infection promptly have improved life expectancy to the 4th decade [8]. Breakthroughs in antibiotics, anti-inflammatories, mucolytics and pancreatic replacement only treat the manifestations of the disease so the ion channel defect remains. In recent years, fundamental knowledge of molecular and cell biology has helped to define the underlying ion channel defect, and increased the prospects of targeted therapy. This article reviews the most promising therapeutic avenues, which include correcting defects in cellular processing, chloride channel function and replacing the defective gene.

http://dx.doi.org/10.1016/j.ejim.2014.09.018 0953-6205/© 2014 European Federation of Internal Medicine. Published by Elsevier B.V. All rights reserved.

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S. Kumar et al. / European Journal of Internal Medicine 25 (2014) 803–807

Fig. 1. The defect in cystic fibrosis. A mutation in the CFTR gene prevents Cl− being secreted and there is unrestrained Na+ absorption causing the ASL to become dehydrated, leading to defective mucous clearance.

2.1. Ion channel modulators These drugs aim to correct the underlying defect in cellular production and/or potentiate chloride movement across the ion channel. Given the heterogeneity of effects on the CFTR by different mutations, ion channel modulators can only be targeted against specific mutations. The most common CFTR malfunctions can be targeted using this strategy which has led to the development of several drugs. One of these has been approved by the FDA and two drugs are the focus of major clinical trials. Drug categories include potentiators which target defective regulation and impaired conductance, correctors that address impaired processing and read-through therapy which overcomes defective production of the CFTR.

3. Potentiators Ivacaftor (Kalydeco), an oral potentiator produced by Vertex pharmaceuticals, is the only drug in its class currently licensed for use in clinical practice. Known investigationally as VX770, it directly affects CFTR mutation G551D by enhancing gating at the cell surface. This increases the time that activated CFTR channels remain open at the cell surface. In vitro studies identified that Ivacaftor increases CFTR channel opening and promotes apical surface fluid and cilia beating [9]. A randomised, double-blind, placebo-controlled trial validated the safety and adverse event profile of Ivacaftor in 39 patients aged 18 years and over who possessed the G551D mutation [10]. Minor side effects such as fever, cough, nausea and rhinorrhoea resolved spontaneously without drug cessation. There was a marked reduction in sweat chloride concentration, with some subjects outside the diagnostic range for CF. Nasal potential difference (significantly more negative in CF due to increased luminal sodium absorption) and lung function improved. Two major long-term randomised placebo-controlled phase 3 trials have been conducted in G551D CF patients. They assessed the efficacy of Ivacaftor in 161 children aged 12 years and over [11]. Mean forced expiratory volume in the first second (FEV1) increased by 10.5% at 24 weeks, with improvement sustained at 48 weeks. Significant improvements in the secondary clinical end points were a 55% reduction in the risk of developing an infective pulmonary exacerbation, improvement in respiratory symptoms assessed by the Cystic Fibrosis Questionnaire revised (CFQ-R) score and weight gain. The mechanism

for weight gain is unclear but may be due to improved CFTR function in the gut [11]. Ivacaftor was discontinued in 13% compared to 6% in the placebo group, due to adverse effects including liver dysfunction, atrioventricular heart block, haemoptysis, pulmonary exacerbation and panic attacks. Next, an open-labelled trial showed that the effects on the FEV1 were maintained at 60 weeks, with 43.5% of patients receiving Ivacaftor, free of pulmonary infection [12]. The randomised, double-blind, placebocontrolled phase 3 ENVISION trial evaluated the safety and efficacy of Ivacaftor in younger children (age 6–11 years) with at least one G551D mutation [13]. The study confirmed that efficacy of Ivacaftor extended to this younger cohort, with a statistically significant increase in FEV1, a decrease in sweat chloride, and weight gain. Beneficial effects of Ivacaftor have been limited to individuals with the G551D class 3 mutation, which represents only 3% of the burden of CF. Although in vitro studies demonstrate that it increases chloride secretion approximately 10-fold in cultured human CF bronchial epithelial cells (HBECs) carrying the dF508 processing mutation, this has not been replicated in vivo [14]. No significant change in FEV1 or sweat chloride concentration has been demonstrated. Failure of Ivacaftor to modify CFTR function may be explained by misfolding of the protein associated with this mutation preventing translocation of CFTR at the cell surface. 4. Correctors Lumacaftor, known investigationally as VX-809, is a channel corrector that allows the dF508 CFTR to bypass proteomic degradation and increases trafficking of the protein to the epithelial cell surface [15]. Its efficacy was validated in cultured human CF bronchial epithelial cells isolated from patients with CF homozygous for dF508 [16]. Lumacaftor improved dF508-CFTR processing in the endoplasmic reticulum and raised chloride secretion to approximately 14% of non-CF human bronchial epithelial cells, a level that corresponds with milder forms of the disease. These encouraging results prompted a 28-day phase 2 trial in adult CF patients with this gene mutation [17]. The drug produced a dose-dependent reduction in sweat chloride levels compared to placebo, but there was no significant improvement in CFTR function in the nasal epithelium by measuring nasal potential difference, nor were there statistically significant changes in lung function or patientreported outcomes.


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Fig. 2. The classification of CFTR mutations. For normal CFTR production, the CFTR gene is transcribed into mRNA and then translated into the CFTR protein within the endoplasmic reticulum. The protein is glycosylated in the Golgi apparatus and transported and incorporated into the cell membrane. In health, the channel opens in response to cAMP and protein kinase A (PKA), permitting Cl− to leave the cell. In Class 1 mutations, there is a large deletion and stop codon mutation that results in either loss of protein production or a truncated protein that is mostly non-functional. Class 2 mutations cause abnormally folded immature proteins that are recognised by the cell and subsequently degraded by the cell proteases before being processed and transported to the cell surface. Class 3 mutations cause changes to the amino acid sequence that result in production of a defective protein that is insensitive to cAMP and PKA. Class 4 mutations lead to production of defective protein with reduced chloride conductance. Class 5 mutations involve a defect in transcription and splicing that causes a reduced amount of functional protein at the surface membrane.

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Combining Ivacaftor and Lumacaftor in patients with a dF508 mutation represented a natural progression. Indeed, in-vitro studies where the two drugs were combined in dF508 respiratory epithelia revealed a near 30% increase in CFTR-mediated chloride transport compared to the wild type [16]. If this dramatic effect were translated to clinical practice, it would represent a major advance in managing CF. This drug combination is undergoing trials in patients with the dF508 mutation. Ahead of publication, the preliminary findings suggest a beneficial effect on lung function [18]. This has provided the impetus for two phase 3 trials involving around 1000 patients with two copies of the dF508 deletion [19]. Furthermore, Vertex has commissioned a Phase 2 study employing this drug combination in those with one copy of the dF508 mutation [20]. Results from these much anticipated trials should be released in 2015. 5. Read-through therapy Approximately 10% of CF is due to nonsense mutations, which are premature stop codons in the mRNA that lead to class I defects in the CFTR protein. Pre-clinical studies demonstrated that high-dose aminoglycosides could induce a translational ‘read-through’ but a lack of potency and side-effects such as renal and ototoxicity led to new compounds with a similar mechanism of action being developed [21]. Ataluren, known investigationally as PTC-124, is a molecule that allows the ribosome to selectively read through these mutations, but not normal termination codons, to generate a functional CFTR protein [22]. In a murine model of CF exhibiting the G542X mutation, Ataluren restored chloride to 24–29% of normal levels [23]. Phase II clinical trials reported an improvement in chloride transport with a good safety profile [24,25]. However, a phase 3 study that recruited over 200 children aged 6 and over, failed to demonstrate significant improvement in lung function at 48 weeks, though a positive trend was observed [26]. It is of interest that FEV1 improved in a subset not simultaneously taking nebulised aminoglycoside antibiotics. On the basis that aminoglycosides may interact with Ataluren, recruitment to a trial of subjects not treated with long term inhaled aminoglycoside antibiotics, has begun. Researchers are mindful of the non-specific actions of Ataluren and accumulation of long term safety data are needed to ensure that it does not induce genetic aberration [25]. 6. Gene therapy The development of new drugs that modulate ion channels or potentiate the defective CFTR protein is exciting, but they can only offer treatment to a small number of patients due to the multitude of CFTR mutations. Gene therapy is an attractive proposition with the prospect of offering a universal cure. However, research over the past 20 years has been hampered by various difficulties, especially identifying an effective way of transferring the corrected gene to cells of the host. Currently, there are many different strategies of gene transfer, but no data exist on the clinical efficacy of gene therapy. The first clinical trial developed by the UK Cystic Fibrosis Gene Therapy Consortium will provide data regarding the clinical applicability of gene therapy in CF. Two transfer methods have been investigated, viral and non-viral vectors.

host's innate defence mechanisms. The advantage of viral vectors over lipid vectors is that once they enter the cell, degradation by lysosomes is less likely. Gene therapy was initially studied in human nasal epithelium since it has a cellular composition not dissimilar to the conducting airway. Initial studies using adenovirus showed a partial correction in the chloride channel abnormality [28]. Similar results were observed after delivering the viral vector to lung tissue, with a transient inflammatory response observed in those receiving a higher dose, confirming its safety [29]. It was hoped that serial dosing of the gene would lead to increasing chloride levels, but although there was a positive response initially, the level of chloride correction decreased through the development of serum antibodies to the viral vector [30]. Adeno-associated virus (AAV) has been used as a vector on the basis that it is less immunogenic, provoking a milder inflammatory response. Although the virus was better able to survive, studies showed that it was less effective in crossing the cell membrane, so less genetic material was transferred [31]. Moreover, repeated administration produced a limited response due to antibody production. Lentiviruses have also been utilised as gene vectors because they evade the host immune response effectively. For successful gene transfer, modifications to the viral surface proteins are required. The UK consortium is currently investigating a modified form of the lentivirus for entry into clinical trials (WAVE 2). This novel vector permits persistent gene expression in human ex vivo models without chronic toxicity [32].

8. Non-viral vectors Non-viral vectors are cationic and rely on endocytosis to cross the cell membrane on the apical surface. Early non-viral vectors caused partial correction of chloride levels on interaction with nasal epithelium with few safety concerns [33,34]. Repeated administration demonstrated that, unlike viral vectors, there was no immune response or reduced efficacy with subsequent dose [35]. Similar studies involving administration of a healthy CFTR gene complexed to a cationic lipid (GL67) revealed 25% restoration in chloride channel function, reduced inflammatory cells in the sputum and diminished bacterial adherence [36]. Nevertheless, these synthetic vectors do provoke an innate immune system response. This is because the plasmids are derived from bacteria or recombinant viruses, and DNA from these organisms contains unmethylated CPG dinucleotide (CpG) motifs. The presence of a single CpG motif can trigger an acute inflammatory response via the toll-like receptor immune system [37]. More recently, plasmids from which CpG motifs have been removed have been produced which permits sustained transgene expression without inflammation. An example of this is pGM169 introduced to the cationic lipid vector, GL67A, which demonstrates low toxicity and efficient gene transfer into airway epithelial cells. Repeated administration has not been associated with an immune response [36,38]. The UK consortium is currently investigating its efficacy in a double blind study, the largest gene therapy trial in CF to date [39]. The gene is to be delivered via this vehicle on a monthly basis by nebuliser for a year. The results from this landmark trial are due in early 2015.

7. Viral vectors 9. Final remarks and conclusions In CF, the airway is initially normal, and at this stage, the expectation would be that gene transfer across near-normal epithelium would be straightforward. In order to introduce the gene, the vector must have the capacity to overcome these defence mechanisms to enter cells, and be transported to the cell nucleus where transcription occurs to produce the functional CFTR protein. In practice, innate immunological mechanisms of the lungs have proved a significant barrier [27]. Viruses have been considered suitable vectors for delivering the gene because they have developed mechanisms that overcome the

Earlier diagnosis through screening, symptom-directed treatment, effective and intensive multi-disciplinary care and vigilance have led to a dramatic increase in quality of life and life expectancy of CF patients. Recent advances in the treatment of CF have focused on correcting the defective CFTR protein. New drug developments including CFTR protein modulators and gene therapy create hope for treatments of the basic CFTR defect as the prospect of a definitive cure increases.


10. Learning points • Cystic fibrosis is the commonest autosomal recessive condition in Western societies. • This disorder is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene located on chromosome 7. • It is a progressive, multi-system disease that primarily affects the pulmonary, pancreatic and gastrointestinal systems. • Little less than 40 years ago, life expectancy was limited to the teenage years but advances in symptom-directed therapy have improved median survival to around 40 years. • Current research has focused on targeting the underlying gene defect. This has led to the development of drugs that aim to correct cellular processing and/or potentiate chloride movement across the ion channel. • Advances in gene therapy aim to deliver functional CFTR directly to the lungs to replace the defective protein. • New drugs and gene therapy are being investigated in major clinical trials raising hope for a definitive cure. Funding None declared. Ethical approval Not applicable. Contributorship SK and AT co-wrote the first draft of the manuscript. AS added to and edited this draft. All authors contributed equally to the final draft. SK and AT conceived the review. Conflict of interests The authors state that they have no conflicts of interest. Acknowledgements None. References [1] Farrell PM. The prevalence of cystic fibrosis in the European Union. Journal of cystic fibrosis: official journal of the European Cystic Fibrosis Society 2008;7(5):450–3. [2] Dodge JA, Morison S, Lewis PA, Coles EC, Geddes D, Russell G, et al. Incidence, population, and survival of cystic fibrosis in the UK, 1968-95. UK Cystic Fibrosis Survey Management Committee. Archives of disease in childhood 1997;77(6):493–6. [3] Claustres M, Guittard C, Bozon D, Chevalier F, Verlingue C, Ferec C, et al. Spectrum of CFTR mutations in cystic fibrosis and in congenital absence of the vas deferens in France. Human mutation 2000;16(2):143–56. [4] Knowles MR, Durie PR. What is cystic fibrosis? The New England journal of medicine 2002;347(6):439–42. [5] UK Cystic Fibrosis Trust Registry. Cystic Fibrosis annual data report 2012. https:// wwwcysticfibrosisorguk/media/316754/Registry%20Report%20-%20Summary% 202012pdf; 2013. [6] Proesmans M, Vermeulen F, De Boeck K. What's new in cystic fibrosis? From treating symptoms to correction of the basic defect. European journal of pediatrics 2008; 167(8):839–49. [7] De Boeck K, Zolin A, Cuppens H, Olesen HV, Viviani L. The relative frequency of CFTR mutation classes in European patients with cystic fibrosis. Journal of cystic fibrosis: official journal of the European Cystic Fibrosis Society 2014. [8] O'Sullivan BP, Freedman SD. Cystic fibrosis. Lancet 2009;373(9678):1891–904. [9] Van Goor F, Hadida S, Grootenhuis PD, Burton B, Cao D, Neuberger T, et al. Rescue of CF airway epithelial cell function in vitro by a CFTR potentiator, VX-770. Proceedings of the National Academy of Sciences of the United States of America 2009;106(44): 18825–30. [10] Accurso FJ, Rowe SM, Clancy JP, Boyle MP, Dunitz JM, Durie PR, et al. Effect of VX-770 in persons with cystic fibrosis and the G551D-CFTR mutation. The New England journal of medicine 2010;363(21):1991–2003. [11] Ramsey BW, Davies J, McElvaney NG, Tullis E, Bell SC, Drevinek P, et al. A CFTR potentiator in patients with cystic fibrosis and the G551D mutation. The New England journal of medicine 2011;365(18):1663–72.

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