Cystic Fibrosis Gene Research Articles

Lack of applicability of the Enterocyte Chloride ion secretion paradigm to the Pathology of Cystic Fibrosis

This review examines of the concept of a defective chloride channel in epithelial cells being a major cause of cystic fi brotic pathophysiology. The central concept of the defective chloride ion channel paradigm is that faulty CFTR protein or failed delivery of CFTR protein to the mucosal membrane of epithelial cells is the basis of cystic fi brosis. Defective placement or function of CFTR prevents hydration of bronchial mucus that is normally caused by epithelial cells; these are capable through chloride ion secretion of transporting fl uid to the mucosal surface. This concept relies heavily on a paradigm taken from intestinal physiology-namely that the intestinal epithelial cell secretes chloride ion and fl uid and that this has conferred heterozygote selective advantage in carriers of the cystic fibrosis gene. This present review examines the evidence for that hypothesis and assembles evidence from past studies that it is the smooth muscle cell that is of greater relevance. This review does not aim to provide an overview of current research into cystic fi brosis. The intention is to provide an overview of past research that led to the concept of a failure of epithelial cells to hydrate bronchial mucus because of compromised CFTR function. It is important to present all past evidence for aspects of the chloride secretion hypothesis and its associated heterozygote advantage concept so that the important evidential milestones can be re-assessed.

Chest CT Features of Cystic Fibrosis in Korea: Comparison with Non-Cystic Fibrosis Diseases.

ObjectiveCystic fibrosis (CF) is a rare congenital disease in Korea, and its clinical and imaging findings are unclear. The objective of our study was to describe the clinical and CT features of CF in Korea and compare its features with those of other diseases mimicking CF.Materials and MethodsFrom November 1994 to December 2014, a presumptive diagnosis of CF was made in 23 patients based on clinical or radiological examination. After the exclusion of 10 patients without diagnostic confirmation, 13 patients were included in the study. A diagnosis of CF was made with the CF gene study. CT findings were evaluated for the presence and distribution of parenchymal abnormalities including bronchiectasis, tree-in-bud (TIB) pattern, mucus plugging, consolidation, and mosaic attenuation.ResultsOf the 13 patients, 7 (median age, 15 years) were confirmed as CF, 4 (median age, 19 years) had primary ciliary dyskinesia, 1 had bronchiectasis of unknown cause, and 1 had chronic asthma. CT of patients with CF showed bilateral bronchiectasis, TIB pattern, mosaic attenuation, and mucus plugging in all patients, with upper lung predominance (57%). In CT of the non-CF patients, bilateral bronchiectasis, TIB pattern, mosaic attenuation, and mucus plugging were also predominant features, with lower lung predominance (50%).ConclusionKorean patients with CF showed bilateral bronchiectasis, cellular bronchiolitis, mucus plugging, and mosaic attenuation, which overlapped with those of non-CF patients. CF gene study is recommended for the definitive diagnosis of CF in patients with these clinical and imaging features.

Pseudomonas aeruginosa Airway Infection Recruits and Modulates Neutrophilic Myeloid-Derived Suppressor Cells.

Pseudomonas aeruginosa is an opportunistic pathogen that causes infections mainly in patients with cystic fibrosis (CF) lung disease. Despite innate and adaptive immune responses upon infection, P. aeruginosa is capable of efficiently escaping host defenses, but the underlying immune mechanisms remain poorly understood. Myeloid-derived suppressor cells (MDSCs) are innate immune cells that are functionally characterized by their potential to suppress T- and natural killer (NK)-cell responses. Here we demonstrate, using an airway in vivo infection model, that P. aeruginosa recruits and activates neutrophilic MDSCs, which functionally suppress T-cell responses. We further show that the CF gene defect (CF transmembrane conductance regulator, CFTR) modulates the functionality, but not the recruitment or generation of neutrophilic MDSCs. Collectively, we define a mechanism by which P. aeruginosa airway infection undermines host immunity by modulating neutrophilic MDSCs in vivo.

Cystic fibrosis gene therapy: a mutation-independent treatment.

Since cloning of the disease-causing gene 27 years ago, the development of cystic fibrosis (CF) gene therapy has been pursued. Here, we will summarize key findings with a particular focus on recent developments. Almost 3 decades of research have highlighted the complexity of lung gene transfer and have generated a body of data that has recently led to the completion of a large phase IIB study. This trial has, for the first time, shown that nonviral gene transfer can, albeit modestly, stabilize lung function in CF and provides the impetus for further development of more potent gene transfer agents. Lentiviral vectors, specifically pseudotyped to enable entry into airway epithelial cells have most recently been developed. Persistent expression after a single dose and the ability to be administered repeatedly suggest that these viral vectors hold promise for the treatment of CF; a first-in-man clinical trial will shortly be initiated. Although the development of CF gene therapy has been slower than initially anticipated, recent progress has been encouraging and has renewed the interest of academics and industry to pursue lung gene therapy.

Cystic fibrosis gene modifier SLC26A9 modulates airway response to CFTR-directed therapeutics.

Cystic fibrosis is realizing the promise of personalized medicine. Recent advances in drug development that target the causal CFTR directly result in lung function improvement, but variability in response is demanding better prediction of outcomes to improve management decisions. The genetic modifier SLC26A9 contributes to disease severity in the CF pancreas and intestine at birth and here we assess its relationship with disease severity and therapeutic response in the airways. SLC26A9 association with lung disease was assessed in individuals from the Canadian and French CF Gene Modifier consortia with CFTR-gating mutations and in those homozygous for the common Phe508del mutation. Variability in response to a CFTR-directed therapy attributed to SLC26A9 genotype was assessed in Canadian patients with gating mutations. A primary airway model system determined if SLC26A9 shows modification of Phe508del CFTR function upon treatment with a CFTR corrector.In those with gating mutations that retain cell surface-localized CFTR we show that SLC26A9 modifies lung function while this is not the case in individuals homozygous for Phe508del where cell surface expression is lacking. Treatment response to ivacaftor, which aims to improve CFTR-channel opening probability in patients with gating mutations, shows substantial variability in response, 28% of which can be explained by rs7512462 in SLC26A9 (P = 0.0006). When homozygous Phe508del primary bronchial cells are treated to restore surface CFTR, SLC26A9 likewise modifies treatment response (P = 0.02). Our findings indicate that SLC26A9 airway modification requires CFTR at the cell surface, and that a common variant in SLC26A9 may predict response to CFTR-directed therapeutics.

A randomised, double-blind, placebo-controlled trial of repeated nebulisation of non-viral cystic fibrosis transmembrane conductance regulator (CFTR) gene therapy in patients with cystic fibrosis

BackgroundCystic fibrosis (CF) is a chronic, life-limiting disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene leading to abnormal airway surface ion transport, chronic lung infections, inflammation and eventual respiratory failure. With the exception of the small-molecule potentiator, ivacaftor (Kalydeco®, Vertex Pharmaceuticals, Boston, MA, USA), which is suitable for a small proportion of patients, there are no licensed therapies targeting the basic defect. The UK Cystic Fibrosis Gene Therapy Consortium has taken a cationic lipid-mediatedCFTRgene therapy formulation through preclinical and clinical development.ObjectiveTo determine clinical efficacy of the formulation delivered to the airways over a period of 1 year in patients with CF.DesignThis was a randomised, double-blind, placebo-controlled Phase IIb trial of theCFTRgene–liposome complex pGM169/GL67A. Randomisation was performed via InForm™ version 4.6 (Phase Forward Incorporated, Oracle, CA, USA) and was 1 : 1, except for patients in the mechanistic subgroups (2 : 1). Allocation was blinded by masking nebuliser chambers.SettingsData were collected in the clinical and scientific sites and entered onto a trial-specific InForm, version 4.6 database.ParticipantsPatients with CF aged ≥ 12 years with forced expiratory volume in the first second (FEV1) between 50% and 90% predicted and any combination ofCFTRmutations. The per-protocol group (≥ 9 doses) consisted of 54 patients receiving placebo (62 randomised) and 62 patients receiving gene therapy (78 randomised).InterventionsSubjects received 5 ml of nebulised pGM169/G67A (active) or 0.9% saline (placebo) at 28 (±5)-day intervals over 1 year.Main outcome measuresThe primary end point was the relative change in percentage predicted FEV1over the 12-month period. A number of secondary clinical outcomes were assessed alongside safety measures: other spirometric values; lung clearance index (LCI) assessed by multibreath washout; structural disease on computed tomography (CT) scan; the Cystic Fibrosis Questionnaire – Revised (CFQ-R), a validated quality-of-life questionnaire; exercise capacity and monitoring; systemic and sputum inflammatory markers; and adverse events (AEs). A mechanistic study was performed in a subgroup in whom transgene deoxyribonucleic acid (DNA) and messenger ribonucleic acid (mRNA) was measured alongside nasal and lower airway potential difference.ResultsThere was a significant (p = 0.046) treatment effect (TE) of 3.7% [95% confidence interval (CI) 0.1% to 7.3%] in the primary end point at 12 months and in secondary end points, including forced vital capacity (FVC) (p = 0.031) and CT gas trapping (p = 0.048). Other outcomes, although not reaching statistical significance, favoured active treatment. Effects were noted by 1 month and were irrespective of sex, age orCFTRmutation class. Subjects with a more severe baseline FEV1had a FEV1TE of 6.4% (95% CI 0.8% to 12.1%) and greater changes in many other secondary outcomes. However, the more mildly affected group also demonstrated benefits, particularly in small airway disease markers such as LCI. The active group showed a significantly (p = 0.032) greater bronchial chloride secretory response. No difference in treatment-attributable AEs was seen between the placebo and active groups.ConclusionsMonthly application of the pGM169/GL67A gene therapy formulation was associated with an improvement in lung function, other clinically relevant parameters and bronchial CFTR function, compared with placebo.LimitationsAlthough encouraging, the improvement in FEV1was modest and was not accompanied by detectable improvement in patients’ quality of life.Future workFuture work will focus on attempts to increase efficacy by increasing dose or frequency, the coadministration of a CFTR potentiator, or the use of modified viral vectors capable of repeated administration.Trial registrationClinicalTrials.gov NCT01621867.FundingThis project was funded by the Efficacy and Mechanism Evaluation (EME) programme, a Medical Research Council and National Institute for Health Research partnership.

How Antiparasitic Drugs Work-And Sometimes Stop Working!

How Antiparasitic Drugs Work-And Sometimes Stop Working!

23. SiRNA and CRISPR/Cas9 Mediated Knockout of αENAC

Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis conductance regulator (CFTR) gene, which encodes for a chloride channel. Loss of CFTR upregulates the epithelial sodium channel (ENaC) causing hyperabsorption of sodium, reducing the watery lining of the lung, leading to impaired mucociliary clearance and enabling inflammatory lung damage. Therefore, inhibition of ENaC has been proposed as a treatment in CF. In this study we are assessing the use of siRNA and CRISPR/Cas9 system as complementary methods of achieving disruption of ENaC expression in airway epithelial cells using receptor-targeted nanocomplexes. We sought to compare the efficiency and persistence of silencing, repeatability of delivery and its toxicity. We assessed silencing of the αENaC subunit by transfecting primary CFBE cells growing in Air-Liquid Interface cultures with siRNA-bearing nanocomplexes and achieved 30% silencing at the mRNA level. We then assessed their silencing efficiency in lungs of normal mice (C57BL/6) delivered by oropharyngeal instillation. Following a single dose, the siRNA formulations achieved ~30% knockdown of mouse αENaC. Following repeated delivery (3 doses at 48h intervals) we found out that 48h after the last administration ~50% silencing was achieved, with no adverse effects (as judged by body weight and histology), whilst following a single administration there was still ~30% silencing 7 days later. An algorithm was used to design 6 different guide RNA targets for αENaC. These were used to transfect cells along with Cas9 and showed varied levels of indel mutation rates in the αENaC gene by the T7 endonuclease I assay although two, T3 and T4, were optimal. In order to increase the amount of indels created we subjected the cells (CFTE and HBE) to sequential transfections of the same target at 48h intervals. This showed an accumulation of indels in cells targeted with T3 at a rate of 33.3% in HBE cells. This correlated with a 60% decrease in ENaC mRNA compared to controls and with 40% after a single dose to 65% decrease in protein levels after three doses. The siRNA-mediated silencing showed that we can repeatedly deliver the formulations with no adverse effects and that the effect of a single dose, although transient, can last for at least 1 week. CRISPR/Cas9 also mediated efficient gene disruption of αENaC, but with the advantage that this knockdown will be permanent in cells where the life span is quite long. Another encouraging finding was the accumulation of gene disruption with both systems when delivered by a non-immunogenic vector. This is very important as CF therapy would need repeated administrations. It is reasonable to anticipate that these systems offer new prospects to CF gene therapy.

597. Development of Non-Viral Gene Delivery Systems with Antibacterial Effects Deliverable via Aerosol for Lungs Gene Therapy

Objective: Cystic Fibrosis (CF) is a genetic disorder caused by the absence or malfunction of the Cystic Fibrosis Transmembrane conductance Regulator (CFTR). CF gene therapy would consist in delivering a normal copy of the CFTR gene in the nucleus of respiratory epithelial cells. The presence of bacteria in the pulmonary tract can alter the transported DNA, the transfection efficiency as well as targeted cells. At present, the leading method to target the respiratory epithelium being the nebulisation, the goal of this research project is to develop a formulation that can be delivered as an aerosol exhibiting both a transfection activity and an antibacterial activity. Methods: The formulated complexe was composed of 50% (molar percentage) of cationic lipid (an arseno-phospholipid whose transfection efficiency and antibacterial activity against Gram-positive bacteria have already been demonstrated) and 50% of a silver salt (active against Gram-negative bacteria). The transfection activity was evaluated according to the expression of a luciferase reporter gene into human bronchial epithelial cells grown in liquid culture or at air-liquid interface. The antibacterial activity was determined according to the difference of growth in agar plate between a protected area and an exposed area to the aerosol. Results: Contrary to the aerosolization of the classical lipoplexe, the formulation developed containing a silver salt possesses an antibacterial activity against the Gram-negative bacteria. The transfection activity is maintained post-aerosolization with some similar levels of expression to those observed with the cationic lipid used alone, and this both in liquid culture and at air-liquid interface. Discussion and Conclusion: This study shows that an equimolar combination of two compounds: an arseno-phospholipid and a silver salt (i) is simultaneously effective on bacteria frequently isolated from patients, (ii) has a very low human bronchial epithelial cells toxicity, and (iii) exhibits a transfection efficacy under clinically relevant conditions. Unlike a gene transfer system devoid of an antibacterial activity, such a combination may therefore participate in the eradication of bacteria, thus reducing the stress on the bronchial epithelial cells while promoting the transfection process and finally, the expression of the transgene.

RAAV-CFTRΔR Rescues the Cystic Fibrosis Phenotype in Human Intestinal Organoids and Cystic Fibrosis Mice.

Gene therapy holds promise for a curative mutation-independent treatment applicable to all patients with cystic fibrosis (CF). The various viral vector-based clinical trials conducted in the past have demonstrated safety and tolerance of different vectors, but none have led to a clear and persistent clinical benefit. Recent clinical breakthroughs in recombinant adeno-associated viral vector (rAAV)-based gene therapy encouraged us to reexplore an rAAV approach for CF. We evaluated the preclinical potential of rAAV gene therapy for CF to restore chloride and fluid secretion in two complementary models: intestinal organoids derived from subjects with CF and a CF mouse model, an important milestone toward the development of a clinical rAAV candidate for CF gene therapy. We engineered an rAAV vector containing a truncated CF transmembrane conductance regulator (CFTRΔR) combined with a short promoter (CMV173) to ensure optimal gene expression. A rescue in chloride and fluid secretion after rAAV-CFTRΔR treatment was assessed by forskolin-induced swelling in CF transmembrane conductance regulator (CFTR)-deficient organoids and by nasal potential differences in ΔF508 mice. rAAV-CFTRΔR transduction of human CFTR-deficient organoids resulted in forskolin-induced swelling, indicating a restoration of CFTR function. Nasal potential differences demonstrated a clear response to low chloride and forskolin perfusion in most rAAV-CFTRΔR-treated CF mice. Our study provides robust evidence that rAAV-mediated gene transfer of a truncated CFTR functionally rescues the CF phenotype across the nasal mucosa of CF mice and in patient-derived organoids. These results underscore the clinical potential of rAAV-CFTRΔR in offering a cure for all patients with CF in the future.

CFTR is a tumor suppressor gene in murine and human intestinal cancer.

CFTR, the cystic fibrosis (CF) gene, encodes for the CFTR protein that plays an essential role in anion regulation and tissue homeostasis of various epithelia. In the gastrointestinal (GI) tract CFTR promotes chloride and bicarbonate secretion, playing an essential role in ion and acid-base homeostasis. Cftr has been identified as a candidate driver gene for colorectal cancer (CRC) in several Sleeping Beauty DNA transposon-based forward genetic screens in mice. Further, recent epidemiological and clinical studies indicate that CF patients are at high risk for developing tumors in the colon. To investigate the effects of CFTR dysregulation on GI cancer, we generated Apc(Min) mice that carried an intestinal-specific knockout of Cftr. Our results indicate that Cftr is a tumor suppressor gene in the intestinal tract as Cftr mutant mice developed significantly more tumors in the colon and the entire small intestine. In Apc(+/+) mice aged to ~1 year, Cftr deficiency alone caused the development of intestinal tumors in >60% of mice. Colon organoid formation was significantly increased in organoids created from Cftr mutant mice compared with wild-type controls, suggesting a potential role of Cftr in regulating the intestinal stem cell compartment. Microarray data from the Cftr-deficient colon and the small intestine identified dysregulated genes that belong to groups of immune response, ion channel, intestinal stem cell and other growth signaling regulators. These associated clusters of genes were confirmed by pathway analysis using Ingenuity Pathway Analysis and gene set enrichment analysis (GSEA). We also conducted RNA Seq analysis of tumors from Apc(+/+) Cftr knockout mice and identified sets of genes dysregulated in tumors including altered Wnt β-catenin target genes. Finally we analyzed expression of CFTR in early stage human CRC patients stratified by risk of recurrence and found that loss of expression of CFTR was significantly associated with poor disease-free survival.

Cystic Fibrosis Gene Therapy – Not Low-hanging Fruit

The last 25 years have shown that it has been comparatively slow and difficult to develop cystic fibrosis (CF) gene therapy; the lung is a complex target organ. However, research has steadily progressed and recently it was shown that non-viral gene therapy can stabilise CF lung disease. These data, in addition to the development of potent lentiviral vectors, have renewed interest in CF gene therapy within academia and industry.

Cystic fibrosis transmembrane conductance mutation detection using fluorescent hybridization probes and melting curves

Background/aims Single-point mutations or single-nucleotide polymorphisms, deletions, and insertions in genetic sciences are related to several human diseases, such as cancer, metabolic disorders, some types of mental illness, cardiovascular diseases, diabetes, etc. Consequently, precise, fast, and sensitive detection of these mutations in specific genes has substantial value in disease diagnosis, in the forecast of patients’ responses to treatments, threat of deterioration of diseases, and outcomes. However, the existence of minute differences in structural and conformation dynamic stability from single base or multibase mismatches between the wild type (WT) and its mutated targets makes detection convenient. The common cause of cystic fibrosis (CF) is the deletion of three nucleotides (CTT). This deletion happens in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which involves the last cytosine (C) of isoleucine 507 (isoleucine 507ATC) and the two thymidine oligonucleotide (T) of phenylalanine 508 (phenylalanine 508TTT) codons. The significances of this important deletion are the deletion of phenylalanine at the 508 position of the cystic fibrosis transmembrane conductance regulator protein (ΔF508), an identical codon modification for isoleucine 507 (isoleucine 507ATT), and protein dysfunction. Materials and methods Fluorescence and ultraviolet−visible thermal studies were performed for WT and mutant-type target full systems. The target DNAs used were in the form of short oligonucleotides. The tandem probes system was used for detection of WT and single-nucleotide polymorphism alleles of human 3-bp ΔF508 (TTT) homozygous deletion. The pyrene dye attached to a probe oligonucleotide (15 mer) undergoes an excimer fluorescence intensity change on hybridization of the two probes to the WT compared with mutant-type targets. Results Our results indicate that the system consisting of the target sequence and the two probe oligonucleotides bearing the pyrene dye assemble correctly at the specified target. Once the full system (two probes and target) is arranged under suitable conditions, a red-shift emission and change in fluorescence intensity are seen at an excimer wavelength of 480 nm. Thermal studies also showed significant differences in Tm between mutated and unmutated CF genes. The results suggest that the differences in the fluorescence intensity at 480 nm and the spectrophotometric Tm (s) for the mutated and unmutated CF gene can be attributed to the type of binding of the probe to the target. Conclusion On the basis of the data obtained, we have chosen the probes possessing the highest fluorescence intensity along with the best deletion discrimination detection ability. The system was sensitive to deletion nucleotide polymorphisms and this may help in high-throughput applications in genetic testing and molecular diagnostics.

PNA Molecular Beacons Assembled by Post-Synthetic Click Chemistry Functionalization.

To avoid the tedious synthesis of functionalized peptide nucleic acid (PNA) monomers for probe development, we proposed a simple approach to modify PNA oligomers by post-synthetic on-resin click chemistry. PNA molecular beacons (MBs) were prepared by incorporation of azide-containing monomers into the oligomer by automatic solid-phase peptide synthesis and subsequent derivatization with pyrene moieties by copper-catalyzed azide-alkyne cycloaddition. Two pyrene-based quencher-free PNA molecular beacons, a stemless MB and one possessing a stem-loop structure, targeting a portion of the cystic fibrosis gene, were successfully synthesized by using this method. Fluorescence studies showed that the stem-loop MB exhibited better discrimination of changes in excimer/monomer ratios as compared to the stemless MB construct.

Cohorte DEFI-ALPHA et projet hospitalier de recherche clinique POLYGEN DEFI-ALPHA. Étude des facteurs cliniques, biologiques et génétiques associés à l’apparition et à l’évolution de complications hépatiques chez les enfants présentant un déficit en alpha-1 antitrypsine

The alpha-1 antitrypsin (α1-AT) deficiency, most frequently caused by homozygosity for the Z variant (SERPINA1: c.1096 G>A; Glu342Lys), can give rise to two clinical patterns: (i) respiratory impairment with emphysema (mainly in adulthood) because of a pulmonary quantitative defect in anti-elastase activity; (ii) hepatic impairment (mainly in childhood) due to the misfolding of the PiZ protein which accumulates in hepatocytes thus providing cytotoxicity. To date, the clinical and genetic factors responsible for the development of major hepatic injuries (fibrosis and portal hypertension) during childhood in PiZ patients are not known. The DEFI-ALPHA cohort, created in 2008, aims to inventory and prospectively study all α1-AT deficient children diagnosed and included after occurrence of a hepatic sign. The POLYGEN DEFI-ALPHA PHRC has recently (2013) been added to the project to identify modifiers genes by two complementary approaches: (i) the candidate genes strategy with the SERPINA1, CFTR (cystic fibrosis gene), MAN1B1 and SORL1 genes, these two latter being implied in the degradation of misfolding proteins; (ii) the whole exome sequencing (WES) strategy in families in which the PiZ proband has a PiZ brother or sister free of any hepatic sign. The clinical parameter we want to explain is the apparition of a portal hypertension in PiZ children. In the DEFI-ALPHA project, three criteria will be tested: (i) age of inclusion in the cohort, (ii) the way of inclusion (neo-natal icterus or later hepatic impairment) and (iii) treatment or not with ursodesoxycholic acid and, if so, its duration. Genetically, polymorphisms on the SERPINA1 and MAN1B1 genes have already been associated in the literature with different clinical evolutions of the A1ATD but very inconstantly. Our study thus aims to confirm or not this association. The CFTR and SORL1 genes have never been studied in the α1-AT deficiency. Finally, the whole exome sequencing strategy could allow the discovery of new unexpected modifiers genes in this disease.

Breakthrough at long last for cystic fibrosis gene therapy

Breakthrough at long last for cystic fibrosis gene therapy

New and Emerging Treatments for Cystic Fibrosis.

Recently, a significant number of additional key medications have become licensed in Europe for the treatment of patients with cystic fibrosis (CF), including a number of inhaled antibiotics, such as nebulised aztreonam and dry powder versions of colistin and tobramycin for inhalation; dry powder inhaled mannitol, an agent to improve airway hydration and aid airway clearance; and ivacaftor, an oral therapy that directly acts on dysfunctional CFTR to correct the basic defect encountered in CF patients with the G551D CF gene mutation. The marked success of ivacaftor both in clinical trials and in post-licensing evaluation studies in treating patients with G551D and other gating mutations has greatly encouraged the ongoing development of similar therapies that can directly target the underlying cause of CF. Other therapies, including a number of anti-infectives, anti-inflammatories and replacement pancreatic enzymes, are currently undergoing clinical studies. This article reviews those treatments that have been recently licensed for CF and highlights some of the exciting emerging therapies presently under evaluation in clinical trials. In addition, it discusses some of the potential challenges being encountered by research and clinical teams in developing and delivering treatments for this condition.

Changing the Paradigm - Treating the Basic Defect in Cystic Fibrosis.

Since the first description of Cystic fibrosis (CF) more than 75y ago, significant advances have been made in understanding its pathogenesis and in developing specific therapies. The pace of these developments was further accelerated after the discovery of CF gene in 1989 and since then, CF has been transformed from being a pediatric illness into a chronic life-limiting genetic disorder with survival up to the fourth decade. The development of mutation-specific therapies in the first decade of the 21st century has the potential to change the natural history of CF and has now ushered in the era of 'Precision Medicine'. The ability to revert the basic defect in CF by using Personalized Medicine approach based on each individual's genetic profile will serve as a model for other chronic disorders as well. This review highlights the recent advances in the field of CF research that have led to a paradigm shift in its management and outcomes.

9 Analysis of cystic fibrosis gene mutations in children with cystic fibrosis in a regional center in Eastern Romania

Objective The purpose of this study was to describe the genetic mutations identified in patients of pediatric age with cystic fibrosis (CF) diagnosed in a cystic fibrosis center in north-eastern Romania. Methods We performed a retrospective study which included 37 children with CF, followed between December 2012 and December 2014. In the absence of neonatal screening, diagnosis was based on sweat test in patients who presented respiratory or digestive clinical manifestations. The 32 patients (21 boys and 11 girls) involved were performed genetic tests only to identify 38 mutations using Real time PCR method. Results Following the genetic tests performed, there were identified: homozygous (F508del – 11 patients, G542X – 1 patient, 1898 + 1G>A – 1 patient), heterozygous compounds (F508del/G542X – 4 patients, F508del 7T/9T – 2 patients, F508del 9T/9T – 4 patients, D1152H 7T/7T – 1 patient) and other types of mutations 7T/7T – 8 patients. F508del mutation frequency was 65.62% in the study group. F508del mutation was associated with mixed forms (digestive and pulmonary) of CF in 13 patients and with pulmonary form of CF in 8 patients. Mutation 1898 + 1G>A was associated with a severe form of digestive disease and pulmonary of CF with unfavorable evolution. Conclusion F508del mutation frequency was similar compared to the frequency of this particular mutation in other European countries (70%). In Romania, it is necessary to introduce a genetic screening test for an early FC diagnosis and also for establishing the genetic profile of CF.

WS14.1 Ivacaftor improves exercise capacity in patients with G551D CF gene mutations

Introduction G551D is a CFTR mutation that results in impaired chloride channel function in Cystic Fibrosis (CF). Ivacaftor, a CFTR potentiating agent improves lung function and sweat chloride but its effect on exercise is unproven. Objectives To evaluate exercise capacity in response to correction of the chloride channelopathy with ivacaftor. Methods In a placebo-controlled crossover study of ivacaftor over 4 months with a 3 month open label extension we performed cardiopulmonary exercise tests (CPET) at six time points. 20 patients were included in the study. The primary outcome was VO2max as a percentage of baseline at the end of the crossover study (END X) and 3 month open label extension (OLE). A training program was not included in the study. Results There was a stepwise increase in maximal exercise capacity based on VO2max in the treated group above baseline at END X and 3 month OLE treatment periods. FEV1 and sweat chloride were also improved as expected. EndpointEnd X, mean (95% CI)OLE, mean (95% CI)%Δ absolute exercise time8.0 (1.4–14.6) * 12.1 (5.8–18.4) *** %Δ VO2max, ml/kg/min6.7 (2.7–10.8) ** 12.6 (6.3–18.9) *** %Δ VO2 ml/min7.8 (3.7–12.0) *** 15.6 (9.7–21.4) *** %Δ Watts5.0 (1.4–8.6) ** 10.9 (5.6–16.3) *** *p Conclusion Treatment of G551D patients with ivacaftor has a positive effect on exercise capacity which is independent of training. Improved VO2max has not been previously examined in clinical trials with ivacaftor and may have prognostic implications for patients with the G551D mutation. Supported by a grant from Vertex Inc.