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This study explored the use of computed cardiopulmonography (CCP) to assess lung function in early-stage cystic fibrosis (CF). CCP has two components. The first is a particularly accurate technique for measuring gas exchange. The second is a computational cardiopulmonary model where patient-specific parameters can be estimated from the measurements of gas exchange. Twenty-five participants (14 healthy controls, 11 early-stage CF) were studied with CCP. They were also studied with a standard clinical protocol to measure the lung clearance index (LCI2.5). Ventilation inhomogeneity, as quantified through CCP parameter σlnCl, was significantly greater (P < 0.005) in CF than in controls, and anatomical deadspace relative to predicted functional residual capacity (DS/FRCpred) was significantly more variable (P < 0.002). Participant-specific parameters were used with the CCP model to calculate idealized values for LCI2.5 (iLCI2.5) where extrapulmonary influences on the LCI2.5, such as breathing pattern, had all been standardized. Both LCI2.5 and iLCI2.5 distinguished clearly between CF and control participants. LCI2.5 values were mostly higher than iLCI2.5 values in a manner dependent on the participant's respiratory rate (r = 0.46, P < 0.05). The within-participant reproducibility for iLCI2.5 appeared better than for LCI2.5, but this did not reach statistical significance (F ratio = 2.2, P = 0.056). Both a sensitivity analysis on iLCI2.5 and a regression analysis on LCI2.5 revealed that these depended primarily on an interactive term between CCP parameters of the form σlnCL*(DS/FRC). In conclusion, the LCI2.5 (or iLCI2.5) probably reflects an amalgam of different underlying lung changes in early-stage CF that would require a multiparameter approach, such as potentially CCP, to resolve.NEW & NOTEWORTHY Computed cardiopulmonography is a new technique comprising a highly accurate sensor for measuring respiratory gas exchange coupled with a cardiopulmonary model that is used to identify a set of patient-specific characteristics of the lung. Here, we show that this technique can improve on a standard clinical approach for lung function testing in cystic fibrosis. Most particularly, an approach incorporating multiple model parameters can potentially separate different aspects of pathological change in this disease.

The last decade has witnessed developments in the CF drug pipeline which are both exciting and unprecedented, bringing with them previously unconsidered challenges. The Task Force group was brought together to consider these challenges and possible strategies to address them. Over the last 18 months, we have discussed internally and gathered views from a broad range of individuals representing patient organisations, clinical and research teams, the pharmaceutical industry and regulatory agencies. In this and the accompanying article, we discuss two main areas of focus: i) optimising trial design and delivery for speed and efficiency; ii) drug development for patients with rare CFTR mutations. We propose some strategies to tackle the challenges ahead and highlight areas where further thought is needed. We see this as the start of a process rather than the end and hope herewith to engage the wider community in seeking solutions to improved treatments for all patients with CF.

This article was originally published with the sentence “Thank you for the opportunity to respond to the correspondence by J.G. Nielsen from Innovision about our recent paper”. The authors have since been made aware that J.G. Nielsen sold Innovision ApS (Glamsbjerg, Denmark) prior to the submission of his correspondence and, at the time of writing, has no financial interests in any business relating to lung clearance index technologies. This sentence has now been changed to “Thank you for the opportunity to respond to the correspondence by J.G. Nielsen about our recent paper” in the article itself.

The last decade has witnessed developments in the CF drug pipeline which are both exciting and unprecedented, bringing with them previously unconsidered challenges. The Task Force group came together to consider these challenges and possible strategies to address them. Over the last 18 months, we have discussed internally and gathered views from a broad range of individuals representing patient organizations, clinical and research teams, the pharmaceutical industry and regulatory agencies. In this and the accompanying article, we discuss two main areas of focus: i) optimising trial design and delivery for speed/efficiency; ii) drug development for patients with rare CFTR mutations. We propose some strategies to tackle the challenges ahead and highlight areas where further thought is needed. We see this as the start of a process rather than the end and hope herewith to engage the wider community in seeking solutions to improved treatments for all patients with CF.

Background KIWI (NCT01705145) was a 24-week, single-arm, pharmacokinetics, safety, and efficacy study of ivacaftor in children aged 2 to 5 years with cystic fibrosis (CF) and a CFTR gating mutation. Here, we report the results of KLIMB (NCT01946412), an 84-week, open-label extension of KIWI. Methods Children received age- and weight-based ivacaftor dosages for 84 weeks. The primary outcome was safety. Other outcomes included sweat chloride, growth parameters, and measures of pancreatic function. Results All 33 children who completed KIWI enrolled in KLIMB; 28 completed 84 weeks of treatment. Most adverse events were consistent with those reported during KIWI. Ten (30%) children had transaminase elevations >3 × upper limit of normal (ULN), leading to 1 discontinuation in a child with alanine aminotransferase >8 × ULN. Improvements in sweat chloride, weight, and body mass index z scores and fecal elastase-1 observed during KIWI were maintained during KLIMB; there was no further improvement in these parameters. Conclusions Ivacaftor was generally well tolerated for up to 108 weeks in children aged 2 to 5 years with CF and a gating mutation, with safety consistent with the KIWI study. Improvements in sweat chloride and growth parameters during the initial 24 weeks of treatment were maintained for up to an additional 84 weeks of treatment. Prevalence of raised transaminases remained stable and did not increase with duration of exposure during the open-label extension.

Synthetic biology designed cell-free biosensors are a promising new tool for the detection of clinically relevant biomarkers in infectious diseases. Here, we report that a modular DNA-encoded biosensor in cell-free protein expression systems can be used to measure a bacterial biomarker of Pseudomonas aeruginosa infection from human sputum samples. By optimizing the cell-free system and sample extraction, we demonstrate that the quorum sensing molecule 3-oxo-C12-HSL in sputum samples from cystic fibrosis lungs can be quantitatively measured at nanomolar levels using our cell-free biosensor system, and is comparable to LC-MS measurements of the same samples. This study further illustrates the potential of modular cell-free biosensors as rapid, low-cost detection assays that can inform clinical practice.

Pseudomonas aeruginosa (Pa) utilises quorum sensing (QS) to mediate several virulence factors including growth in biofilms. Intriguing in vitro data suggests QS signalling molecules can be “sensed” at the epithelial surface via the T2R38 (bitter taste) receptor expressed on air- way cilia (Lee RJ, et al. J Clin Invest. 2012;122:4145-59). Activation of this receptor is predicted to lead to changes in ciliary beat frequency and nitric oxide production, which may enhance bacterial clearance. Three common polymorphisms exist in the gene coding for this receptor, altering the amino acid sequence at positions 49, 262, and 296 and correlating with receptor function; the functional allele has proline-alanine-valine (PAV) whereas the non-functional allele has alanine-valine-isoleucine (AVI) at these residues. In vitro response to QS molecules has been shown to be greatest in cells with the PAV/PAV genotype. We hypothesised that the T2R38 receptor may be important in host defence against Pa in people with cystic fibrosis (CF) and that T2R38 genotype may therefore modify infection status and clinical outcomes.

Rapid evaporative ionization mass spectrometry (REIMS) is a new technique that has been shown to accurately classify yeast and bacteria. Thermal stress is applied onto bacterial colonies which results in evaporation and ionisation of metabolites and structural lipids. The mass spectrometer detects and identifies analytes through spectral database comparison (Strittmatter N, et al. Anal Chem. 2014;86:6555-62). We describe the application of REIMS to cystic fibrosis (CF)-related pathogens and show that the technology can identify P. aeruginosa with 100% accuracy. Differences were observed in the phospholipid and rhamnolipid range; derivatives of quorum sensing molecules (QSM) were also detected. These may provide useful biomarkers for a non-invasive diagnostic tool using ambient mass spectrometric (MS) approaches currently in development.

The UK Cystic Fibrosis Gene Therapy Consortium has been working towards clinical gene therapy for patients with cystic fibrosis for several years. We have recently embarked on a large, multi-dose clinical trial of a non-viral, liposome-based formulation powered for the first time to detect clinical benefit. The article describes the details of the protocol.

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.

Tabinda J Burney1,2,Jane C Davies1,2,31Department of Gene therapy, Imperial College London, 2UK CF Gene Therapy Consortium London, 3Department of Paediatric Respiratory Medicine,Royal Brompton and Harefield NHS Foundation Trust, London, UKAbstract: Gene therapy is being developed as a novel treatment for cystic fibrosis (CF), a condition that has hitherto been widely-researched yet for which no treatment exists that halts the progression of lung disease. Gene therapy involves the transfer of correct copies of cystic fibrosis transmembrane conductance regulator (CFTR) DNA to the epithelial cells in the airways. The cloning of the CFTR gene in 1989 led to proof-of-principle studies of CFTR gene transfer in vitro and in animal models. The earliest clinical trials in CF patients were conducted in 1993 and used viral and non-viral gene transfer agents in both the nasal and bronchial airway epithelium. To date, studies have focused largely on molecular or bioelectric (chloride secretion) outcome measures, many demonstrating evidence of CFTR expression, but few have attempted to achieve clinical efficacy. As CF is a lifelong disease, turnover of the airway epithelium necessitates repeat administration. To date, this has been difficult to achieve with viral gene transfer agents due to host recognition leading to loss of expression. The UK Cystic Fibrosis Gene Therapy Consortium (Imperial College London, University of Edinburgh and University of Oxford) is currently working on a large and ambitious program to establish the clinical benefits of CF gene therapy. Wave 1, which has reached the clinic, uses a non-viral vector. A single-dose safety trial is nearing completion and a multi-dose clinical trial is shortly due to start; this will be powered for clinically-relevant changes. Wave 2, more futuristically, will look at the potential of lentiviruses, which have long-lasting expression. This review will summarize the current status of translational research in CF gene therapy.Keywords: cystic fibrosis transmembrane conductance regulator (CFTR) gene, gene expression, gene transfer agents (GTAs), outcome measures

We will be undertaking a phase IIB clinical trial of repeated application of liposome-based gene therapy over a one year period in approximately 100 CF patients (Multidose Trial). In preparation for this, we sought to address two key questions. Firstly, could we define the optimal set of patients in which the therapy could both be delivered (good access to the airways via nebulisation), and in whom any therapeutic effect was measurable (one or more abnormal measures of lung disease). Secondly, in this set of ‘can deliver—can measure’ patients, which biomarker(s) could be powered to be the primary outcome measure for the trial. To address both questions, we undertook a study (Run-in), cross-sectionally assessing ‘can deliver’ and longitudinally assessing a large set of candidate biomarkers for ‘can measure’.192 patients from age 10 upwards, with FEV1 >40% were enrolled at two clinical centres; 154 of these remained in the study after four visits spaced at approximately 4–5 month intervals. Biomarkers assessed cross-sectionally included radionucleotide deposition scans, CT and mucocilary clearance. Longitudinal biomarkers included a large series of serum, sputum and exhaled breath inflammatory markers, lung physiology, exercise-related assays and quality of life assessment. 12 patients were judged too severe for adequate delivery and were excluded. A shortlist of 4 biomarkers was generated based on a) showing a CF/non-CF difference, b) response to course of intravenous antibiotics, and c) coefficients of variation. These four were matched against the remaining 142 patients, and a further seven patients excluded in whom none of these short listed biomarkers was abnormal. 89 patients (3 or 4 biomarkers abnormal) have been definitely included to progress into the Multidose Trial, and a further 46 (1 or 2 biomarkers abnormal) are awaiting the final primary outcome selection. The Run-in study has, therefore, been able to a) select a cohort of ‘optimal’ patients in which to assess gene therapy and b) provide an indication of which may be the more useful biomarkers to use in phase IIB clinical trials of novel therapeutic agents.

A successful discrimination of fish populations is essential for sustainable management and assessment. Otolith shape analysis has been used on several species to reveal their population structure. Our aim was to use the otolith shape of European sprat (Sprattus sprattus) to investigate large- and small-scale geographical variability across the Greater North Sea ecoregion. The otolith shape was extracted from digitalised images and transformed into Wavelet coefficients to be analysed with multivariate statistics. Otolith shape was observed to follow the genetic population structure recently defined for the region, supporting the latest revision of the stock boundaries. Four main groups were identified based on phenotypic variability: (i) Norwegian fjords; (ii) North Sea and offshore Skagerrak–Kattegat; (iii) coastal Skagerrak–Kattegat; and (iv) Uddevalla fjord. However, 4-fold cross-validations based on Linear Discriminant Analysis resulted in low accuracy limiting at the moment the ability to use otolith shape analysis for population identification at an operational basis. Our results show the importance of coastal areas, which might be inhabited by distinct populations of sprat that are currently not acknowledged in the management and assessment. publishedVersion