Repeated nebulisation of non-viral CFTR gene therapy in patients with cystic fibrosis: a randomised, double-blind, placebo-controlled, phase 2b trial

1Cystic fibrosis has been a target for gene therapy since the CFTR gene was cloned in 1989. Lung disease is the main cause of morbidity and mortality in individuals with cystic fibrosis, with a median age at death of 29 years (95% CI 27–31). Early expectations of a rapid breakthrough were based on supposed ease of access to the target respiratory epithelium via inhaled aerosols. These hopes were tempered by the subsequent realisation that the airways are well defended, in keeping with their predominant function as conducting passages, rather than absorptive surfaces.

2Evidence before this studyWe searched PubMed between June 1, 1992, and March 1, 2015, for studies published that included the terms “non-viral, gene therapy, cystic fibrosis” or “liposome, gene therapy, cystic fibrosis”.Added value of this studyWe report the first trial of non-viral CFTR gene therapy for patients with cystic fibrosis that is powered to detect clinically relevant pulmonary changes. Our study has progressed this field of research from phase 1 and 2a studies showing changes in molecular surrogates of CFTR function, to a phase 2b setting assessing changes in lung function in patients with a broad range of CFTR mutations. Additionally, our study shows that monthly repeated application of non-viral gene therapy can be safely administered to the lungs over a 1 year period.Implications of all the evidenceBy providing the first proof of concept that non-viral gene therapy can beneficially affect lung function, follow-up studies can assess optimum dose, dosing interval, and patient stratification at trial entry. Our findings are likely to catalyse earlier translation of more efficient vectors into first-in-man trials.

3Evidence before this study

4We searched PubMed between June 1, 1992, and March 1, 2015, for studies published that included the terms “non-viral, gene therapy, cystic fibrosis” or “liposome, gene therapy, cystic fibrosis”.

5Added value of this study

6We report the first trial of non-viral CFTR gene therapy for patients with cystic fibrosis that is powered to detect clinically relevant pulmonary changes. Our study has progressed this field of research from phase 1 and 2a studies showing changes in molecular surrogates of CFTR function, to a phase 2b setting assessing changes in lung function in patients with a broad range of CFTR mutations. Additionally, our study shows that monthly repeated application of non-viral gene therapy can be safely administered to the lungs over a 1 year period.

7Implications of all the evidence

8By providing the first proof of concept that non-viral gene therapy can beneficially affect lung function, follow-up studies can assess optimum dose, dosing interval, and patient stratification at trial entry. Our findings are likely to catalyse earlier translation of more efficient vectors into first-in-man trials.

9Various vectors for delivery of the CFTR gene into respiratory epithelial cells have been assessed. Viral approaches, including adenoviruses, adeno-associated viruses, and retroviruses, have faltered because of inefficient transduction from the luminal surface and immune responses restricting the efficacy of repeated application. As such, research from the UK Cystic Fibrosis Gene Therapy Consortium has initially focused on non-viral vectors. Formulation and delivery of plasmid DNA–liposome complexes have been refined in a large series of preclinical studies,, and safety,, molecular efficacy, and practical doses have been assessed in several phase 1 and 2a studies in patients with cystic fibrosis., We did this study to assess the clinical efficacy of the non-viral CFTR gene–liposome complex pGM169/GL67A after repeated delivery to the airways.

10We did this randomised, double-blind, placebo-controlled, phase 2b trial in two cystic fibrosis centres with patients recruited from 18 sites in the UK. Eligible participants had diagnosed cystic fibrosis, were aged 12 years or older, had a forced expiratory volume in 1 s (FEV1) of 50–90% predicted, and had any combination of CFTR mutations.

11The protocol was approved by the National Research Ethics Committee and the local Research Committees at the two dosing sites and the 16 other referral centres. Each patient, or a parent, provided written informed consent, and children provided assent.

12We randomly assigned patients (1:1), via a computer-based randomisation system, to receive nebulised pGM169/GL67A or 0·9% saline (placebo). Randomisation was stratified by % predicted FEV1 (<70 vs ≥70%), age (<18 vs ≥18 years), inclusion in the mechanistic substudy, and dosing site (London or Edinburgh). Participants in the mechanistic substudy were randomly assigned (2:1) to receive nebulised pGM169/GL67A or placebo, and could participate as part of either a nasal or bronchoscopy group, or both. Participants and investigators were masked to treatment allocation, with the randomisation code known only by pharmacy staff at the two dosing sites.

13Patients received 5 mL of either 0·9% saline or pGM169/GL67A complex nebulised through a Trudell AeroEclipse II device (Trudell Medical International, London, ON, Canada) at 28 day intervals (plus or minus 5 days) for 12 months. Each 5 mL dose of pGM169/GL67A contained 13·3 mg of plasmid DNA and 75 mg of the GL67A lipid mixture. Routine treatments were continued throughout the study, except for DNase, which was withheld for 24 h before and after dosing. In addition to the nebulised dose, patients in the nasal group of the mechanistic substudy received 2 mL of placebo or pGM169/GL67A divided between nasal cavities via a nasal spray device at the time of each lung dose. Patients in the bronchoscopy group followed the standard protocol, but also underwent a bronchoscopy under general anaesthesia before the first dose and 28 days (plus or minus 5 days) after the final dose.

14The primary efficacy endpoint was the relative change in % predicted FEV1, calculated from the mean of two baseline values (at screening and before dosing on day of the first dose) to the mean of two values (2 and 4 weeks after last dose) at study completion. Secondary outcomes included additional measurements of lung function, CT scans, and Cystic Fibrosis Questionnaire-Revised (CFQ-R) scores. Exploratory endpoints included exercise testing, activity monitoring, and sputum inflammatory markers. Mechanistic endpoints were nasal or bronchial vector-specific DNA, mRNA, and electrophysiological assessment of CFTR function. We did extensive safety assessments.

15The statistical analyses were prespecified in a statistical analysis plan. With use of pilot data, we estimated the standard deviation of the relative change in % predicted FEV1 in the target cystic fibrosis population to be 10% over 12 months. A total sample size of 120 assessable patients would provide 90% power to detect a 6% difference between groups in the mean change from baseline at a two-sided 5% significance level. This power calculation was conservative because covariate adjustment can be expected to increase statistical power. We did analyses in the per-protocol population (primary analysis), predefined as participants who received at least nine doses of pGM169/GL67A or placebo, and in the intention-to-treat population, who received at least one dose of pGM169/GL67A or placebo.

16We compared outcomes between groups with an ANCOVA model, with inclusion of the relevant baseline value, treatment allocation, and stratification factors (baseline predicted FEV1, age, dosing site, inclusion in substudy). Results are reported as adjusted mean differences with corresponding 95% CIs. We assessed subgroup effects by including the relevant interaction term in the ANCOVA model. To allow results from different endpoints to be plotted on a common scale, the estimated treatment effects were standardised and presented as multiples of the underlying SD. No adjustment was made to the p values to allow for multiplicity because the secondary endpoints were supportive and the corresponding p values were interpreted conservatively. We assessed bronchial and nasal biomarkers with a Mann–Whitney U test. A two-sided p value less than 0·05 was considered statistically significant.

17The trial was overseen by an independent Data Monitoring and Ethics Committee and a Trial Steering Committee. This trial is registered with ClinicalTrials.gov, number NCT01621867.

18The funder of the study had no role in study design, data collection, data analysis, data interpretation, or writing of the report. The corresponding author had full access to all the data in the study and had final responsibility for the decision to submit for publication.

19Figure 1 shows the trial profile. Between June 12, 2012, and June 24, 2013, we randomly assigned 140 patients to receive placebo (n=62) or pGM169/GL67A (n=78), of whom 136 (97%) patients comprised the intention-to-treat population and 116 (83%) patients comprised the per-protocol population (figure 1). Reasons for discontinuation in the intention-to-treat population were similar between groups (appendix). Baseline characteristics were similar between the two groups (table 1). Unless indicated otherwise, all subsequent details relate to the per-protocol population.

20114 (98%) patients had paired pre-treatment and post-treatment measurements of % predicted FEV1. Of the two patients (both in the placebo group) who did not have paired measurements, one patient could not do the test because of a surgery-related pneumothorax and one withdrew because of time commitments and was unavailable for follow-up measurements. We recorded a significant ANCOVA-adjusted treatment effect in the pGM169/GL67A group versus placebo at 12 months' follow-up (3·7%, 95% CI 0·1–7·3; p=0·046; figure 2) The relative changes within each of the individual groups were −4·0% (95% CI −6·6 to −1·4) in the placebo group and −0·4% (−2·8 to 2·1) in the pGM169/GL67A group (figure 2). Post-hoc analysis showed that 21 (18%) patients (n=6 in the placebo group and n=15 in the pGM169/GL67A group) had an improvement in % predicted FEV1 of 5% or more of their individual baseline values. For comparison, the treatment effect in patients in the intention-to-treat population who had spirometry measurements both before dosing and within the protocol-defined window after their final dose (n=56 in the placebo group and n=65 in the pGM169/GL67A group) was 3·6% (95% CI 0·2–7·0; p=0·039), with the 20 patients included in the intention-to-treat, but not per-protocol, analysis, receiving a mean of 3·7 doses (SD 1·9).

21Figure 3 summarises changes in a range of secondary outcomes. The treatment effect was significant for FVC (p=0·031; appendix) and CT gas trapping (p=0·048), but not for other measures of lung function, imaging, and quality of life (figure 3). We assessed whether a responder subgroup could be identified; the appendix summarises the prespecified subgroups. We noted no significant differences in the primary outcome treatment effect with respect to sex, age, CFTR mutation (phe508del homozygous vs other), Pseudomonas colonisation, predominant smaller or larger airway disease on CT at presentation, concurrent drugs, or treatment-associated adverse events (appendix). Although some subgroups had larger treatment effects than others, these results were typically due to a greater decline in FEV1 in the placebo group, rather than to any difference of effect in the pGM169/GL67A group (appendix). Stratification by baseline % predicted FEV1 suggested a difference, albeit non-significant, in treatment effect between patients with more severe disease (FEV1 49·6–69·2% predicted), who had a treatment effect of 6·4% (95% CI 0·8–12·1), and those with less severe disease (69·6–89·9% predicted), who had a treatment effect of 0·2% (−4·6 to 4·9; pinteraction=0·065; appendix). In patients with more severe disease, post-trial and pre-trial changes in both the placebo group (−4·9%) and the pGM169/GL67A group (1·5%) contributed to the treatment effect. Secondary outcomes showed a similar trend favouring the more severe category (appendix).

22Patients in both treatment groups received a median of three (IQR one to five) courses of oral or intravenous antibiotics during the trial. Specifically, we assessed co-administered antibiotics during the critical analysis period from dose 11 to the end of the trial. Numbers of patients receiving any additional antibiotics were 26 (48%) in the placebo group and 30 (51%) in the pGM169/GL67A group (χ2 p=0·774). Thus, the observed FEV1 treatment effect was considered to be independent of concurrent antibiotic courses.

23No clinically relevant pattern of changes could be distinguished in the exploratory outcomes of activity and exercise monitoring and serum and sputum inflammatory markers (appendix). In the bronchoscopy group of the substudy, vector-specific DNA increased in 12 (86%) of 14 patients in the pGM169/GL67A group and was below the limit of quantification in all (n=7) placebo samples (p=0·001; figure 4A); vector-specific mRNA was below the level of sensitivity in both groups (appendix). Changes in basal post-trial and pre-trial potential difference values did not differ significantly in either group (appendix). Figure 4B shows bronchial chloride responses using the mean of all interpretable tracings for each patient; a negative value indicates a change in the non-cystic fibrosis direction. Patients in the placebo group (n=7) had a median change (post-trial minus pre-trial) of 3·1 mV (range 9·3 to −1·2) and those in the pGM169/GL67A group (n=10) had a change of −1·3 mV (4·0 to −5·8; p=0·032; figure 4B). Five (50%) of ten patients in the pGM169/GL67A group had values that were more negative than the largest response in the placebo group (figure 4). In the same analysis with only the most negative value recorded for each patient at any timepoint, patients in the placebo group had a median post-trial minus pre-trial change of 2·6 mV (range 9·3 to −1·2) and those in the pGM169/GL67A group had a change of −2·8 mV (4·0 to −16·8 mV; p=0·088; figure 4C). Six (60%) patients in the pGM169/GL67A group had values that were more negative than the largest response in the placebo group (figure 4). The appendix shows absolute bronchial potential difference values.

24In patients in the nasal group of the substudy, vector-specific DNA increased in all the 17 patients given pGM169/GL67A. Despite apparent pGM169 contamination in some samples, the change in pGM169 concentrations differed significantly between the groups (appendix); no vector-specific mRNA was quantifiable in either group. We noted no significant changes in the baseline, zero chloride, or isoprenaline responses (appendix). Four (29%) of 14 pGM169/GL67A patients had mean post-trial minus pre-trial treatment responses (ranging from −3·4 mV to −7·0 mV) that were more negative than the largest response in the placebo group (n=6; appendix). The appendix shows absolute nasal potential difference values.

25All patients had adverse events, with no significant difference between groups for either total events or within the nine predefined adverse event categories (table 2). One patient in the placebo group and one patient in the pGM169/GL67A group discontinued study treatment because of adverse events (fatigue and increased respiratory symptoms and flu-like symptoms, respectively). We recorded six serious adverse events, all in the pGM169/GL67A group (appendix). Neither the Data Monitoring and Ethics Committee nor the Trial Steering Committee regarded any serious adverse event as related to study drug; however, one event was considered to be possibly related to a trial procedure (bronchoscopy). We noted no clinically relevant changes in haematology, biochemistry, conversion of anti-CFTR T cells, anti-DNA antibodies, histology, or lipid staining (appendix) and no patients died during the study.

26We report the first trial of non-viral based gene therapy for cystic fibrosis, powered to detect clinically relevant pulmonary changes. After monthly dosing for 1 year, we recorded evidence of a beneficial effect of gene therapy versus placebo on FEV1. No effect of sex, age, or whether patients were homozygous for the most common F508del CFTR mutation could be detected. No clinically important adverse events attributable to treatment with pGM169/GL67A were reported.

27Although these findings are encouraging, they should be put into perspective. We noted a stabilisation of FEV1 in the pGM169/GL67A group rather than an improvement. This stabilisation took place over a 1 year period and further work will be needed to see if this effect is maintained. The reduction in FEV1 in the placebo group was within the range reported in some other prospective trials,, and is consistent with a median survival of 29 years, but is greater than would be expected from registry data. Three factors are likely to have influenced this difference. First, the requirement for clinical stability at trial entry meant that patients might have been at their optimum respiratory health at this stage. Second, the enthusiasm of patients to enter the trial, accompanied by a focus on self-care, might have resulted in short-term improvements in lung function during the recruitment period. Both factors are likely to lead to a subsequent decline in lung function as patients regress to their mean values. Third, we included all available data, whether from stable patients or those with exacerbations, by contrast with registry data, which focuses on measurements obtained at annual review. Stabilisation of lung disease in itself is a worthwhile aim and we would caution against the bar being set too high for novel therapeutics in cystic fibrosis populations with an unselected range of mutations. The large response to ivacaftor in patients with class III mutations takes place in the context of correctly localised CFTR protein. By contrast, much smaller improvements in lung function were shown in the ivacaftor–lumacaftor trial for the most common mutation (phe508del) in which the CFTR protein is misfolded and mislocalised.

28The response in our study was heterogeneous, with apparent responders and non-responders. The data suggest that an approximate doubling of treatment effect was achieved in patients with more severe disease stratified by baseline FEV1, supported by trends in other clinically relevant secondary measures. A larger trial with a stratified trial entry design, powered to assess subgroups, and that addresses the mechanisms of response heterogeneity, will be important to verify or refute these data. This differential response could relate to the dose deposited in the airways; in patients with lower baseline FEV1 the relatively more obstructed smaller airways result in a larger proportion of the 5 mL dose being deposited in the larger airways. In pre-trial studies we assessed airway deposition in patients with cystic fibrosis with varying FEV1 severity with technetium-99m labelled human serum albumin of similar droplet size (3–4 μm, using a different nebuliser system) to the pGM169/GL67A formulation. Bronchial airway (generations 2–8) fractional deposition was 2·9% of delivered dose (standard error of the mean [SEM] 0·2; n=33) in patients with 70–90% predicted FEV1 and roughly twice as great (6·0%, SEM 1·0; n=23) in those with 50–70% predicted FEV1. An additional contributory factor to this enhanced efficacy might be the increased mitotic rate of more severely affected tissues, which decreases the proportion of time that the nuclear membrane is intact, the membrane acting as a barrier to plasmid DNA entry to the nucleus.

29We cannot rule out that the changes recorded in the present study are the result of a non-specific response to the pGM169/GL67A formulation. The placebo was 0·9% saline rather than a scrambled or CFTR-deleted plasmid–liposome complex. We selected 0·9% saline partly on the basis of pragmatic financial considerations, but mainly for ethical considerations, not wishing to expose patients with cystic fibrosis to first-in-man repeated pulmonary dosing of an untested product that might direct the expression of an immunologically active peptide or novel non-coding RNA molecule with deleterious biological functions. Furthermore, we wanted to compare progression on therapy with the natural history of the disease. In terms of alternative explanations for the effects we noted, we know of no evidence that monthly nebulisation of 0·9% saline is deleterious to lung function, nor that liposome alone produces physiological improvements in either patients without, or those with cystic fibrosis. Delivery of non-CFTR encoding plasmid DNAs to the human airways has not been associated with a gain in CFTR chloride-channel function, nor improvement in any cystic fibrosis-related assay,, and plasmid DNA is generally associated with pro-inflammatory, rather than non-specific, beneficial effects. We did not identify any pathophysiological changes in the airways, such as inflammation or remodelling, nor any changes in bacterial species that might otherwise explain the outcomes. Nevertheless, we cannot exclude that DNA–liposome complexes augment host defences, stimulate mucus clearance, or enhance bacterial killing to an extent undetectable on semi-quantitative routine culture.

30Results showing more robust changes in molecular CFTR surrogates would have been reassuring. Despite extensive optimisation of quantitative realtime-PCR assays, the pGM169-derived mRNA assay has poor sensitivity and is adversely affected by the inclusion of high levels of total RNA or modest concentrations of pGM169 plasmid DNA. In ovine studies we have shown that a 20 mL nebulised dose of pGM169/GL67A, four times that used in the present trial, is the lower threshold for reproducible detection of mRNA with this assay in airway tissue samples (unpublished). Thus, our inability to detect pGM169-derived mRNA after delivery of 5 mL of pGM169/GL67A to the human airways, although disappointing, was not surprising. In human tissues, we have noted the low sensitivity of assays assessing vector-specific mRNA from human samples in vivo,,, and have noted the greater sensitivity of detection of electrophysiological changes, consistent with findings in this study.,,

31The ratio of area sampled to area dosed is small. Although we recorded significant chloride secretory changes in the bronchial, but not the nasal, epithelium, we caution against placing undue weight on either observation. The size of the groups in the mechanistic substudy was limited by both the practicality of the procedures and the acceptability to patients of the additional invasive tests, leading to low statistical power for these measures. We would instead conclude that modest variable changes can be shown with currently available assays that remain insufficiently sensitive to detect changes in low levels of CFTR function when assessed in vivo in humans; further optimisation in these or other assays is needed.

32Although we are encouraged by the first demonstration of a significant beneficial effect in lung function compared with placebo associated with gene therapy in patients with cystic fibrosis, the mean difference was modest, only recorded in some individuals, and at the lower end of the range of results seen in clinical trials which result in changes in patient-related care., We did not formally assess infective exacerbations in view of the fairly small patient numbers in our study, but use of antibiotic courses as a surrogate identified no obvious treatment advantage. The treatment effect is consistent with a clinically meaningful benefit from the perspective of the European Medicine Agency; however, further improvements in efficacy and consistency of response to the current formulation, or its combination with CFTR potentiators, are needed before gene therapy is suitable for clinical practice. Furthermore, our findings should encourage the rapid introduction of more potent gene transfer vectors into early phase trials, now that much of the groundwork has been established.

33The data reported here provide the first proof of concept that repeated administration of non-viral CFTR gene therapy can safely change clinically relevant parameters, providing another step along the path of translational cystic fibrosis gene therapy.

34For the UK Cystic Fibrosis Gene Therapy Consortium see http://www.cfgenetherapy.org.uk

35For the UK Cystic Fibrosis Gene Therapy Consortium see http://www.cfgenetherapy.org.uk

36This online publication has been corrected. The corrected version first appeared at thelancet.com/respiratory on September 7, 2015

37This online publication has been corrected. The corrected version first appeared at thelancet.com/respiratory on September 7, 2015