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

BACKGROUND: Lung delivery of plasmid DNA encoding the CFTR gene complexed with a cationic liposome is a potential treatment option for patients with cystic fibrosis. We aimed to assess the efficacy of non-viral CFTR gene therapy in patients with cystic fibrosis. METHODS: We did this randomised, double-blind, placebo-controlled, phase 2b trial in two cystic fibrosis centres with patients recruited from 18 sites in the UK. Patients (aged ≥12 years) with a forced expiratory volume in 1 s (FEV1) of 50-90% predicted and any combination of CFTR mutations, were randomly assigned, via a computer-based randomisation system, to receive 5 mL of either nebulised pGM169/GL67A gene-liposome complex or 0.9% saline (placebo) every 28 days (plus or minus 5 days) for 1 year. 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 and investigators were masked to treatment allocation. The primary endpoint was the relative change in % predicted FEV1. The primary analysis was per protocol. This trial is registered with ClinicalTrials.gov, number NCT01621867. FINDINGS: 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 116 (83%) patients comprised the per-protocol population. We noted a significant, albeit modest, 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). This outcome was associated with a stabilisation of lung function in the pGM169/GL67A group compared with a decline in the placebo group. We recorded no significant difference in treatment-attributable adverse events between groups. INTERPRETATION: Monthly application of the pGM169/GL67A gene therapy formulation was associated with a significant, albeit modest, benefit in FEV1 compared with placebo at 1 year, indicating a stabilisation of lung function in the treatment group. Further improvements in efficacy and consistency of response to the current formulation are needed before gene therapy is suitable for clinical care; however, our findings should also encourage the rapid introduction of more potent gene transfer vectors into early phase trials. FUNDING: Medical Research Council/National Institute for Health Research Efficacy and Mechanism Evaluation Programme.

Low-frequency ultrasound increases non-viral gene transfer to the mouse lung.

The aim of the study was to assess if low-frequency ultrasound (US), in the range of 30-35 kHz, increases non-viral gene transfer to the mouse lung. US is greatly attenuated in the lung due to large energy losses at the air/tissue interfaces. The advantages of low-frequency US, compared with high-frequency US are: (i) increased cavitation (responsible for the formation of transient pores in the cell membrane) and (ii) reduced energy losses during lung penetration. Cationic lipid GL67/plasmid DNA (pDNA), polyethylenimine (PEI)/pDNA and naked pDNA were delivered via intranasal instillation and the animals were then exposed to US (sonoporation) at 0.07 or 0.1 MPa for 10 min. Under these conditions, US did not enhance GL67 or PEI-mediated transfection. It did, however, increase naked pDNA gene transfer by approximately 4 folds. Importantly, this was achieved in the absence of microbubbles, which are crucial for the commonly used high-frequency (1 MHz) sonoporation but may not be able to withstand nebulization in a clinically relevant setup. Lung hemorrhage was also assessed and shown to increase with US pressure in a dose-dependent manner. We have thus, established that low-frequency US can enhance lung gene transfer with naked pDNA and this enhancement is more effective than the previously reported 1 MHz US.

Identification and functional characterization of cytoplasmic determinants of plasmid DNA nuclear import.

Import of exogenous plasmid DNA (pDNA) into mammalian cell nuclei represents a key intracellular obstacle to efficient non-viral gene delivery. This includes access of the pDNA to the nuclei of non-dividing cells where the presence of an intact nuclear membrane is limiting for gene transfer. Here we identify, isolate, and characterize, cytoplasmic determinants of pDNA nuclear import into digitonin-permeabilized HeLa cells. Depletion of putative DNA-binding proteins, on the basis of their ability to bind immobilized pDNA, abolished pDNA nuclear import supporting the critical role of cytoplasmic factors in this process. Elution of pDNA-bound proteins, followed by two-dimensional sodium dodecyl polyacrylamide gel electrophoresis identified several candidate DNA shuttle proteins. We show that two of these, NM23-H2, a ubiquitous c-Myc transcription-activating nucleoside diphosphate kinase, and the core histone H2B can both reconstitute pDNA nuclear import. Further, we demonstrate a significant increase in gene transfer in non-dividing HeLa cells transiently transfected with pDNA containing binding sequences from two of the DNA shuttle proteins, NM23-H2 and the homeobox transcription factor Chx10. These data support the hypothesis that exogenous pDNA binds to cytoplasmic shuttle proteins and is then translocated to the nucleus using the minimal import machinery. Importantly, increasing the binding of pDNA to shuttle proteins by re-engineering reporter plasmids with shuttle binding sequences enhances gene transfer. Increasing the potential for exogenously added pDNA to bind intracellular transport cofactors may enhance the potency of non-viral gene transfer.

In vivo imaging of gene transfer to the respiratory tract.

Imaging of in vivo gene expression using luciferase expression in various organs has been used for several years. In contrast to other organs, in vivo imaging of the lung, particularly after non-viral gene transfer has not been extensively studied. The aim of this study was to address several questions: (1) Does in vivo light emission correlate with standard tissue homogenate-based luciferase detection in a dose-dependent manner? Recombinant Sendai virus (SeV) transduces airway epithelial cells very efficiently and was used to address this question, (2) Is the sensitivity of the assay sufficient to detect non-viral gene transfer? We treated mice with SeV-Lux vector using our standard "sniffing" protocol, a method that predominantly results in lung deposition. Dose-related in vivo light emission was visible in all animals. Importantly, there was a significant correlation (r>0.90, p<0.0001) between the in vivo and ex vivo assays in both the left and right lung. We next transfected the nasal epithelium via nasal perfusion or the lungs ("sniffing") of mice with a luciferase plasmid (pCIKLux) complexed to the cationic lipid GL67 (n=25-27/group) and imaged luciferase expression in vivo 24h after transfection. Gene expression was detectable in both organs. Correlation between the in vivo and ex vivo assays was significant (r=0.52, p<0.005) in the left, but not the right lung. The correlation in the nose was weaker (r=0.45, p<0.05). To our knowledge these studies show for the first time that this non-invasive method of assessing pulmonary gene transfer is viable for evaluating non-viral gene transfer agents.

Small chronic pneumothoraces and pulmonary parenchymal abnormalities after bone marrow transplantation.

The features of 4 allogeneic bone marrow transplant patients with an unusual late-onset complication of pulmonary abnormalities with small chronic pneumothoraces are described. Thin-section computed tomography demonstrated upper zone fibrotic changes and diffuse abnormalities suggestive of constrictive obliterative bronchiolitis. An important feature of the pneumothoraces was that they tended to be recurrent and small. We suggest that awareness of this unusual association may be useful in the radiologic evaluation of late-onset pulmonary complications after allogeneic bone marrow transplantation.

Reduction of persistent air leak with endoscopic valve implants.

The standard management of air leaks due to persistent bronchopleural fistula involves chest drainage and occasionally pleurodesis, with intractable cases requiring surgical decortication or surgical repair. However, some of these patients may be at high risk for surgery, particularly if they have already had thoracic surgery or have other medical problems; for this group there is a need for less invasive methods of stopping or reducing air leaks. Emphasys endobronchial valves (EBV) are occlusive devices designed primarily for endoscopic lung volume reduction in emphysema. Because the device is a one-way inspiratory airway blocker, it is possible that it could be used in controlling persistent air leaks while maintaining the drainage of secretions. Two cases are reported of persistent air leaks that were managed by endoscopic occlusion with EBV. In one case complete stoppage of the air leak was achieved with immediate clinical benefits. The second patient died 5 days after treatment from additional complications apparently not related to the procedure. Endobronchial blockage may be a useful salvage procedure for patients with persistent air leak for whom there is no other treatment available.

Airway glucose concentrations and effect on growth of respiratory pathogens in cystic fibrosis.

BACKGROUND: Pulmonary decline accelerates in cystic fibrosis-related diabetes (CFRD) proportional to severity of glucose intolerance, but mechanisms are unclear. In people without CF, airway glucose (AG) concentrations are elevated when blood glucose (BG)> or =8 mmol L(-1) (airway threshold), and are associated with acquisition of respiratory infection. METHODS: To determine the relationship between BG and AG, 40 CF patients underwent paired BG and AG (nasal) measurements. Daily time with BG>airway threshold was compared in 10 CFRD, 10 CF patients with normal glucose tolerance (CF-NGT) and 10 healthy volunteers by continuous BG monitoring. The effect of glucose at airway concentrations on bacterial growth was determined in vitro by optical densitometry. RESULTS: AG was present more frequently (85%-vs.-19%, p<0.0001) and at higher concentrations (0.5-3 mmol L(-1)-vs.-0.5-1 mmol L(-1), p<0.0001) when BG was > or =8 mmol L(-1)-vs.-<8 mmol L(-1). Daily time with BG> or =8 mmol L(-1) was CFRD (49+/-25%), CF-NGT (6+/-5%), healthy volunteers (1+/-3%), p<0.0001. Staphylococcus aureus growth increased at > or =0.5 mmol L(-1) (p=0.006) and Pseudomonas aeruginosa growth above 1-4 mmol L(-1) glucose (p=0.039). CONCLUSIONS: BG> or =8 mmol L(-1) predicted elevated AG concentrations in CF, at least in nasal secretions. CFRD patients spent approximately 50% day with BG>airway threshold, implying persistently elevated AG concentrations. Further studies are required to determine whether elevated airway glucose concentrations contribute to accelerated pulmonary decline in CFRD.

Evaluation of the porcine ameroid constrictor model of myocardial ischemia for therapeutic angiogenesis studies.

The porcine ameroid model of chronic myocardial ischemia has been widely used for the evaluation of coronary collateralization development. The impact of target vessel occlusion on the presence of myocardial ischemia, and the relationship between morphological, functional, and hemodynamic measurements in the context of therapeutic angiogenesis studies, however, has not been studied thus far. The authors therefore performed a systematic analysis of 94 animals undergoing ameroid constrictor placement around the left circumflex coronary artery (LCX) and, furthermore, a comprehensive evaluation including echocardiography and coronary angiography 26 +/- 1 (mean +/- SEM) days after ameroid placement. Complete LCX occlusion was observed in 34/94 animals (36%) and identified those with myocardial ischemia of the lateral wall, both at rest and under pharmacological stress. By applying a set of angiographic criteria (TIMI or= 1), another 27/94 animals with myocardial ischemia under conditions of pharmacological stress conditions could be identified. Interestingly, echocardiographic parameters of regional and global myocardial function were not correlated with myocardial blood flow or the degree of ischemia. There was no relationship between the extent of coronary collateralization, as assessed by angiography, echocardiographic parameters, or myocardial blood flow. The authors therefore conclude that complete occlusion of the ameroid instrumented coronary artery is not a prerequisite for successfully establishing the pathophysiology of myocardial ischemia. Defined angiographic criteria are important in identifying ischemic animals, thus reducing total animal numbers. Angiographic assessment of the degree of coronary collateralization, however, is not associated with myocardial blood flow or function and should not be used as a primary outcome measure of therapeutic angiogenesis studies in this model.

Bronchoscopic lung volume reduction for end-stage emphysema: report on the first 98 patients.

OBJECTIVES: To report the first multicenter experience on the treatment of end-stage emphysema using an endobronchial valve (EBV) [Emphasys EBV; Emphasys Medical; Redwood City, CA]. DESIGN: Retrospective analysis from prospective multicenter registry. PATIENTS AND INTERVENTIONS: This is a study of the use of EBVs in the treatment of end-stage emphysema at nine centers in seven countries. Ninety-eight patients with mean FEV(1) of 0.9 +/- 0.3 L (30.1 +/- 10.7% of predicted) [+/- SD] and residual volume (RV) of 5.1 +/- 1.3 L (244.3 +/- 0.3% of predicted) were treated over a period of 20 months. Spirometry, plethysmography, and diffusing capacity of the lung for carbon monoxide (Dlco) and exercise tolerance testing were performed at 30 days and 90 days after the procedure. RESULTS: RV decreased by 4.9 +/- 17.4% (p = 0.025), FEV(1) increased by 10.7 +/- 26.2% (p = 0.007), FVC increased by 9.0 +/- 23.9% (p = 0.024), and 6-min walk distance increased by 23.0 + 55.3% (p = 0.001). There was a trend toward improvement in Dlco, but this did not reach statistical significance (17.2 +/- 52.0%, p = 0.063). Patients treated unilaterally showed a trend toward greater improvement than those treated bilaterally. A similar trend toward improvement was observed in patients who had one entire lobe treated compared to those with just one or two bronchopulmonary segments treated. Eight patients (8.2%) had serious complications in the first 90 days, including one death (1.0%). CONCLUSION: This multicenter analysis confirms that improvement in pulmonary function and exercise tolerance can be achieved in emphysematous patients using EBVs. Future efforts should be directed to determining how to select those patients who would benefit most from this procedure and the best endobronchial treatment strategy.

Relationship between glycosylated haemoglobin and mean plasma glucose concentration in cystic fibrosis.

BACKGROUND: HbA(1c) is recommended for monitoring glycaemic control in people with cystic fibrosis-related diabetes (CFRD). However the relationship between HbA(1c) and mean plasma glucose concentration (MPG) has not been established in CFRD, as in other forms of diabetes mellitus. METHODS: 20 people (13 male, 29.7+/-8.8 years, 10 CFRD) with cystic fibrosis (CF) underwent HbA(1c) measurement and 48 h continuous glucose monitoring for estimation of MPG. The relationship between HbA(1c) and MPG was established and compared to the reported relationship for type 1 diabetes. RESULTS: HbA(1c) was strongly correlated with MPG (R(2)=0.888, p<0.0001) in CF. The relationship of MPG to HbA(1c) was described by the equation MPG=(1.47xHbA(1c))-1.15, giving a 1.47 mmol L(-1) change in MPG per 1% change in HbA(1c). This equation predicts that MPG in people with CF and HbA(1c) <7.0% will be similar to MPG in people with type 1 diabetes who achieve the same HbA(1c) target. CONCLUSIONS: These results imply that HbA(1c)<7.0% will predict good blood glucose control in CF as in type 1 diabetes. However, although HbA(1c) predicts complications in type 1 diabetes, further studies are required to establish the relationship between HbA(1c) and diabetic complications in people with CFRD.

Exploring the mechanisms of macrolides in cystic fibrosis.

Several studies have reported clinical improvements in cystic fibrosis (CF) patients on macrolide antibiotics although the mechanism of action remains unclear. We conducted an open-label study of azithromycin (500 mg daily for 2 weeks) in 9 adult CF patients to explore 3 possible mechanisms: up-regulation of the multi-drug resistance (MDR) or cystic fibrosis transmembrane regulator (CFTR) proteins, correction of epithelial ion transport and reduced bacterial adherence. End-points included nasal potential difference (PD) measurements, nasal epithelial MDR and CFTR mRNA levels and Pseudomonas aeruginosa adherence to nasal epithelium. Forced expiratory volume in the 1st second (FEV(1)) increased significantly after 2 weeks of azithromycin (pre- 41.1 [5.0]%; post- 44.6 [5.8]%; P<0.05), although improvements in forced vital capacity (FVC) did not reach significance (pre- 61.3 [4.0]%; post- 67.1 [5.4]%, NS). Before treatment all subjects had nasal PD measurements characteristic of CF. Treatment led to no significant group differences in any measures of either sodium absorption or chloride secretion. Neither CFTR nor MDR mRNA levels had altered significantly and the adherence of P. aeruginosa did not decrease. We conclude that these are unlikely to be significant contributing mechanisms accounting for the consistent beneficial results observed in clinical trials of macrolides in CF.

Vascular oligonucleotide transfer facilitated by a polymer-coated stent.

To evaluate the potential of clinically used phosphorylcholine (PC)-coated stents for their ability to load and release small decoy oligonucleotides (ODNs). Stents were loaded with 41 +/- 6 microg ODNs. Ex vivo deployment of ODN-loaded stents in explanted rabbit aortas showed significant vascular ODN transfer, with 18 +/- 12% of intimal or medial cell nuclei containing ODNs. In proof-of-principle in vivo experiments (using the double-injury rabbit model) there was no difference in fluorescent signal intensity between animals receiving ODNloaded stents or controls. However, a significant increase in signal intensity was detected in the kidneys of animals receiving ODN-loaded stents. PC-coated stents can be loaded with ODNs. Despite successful ex vivo ODN deposition and nuclear uptake in the vessel wall, in vivo vascular ODN transfer was not achieved. Rapid intravascular release of ODN before implantation and potential vascular barriers for gene transfer are most likely responsible for the currently unsatisfactory in vivo release kinetics.

The efficacy of a 'master switch gene' HIF-1alpha in a porcine model of chronic myocardial ischaemia.

AIMS: Therapeutic angiogenesis is a potential new treatment for patients unsuitable for conventional revascularization strategies. We investigated angiogenesis via a 'master switch gene' hypoxia inducible factor (HIF-1alpha). METHODS AND RESULTS: Ameroid occluders were placed around the left circumflex coronary artery of 74 pigs. Three weeks later, pigs were randomized to receive (i) adenovirus encoding HIF-1alpha (Ad2/HIF-1alpha VP-16 10(10) particles); (ii) plasmid DNA encoding HIF-1alpha (pHIF-1alpha NFkappaB 500 microg); (iii) pHIF-1alpha NFkappaB 2500 microg; and (iv) adenoviral control (Ad2/CMV-empty vector 10(10) particles). Twenty injections (50 microL each) were administered epicardially via re-thoracotomy. Three weeks after gene delivery significant (ANOVA P=0.02) changes in myocardial perfusion during stress were seen in the area adjacent to injections. Post hoc testing (Bonferroni) demonstrated that the AdHIF-1alpha group was significantly (P=0.02) different from the Ad2/control. There were also significant (ANOVA P=0.02) differences in resting left ventricular (LV) function. Post hoc (Bonferroni) showed that the AdHIF-1alpha group was significantly different from the Ad2/control (P=0.03). No significant changes in any parameter were seen with plasmid HIF-1alpha. There were no differences in collateralization or capillary growth. CONCLUSION: Ad2/HIF-1alpha increased myocardial perfusion and improved LV function. Plasmid HIF-1alpha was not associated with improvements in any bioactivity endpoints.

Potential difference measurements in the lower airway of children with and without cystic fibrosis.

Nasal potential difference measurements are valuable endpoint assays in clinical studies of novel treatments for cystic fibrosis (CF). Similar measurements made on the lower airway via the bronchoscope have been successful in adults, but have not been reported in children, the group most likely to benefit from such therapies. Here we report the design and validation of a small, single-lumen catheter technique allowing baseline potential difference and chloride secretion to be assessed in the distal airways of children as young as 1 year of age. Tracheal baseline values were significantly higher in children with CF than those without, although this was not the case more distally. In airways between the third and seventh generation, perfusion with a zero chloride solution containing isoprenaline led to a significant change in potential difference in children without CF, whereas no change was seen in those with CF. This measure provided a reliable distinguishing test between the two disease groups. We confirm that invasive bronchoscopic techniques can be performed safely and reliably in small children. Potential difference measurements could form a useful functional endpoint assay for future studies of either the CFTR gene or protein-based therapies in future trials in the pediatric age group.

Effect of bronchoscopic lung volume reduction on dynamic hyperinflation and exercise in emphysema.

Endobronchial valve placement improves pulmonary function in some patients with chronic obstructive pulmonary disease, but its effects on exercise physiology have not been investigated. In 19 patients with a mean (SD) FEV(1) of 28.4 (11.9)% predicted, studied before and 4 weeks after unilateral valve insertion, functional residual capacity decreased from 7.1 (1.5) to 6.6 (1.7) L (p = 0.03) and diffusing capacity rose from 3.3 (1.1) to 3.7 (1.2) mmol . minute(-1) . kPa(-1) (p = 0.03). Cycle endurance time at 80% of peak workload increased from 227 (129) to 315 (195) seconds (p = 0.03). This was associated with a reduction in end-expiratory lung volume at peak exercise from 7.6 (1.6) to 7.2 (1.7) L (p = 0.03). Using stepwise logistic regression analysis, a model containing changes in transfer factor and resting inspiratory capacity explained 81% of the variation in change in exercise time (p < 0.0001). The same variables were retained if the five patients with radiologic atelectasis were excluded from analysis. In a subgroup of patients in whom invasive measurements were performed, improvement in exercise capacity was associated with a reduction in lung compliance (r(2) = 0.43; p = 0.03) and isotime esophageal pressure-time product (r(2) = 0.47; p = 0.03). Endobronchial valve placement can improve lung volumes and gas transfer in patients with chronic obstructive pulmonary disease and prolong exercise time by reducing dynamic hyperinflation.

The effect of varying tonicity on nasal epithelial ion transport in cystic fibrosis.

There is reasonable evidence that the fluid layer of the airway epithelium is exposed to changes in tonicity. The inspiration of cool, dry air causes an increased tonicity, whereas this tonicity may be decreased by glandular secretions. We hypothesized that the cystic fibrosis transmembrane conductance regulator (CFTR) is involved in the responses to changes in tonicity and that these may be altered in cystic fibrosis (CF). Using nasal potential difference (PD) protocols in 8 subjects with CF and 10 subjects without CF, we investigated the effects of hyper- and hypotonicity on ion transport processes. We found significant differences between the two groups. In response to a hypertonic challenge (mannitol 500 mM), there was a decreased PD in both groups, suggesting decreased sodium absorption. However, after the prior inhibition of sodium transport using amiloride, there was an increased PD in the non-CF group alone, suggesting CFTR-mediated chloride secretion in response to luminal hypertonicity. For the hypotonic solution, we found that hypotonicity inhibited CFTR-mediated chloride secretion in the non-CF group. These data suggest that CFTR plays a role in the recognition and regulation of airway fluid tonicity.

Advances in cystic fibrosis gene therapy.

PURPOSE OF REVIEW: The first cystic fibrosis gene therapy trials were carried out in 1993, and although proof-of-principle for gene transfer to the lungs was established, efficiency was generally low. The authors review the most recent advances in preclinical airway gene transfer and summarize the results from the latest clinical trials. RECENT FINDINGS: Recent clinical trials report encouraging results. Repeat administration of adeno-associated virus to the lung was safe. Nonviral nanoparticles used, for the first time, in the nose of cystic fibrosis patients were also safe and led to partial correction of the chloride transport defect in nasal epithelium. Important advances have been made in preclinical research, including the development of new viral and nonviral gene transfer agents and improved plasmid DNA. In addition, physical delivery methods, such a magnetofection and electroporation, are being assessed to improve nonviral gene transfer. SUMMARY: Considerable progress has been made in understanding and overcoming the problems associated with gene transfer to airway epithelial cells, the target cells for cystic fibrosis gene therapy. It has also been recognized that novel preclinical and clinical assays are crucial for the success of cystic fibrosis gene therapy, and considerable effort is currently being put into assay development and trial designs.

Critical appraisal of the mouse model of myocardial infarction.

In order to critically evaluate the utility of a mouse model of myocardial infarction (MI) for therapeutic studies, we investigated survival, haemodynamic measurements and histopathology in mice with an occluding suture placed at one of three distinct sites along the left anterior descending coronary artery. The suture was placed at the atrioventricular juncture (High), or at two sites more distally towards the base (Middle and Low). In the High group, only 33% of animals survived 7 days after MI (P < 0.05 compared to all other groups). Only the Middle group had significantly reduced haemodynamics compared to sham-operated animals (maximum left ventricular pressure: 55.9 +/- 3.5 versus 80.8 +/- 5.1 mmHg, maximum change in pressure over time : 2003 +/- 172 versus 4402 +/- 491, P < 0.01). Histological examination showed morphological changes in all MI groups. The Middle group had larger lesions than the Low group (P < 0.05). Lesions in the anterior and lateral walls correlated, albeit weakly, with cardiac function. Power calculations indicated that, despite a certain amount of intragroup variation, the Middle Suture model may be useful for therapeutic studies to assess the effects of treatment on cardiac function and overall lesion size.