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.

The safety profile of a cationic lipid-mediated cystic fibrosis gene transfer agent following repeated monthly aerosol administration to sheep.

Clinically effective gene therapy for Cystic Fibrosis has been a goal for over 20 years. A plasmid vector (pGM169) that generates persistent expression and reduced host inflammatory responses in mice has raised prospects for translation to the clinic. The UK CF Gene Therapy Consortium is currently evaluating long-term repeated delivery of pGM169 complexed with the cationic lipid GL67A in a large Multidose Trial. This regulatory-compliant evaluation of aerosol administration of nine doses of pGM169/GL67A at monthly intervals, to the sheep lung, was performed in preparation for the Multidose Trial. All sheep tolerated treatment well with no adverse effects on haematology, serum chemistry, lung function or histopathology. Acute responses were observed in relation to bronchoalveolar cellularity comprising increased neutrophils and macrophage numbers 1 day post-delivery but these increases were transient and returned to baseline. Importantly there was no cumulative inflammatory effect or lung remodelling with successive doses. Molecular analysis confirmed delivery of pGM169 DNA to the airways and pGM169-specific mRNA was detected in bronchial brushing samples at day 1 following doses 1, 5 and 9. In conclusion, nine doses of pGM169/GL67A were well tolerated with no significant evidence of toxicity that would preclude adoption of a similar strategy in CF patients.

An immunocytochemical assay to detect human CFTR expression following gene transfer.

BACKGROUND: To assess gene therapy treatment for cystic fibrosis (CF) in clinical trials it is essential to develop robust assays that can accurately detect transgene expression in human airway epithelial cells. Our aim was to develop a reproducible immunocytochemical assay for human CFTR protein which can measure both endogenous CFTR levels and augmented CFTR expression after gene delivery. METHODS: We characterised an antibody (G449) which satisfied the criteria for use in clinical trials. We optimised our immunocytochemistry method and identified G449 dilutions at which endogenous CFTR levels were negligible in CF samples, thus enhancing detection of transgenic CFTR protein. After developing a transfection technique for brushed human nasal epithelial cells, we transfected non-CF and CF cells with a clinically relevant CpG-free plasmid encoding human CFTR. RESULTS: The optimised immunocytochemistry method gave improved discrimination between CF and non-CF samples. Transfection of a CFTR expression vector into primary nasal epithelial cells resulted in detectable RNA and protein expression. CFTR protein was present in 0.05-10% of non-CF cells and 0.02-0.8% of CF cells. CONCLUSION: We have developed a sensitive, clinically relevant immunocytochemical assay for CFTR protein and have used it to detect transgene-expressed CFTR in transfected human primary airway epithelial cells.

Optimizing aerosol gene delivery and expression in the ovine lung.

We have developed the sheep as a large animal model for optimizing cystic fibrosis gene therapy protocols. We administered aerosolized gene transfer agents (GTAs) to the ovine lung in order to test the delivery, efficacy, and safety of GTAs using a clinically relevant nebulizer. A preliminary study demonstrated GTA distribution and reporter gene expression throughout the lung after aerosol administration of plasmid DNA (pDNA):GL67 and pDNA:PEI complexes. A more comprehensive study examined the dose-response relationship for pDNA:PEI and assessed the influence of adjunct therapeutic agents. We found that the sheep model can differentiate between doses of GTA and that the anticholinergic, glycopyrrolate, enhanced transgene expression. Dose-related toxicity of GTA was reduced by aerosol administration compared to direct instillation. This large animal model will allow us to move toward clinical studies with greater confidence.

Local lung responses following endobronchial elastase and lipopolysaccharide instillation in sheep.

Chronic lipopolysaccharide (LPS) exposure may contribute to the pathogenesis of a number of lung diseases including COPD and emphysema. We sought to develop a large-animal model of emphysema using repeated LPS administration into sheep lung segments. An experimental protocol was designed to facilitate comparisons with elastase-treated and control segments within the same lung of individual sheep. Histopathologic evaluation of segments treated with LPS demonstrated low-grade inflammation characterized by an increase in the number of intra-alveolar macrophages and lymphocytes. Treated segments demonstrated a significant reduction in airspace surface area (ASA), an increase in percent disrupted alveolar attachments and the distance between normal alveolar attachments, and a reduction in the number of normal alveolar attachments surrounding nonrespiratory bronchioles. Coefficient of variation of individual ASA measurements in elastase-treated segments was indicative of a heterogeneous parenchymal response, in contrast to that associated with chronic LPS treatment. Our results demonstrate that chronic LPS treatment of individual lung segments in sheep induces microscopic emphysema qualitatively and quantitatively consistent with both accepted pathologic definitions of this condition and with that produced by airway instillation of elastolytic enzymes. Development of this phenotype is associated with evidence of downregulated activation of transforming growth factor beta.

Trappin ovine molecule (TOM), the ovine ortholog of elafin, is an acute phase reactant in the lung.

As large animal models continue to play an important role in translating lung-directed therapeutic strategies from laboratory animals to humans, there is an increasing interest in the analysis of endogenous regulators of inflammation at both a genomic and a therapeutic level. To this end, we have sought to characterize the ovine ortholog of elafin, an important regulator of inflammation in humans. We have isolated both the elafin cDNA and gene, which have a similar structure to other species' orthologs. Interestingly, we have isolated two alleles for ovine elafin, which contain a very high number of transglutamination repeats, thought to be important in binding elafin to the interstitium. The mainly mucosal mRNA distribution for ovine elafin suggests that ovine elafin may, like its human ortholog, have functions in innate immunity. This is supported by analysis of elafin and the related protein secretory leukocyte protease inhibitor (SLPI) in ovine bronchoalveolar fluid in response to locally administered lipopolysaccharide and confirmation of them acting as "alarm" antiproteases. We have also cloned the ovine elafin cDNA into an adenoviral vector and have demonstrated correct processing of the secreted protein as well as biological activity. Overexpression of ovine elafin in a lung-derived epithelial cell line has a protective effect against the enzymes human neutrophil and porcine pancreatic elastase. The identification of the ovine elafin gene and its translated protein are important in developing practical strategies aimed at regulating inflammation in the large mammalian lung.

Transfection efficiency and toxicity following delivery of naked plasmid DNA and cationic lipid-DNA complexes to ovine lung segments.

We defined, using a novel large animal model system, the acute pathologic response to localized pulmonary administration of either naked plasmid DNA (pDNA) or cationic lipid-pDNA complexes (pDNA:GL67) and related such responses to concomitant indicators of transfection efficiency, namely levels of chloramphenicol acetyl transferase (CAT) protein and mRNA in specific lung tissue compartments. We instilled doses of 0.2, 1, and 5 mg pDNA to spatially distinct lung segments in six anesthetized sheep and doses of 0.2, 1, and 5 mg pDNA:GL67 to a further six sheep. Twenty-four hours after gene delivery the sheep were euthanized and necropsy examination with sampling of relevant tissues was carried out. Levels of plasmid-derived CAT-specific mRNA and CAT protein in samples derived from segments treated with either pDNA or pDNA:GL67 increased in relation to the administered dose. Levels of mRNA and protein expression were greater for pDNA:GL67 than for pDNA alone. A significant correlation was observed between mRNA and protein expression in samples derived from airways treated with pDNA:GL67. Histopathological changes following administration of both pDNA and pDNA:GL67 were characterized by a neutrophilic inflammation predominantly oriented on airways. The severity of the inflammatory response appeared to correlate with the administered dose of DNA and was generally more severe for pDNA:GL67.