A Phase I/IIa Safety and Efficacy Study of Nebulized Liposome-mediated Gene Therapy for Cystic Fibrosis Supports a Multidose Trial.

The vast majority of treatments for cystic fibrosis (CF) target the downstream consequences of the disease and are incompletely effective. The success of the CF transmembrane conductance regulator (CFTR) potentiator ivacaftor has illustrated the clinical benefits arising from restoration of CFTR protein function (1). This agent is applicable as a monotherapy for a minority of patients with specific, rare mutations. CFTR gene therapy, a mutationindependent alternative, has demonstrated proof of principle for gene transfer in animal models and human trials, but only one study (using a viral vector) has unsuccessfully assessed whether clinical outcomes can be improved (2). In preparation for a phase IIb clinical trial of repeatedly administered, nonviral, liposome-mediated CFTR gene transfer assessing clinically meaningful outcomes (3), the UK CF Gene Therapy Consortium (www.cfgenetherapy.org.uk) undertook a single-application safety and dose-ranging study (NCT00789867). Some of the results of these studies have been previously reported in the form of abstracts (4, 5). The chosen plasmid DNA expresses CFTR under the control of the human cytomegalovirus enhancer/elongation factor 1a sequence (6), a modified EF1a promoter aiming for extended duration of expression (7), and was rendered CpG-free to minimize a host inflammatory response (6). The cationic lipid, GL67A, was chosen on the basis of extensive preclinical testing (8). After informed consent, adult patients with CF received a single nebulized 1/2 nasal dose of pGM169/GL67A. Reconstitution and preparation of pGM169/GL67A was undertaken on the day, and doses were delivered in sealed negative-pressure cubicles after pretreatment with inhaled salbutamol (albuterol). Preplanned adjunctive therapies including ibuprofen, prednisolone, or paracetamol were administered to some patients. The primary outcome of the clinical study was safety; assessment included examination, standard hematology/ biochemistry, adverse events, spirometry, lung clearance index, chest computed tomography scan, gas transfer, bronchial biopsy histology, and immune markers. pGM169-specific DNA and mRNA were measured on nasal and lower airway brushings, with potential difference also measured bronchoscopically and nasally. For the latter, “responders” were defined as demonstrating chloride secretion 5 mV or more greater than their mean predose value, and greater than any of their predose responses. A total of 35 subjects (Tables 1 and 2) received a nebulized dose (5 ml, n = 8; 10 ml, n = 10; 20 ml, n = 17) via an AeroEclipse II (Trudell Medical International, London, ON, Canada) breathactuated nebulizer (9). Three subjects undertook slow delivery (z75 vs. 25 min for each 5 ml). Standard spray devices were used for nasal delivery (2 ml, n = 21). According to pre-/post-device weighing, a mean (SD) of 88.7% (2.9%) of expected nebulized dose and 94.5% (15.0%) of expected nasal dose was delivered. There were two serious adverse events: one occurred after the predosing bronchoscopy (swelling of the uvula related to intubation) and led to observation overnight in hospital, and the other was an episode of pancreatitis occurring around Day 10 after dosing (10 ml nebulized cohort). The subject was exocrine pancreatic sufficient and had likely experienced previous similar, but undiagnosed, episodes. Overall, in the trial, 94.3% of subjects experienced at least one adverse event, the majority of which were mild to moderate in severity and resolved spontaneously or with standard antipyretics. The most common occurred on the day of dosing and largely resolved within 24–48 hours (Tables 1 and 2): Typically, within the first few hours after dosing, a mild, self-limiting influenza-like systemic response was seen, most frequently in the 20-ml patients. This was not affected by slow delivery or coadministration of ibuprofen or prednisolone but was clearly dose-related and reduced by paracetamol. Symptoms of headache and/or tiredness were reported by 82, 70, and 13%, and raised serum inflammatory markers were recorded in 100, 60, and 63% of the 20-, 10-, and 5-ml groups respectively, with dose-related trends in maximal values. No patient dosed with 5 ml had a temperature higher than 388C (Table 2). A relatively asymptomatic, dose-related, restrictive drop in spirometry was also observed, with no change in respiratory rate or oxygen saturation. No patient dosed with 5 ml showed a more than 20% relative fall in FEV1 (Table 2). The 20-ml group showed a small, significant (P, 0.05) mean (SD) drop in gas transfer (transfer factor for carbon monoxide corrected for alveolar volume and hemoglobin concentration) on Day 2 of 4.5% (6.0%), which returned to baseline values by Day 14. No changes were seen in the other cohorts. Two of the 20-ml patients had small areas of ground glass opacity reported on their Day 2 chest computed tomography scans, which resolved by Day 14. No significant changes were seen in endobronchial histology (20 ml; n = 10). Consistent with the proposed excretion route for lipids, small but significant serum creatinine rises within the normal range could be detected 8 hours after dosing in the 20and 10-ml groups, but not the 5-ml cohort; there were no other biochemical changes. Bilirubin rose on Day 1 in all dosing groups, as with creatinine, remaining within the normal range, and normalized by Day 2. There was no evidence of immune responses based on double-stranded DNA antibodies or human CFTR-specific T cells. Lung clearance index, a sensitive marker of pulmonary dysfunction (10), was included as a safety assay. Fourteen 20-ml patients with paired predosing and 28-day postdosing values showed a small but significant increase (i.e., a deterioration; Figure 1A). In contrast, and unexpectedly, on post hoc analysis, 11 of 14 patients in the lower-dosing groups (5 and 10 ml) showed a small but significant improvement (Figure 1A). With respect to bronchial samples, 10 patients (all 20 ml) had paired preand postdosing bronchoscopies. pGM169-specific DNA was detected in all bronchial brushing samples at levels z1000-fold Supported by the UK Cystic Fibrosis Trust and the National Institute for Health Research Respiratory Biomedical Research Unit at the Royal Brompton & Harefield NHS Foundation Trust and Imperial College London and the Biomedical Research Centre, based at Imperial College Healthcare NHS Trust and Imperial College London.