Cystic Fibrosis (CF) is life threatening single gene disorder that affects the lungs, pancreas, intestines and reproductive tract. All cases of CF can be ascribed to the inheritance from both parents of a mutant allele of the cystic fibrosis transmembrane conductance regulator gene, CFTR. The CFTR protein regulates the flow of water and ions across secretory epithelia. Whereas the effects of CFTR deficiency in the pancreas and intestines are relatively well managed, the repeated lung infections and progressive loss of lung function are not so. Gene therapy has the potential to halt this progression if safely and effectively delivered to the lung. Pilot studies in the mid 90’s suggested that this might be possible, but expression was transient. In a ten year collaborative programme, the UK CF Gene Therapy Consortium has made significant progress on several fronts to improve the safety and efficacy of these original formulations. Here, I will describe that progress towards the clinical, the results from a Phase 1 dose-ranging study and the status of a follow-on multi-dose Phase 2 study. Results – In a Phase 1 dose-ranging trial to assess safety, a single dose of pGM169/GL67A was administered to CF patients by spray to the nasal epithelium and by nebulisation to lower airways. pGM169 is a CpG-free plasmid encoding a human CFTR cDNA driven by a CMV enhancer and human elongation factor 1 alpha (hCEFI) promoter. GL67A is a lipid gene transfer agent produced by Genzyme. The gene therapy formulation is prepared simply by mixing the two components allowing the lipid to complex with the plasmid DNA. CFTR transgene expression in the airways was measured using QRT-PCR on epithelial cells harvested by brushing. Nasal and bronchial potential differences (PD) were also measured. Thirty-five adult CF patients were recruited with a baseline FEV1 (forced expiratory volume in 1 sec; a standard measure of lung function) of at least 60% predicted. Each was nebulised with 20 mL (n=17), 10 mL (n=10) or 5 mL (n=8) of pGM169/GL67A. A transient drop in FEV1 was observed over the next 6 h (mean [SD]: 20 mL 25.7[10.2]%; 10 mL 17.7[9.9]%; 5 mL 13.0[4.4]%); this was dose-related, well-tolerated, not accompanied by desaturation, showed a restrictive pattern and did not respond to bronchodilators. A similarly dose-related systemic inflammatory response was also noted, the symptoms of which responded well to standard anti-pyretics. Generally this was confined to the first 24-48 hours after dosing. A rise in white blood cell count was observed which correlated significantly with the magnitude of the drop in FEV1, implying that the acute lung function effect may be an inflammatory phenomenon. Interestingly, none of a subset of 6 patients (4 from the 10 mL dose and 2 from the 5 mL dose) given 4 g paracetamol over an 18 hour period after dosing developed a fever: these subjects also displayed less marked systemic inflammatory responses. This phenomenon may be related to the pulmonary inflammation attenuation effects of paracetamol observed in influenza-infected mice and reported by Lauder et al [Thorax (2011) 66:368]. Pre- and post-dosing (day 6 or day 14) bronchoscopies were conducted on 10 patients. QRT-PCR detected CFTR transgene expression in samples from some of this cohort In bronchial PD measurements, the majority of the patients’ zero chloride responses to isoproterenol increased towards non-CF values after dosing. Eleven of the 19 patients receiving a 2 mL nasal dose responded when assessed by nasal PD measurement: a positive response was defined as a chloride secretory response outside the range of the subject’s pre-dose measurements. Intriguingly, PD was noticeably more often positive when measured at later time points after dosing. In the two best responders, maximal zero chloride responses exceeded 10 mV (clearly within the normal non-CF range) and persisted to days 63 and 91 respectively. This is concordant with the plasmid’s engineered ability to build up transgene expression slowly, but maintain peak expression for an extended period. Transgene expression was seen in nasal and lower airways, and restoration of CFTR function to the non-CF range was observed by PD measurement out to 91 days following a single nasal dose. Conclusions/Implications – These data, together with our supportive toxicology results, encourage us to embark on a repeated dosing clinical trial. We regard the pulmonary and systemic side effects observed after the 20 mL nebulised dose as undesirable in a repeated administration context. Those seen with 10 mL were more acceptable: 5 mL produced only small effects. Simple antipyretic treatment moderates the symptoms, and the repeated dosing clinical trial will use 5 mL.