Christopher Boyd

534. Preparation for a First-in-Man Lentivirus Trial in Cystic Fibrosis Patients

Background: We have recently shown that non-viral gene therapy can stabilise the decline of lung function in cystic fibrosis (CF) patients. However, the effect was modest, and it is important to develop more potent gene transfer agents in parallel. F/HN-pseudotyped lentiviral vectors are more efficient for lung gene transfer than non-viral vectors in pre-clinical models. In preparation for a first-in-man CF trial using the lentiviral vector we have undertaken key translational pre-clinical studies. Methods: Regulatory-compliant vectors carrying a range of promoter/enhancer elements were assessed in mice and human air liquid interface cultures to select the lead candidate; CFTR expression and function were assessed in CF models (knockout mice and human intestinal organoids) using this lead candidate vector. Toxicity was assessed and “benchmarked” against the leading non-viral formulation recently used in a Phase IIb clinical trial. Integration site profiles were mapped and transduction efficiency determined to inform clinical trial dose-ranging. The impact of pre-existing and acquired immunity against the vector and vector stability in several clinically relevant delivery devices was assessed. Results: A hybrid promoter consisting of the elongation factor 1α promoter and the CMV enhancer was most efficacious in both murine lungs and human air liquid interface cultures. The efficacy, toxicity and integration site profile supports further progression towards clinical trial and pre-existing and acquired immune responses do not interfere with vector efficacy. The lead rSIV.F/HN candidate expresses functional CFTR and the vector is stable in clinically relevant delivery devices. Conclusions: The data support progression of the F/HN pseudotyped lentiviral vector into a first-in-man CF trial due to start in Q2 2017. Regulatory-compliant toxicology studies are currently being performed.

Longitudinal assessment of biomarkers for clinical trials of novel therapeutic agents: the run-in study

We will be undertaking a phase IIB clinical trial of repeated application of liposome-based gene therapy over a one year period in approximately 100 CF patients (Multidose Trial). In preparation for this, we sought to address two key questions. Firstly, could we define the optimal set of patients in which the therapy could both be delivered (good access to the airways via nebulisation), and in whom any therapeutic effect was measurable (one or more abnormal measures of lung disease). Secondly, in this set of ‘can deliver—can measure’ patients, which biomarker(s) could be powered to be the primary outcome measure for the trial. To address both questions, we undertook a study (Run-in), cross-sectionally assessing ‘can deliver’ and longitudinally assessing a large set of candidate biomarkers for ‘can measure’.192 patients from age 10 upwards, with FEV1 >40% were enrolled at two clinical centres; 154 of these remained in the study after four visits spaced at approximately 4–5 month intervals. Biomarkers assessed cross-sectionally included radionucleotide deposition scans, CT and mucocilary clearance. Longitudinal biomarkers included a large series of serum, sputum and exhaled breath inflammatory markers, lung physiology, exercise-related assays and quality of life assessment. 12 patients were judged too severe for adequate delivery and were excluded. A shortlist of 4 biomarkers was generated based on a) showing a CF/non-CF difference, b) response to course of intravenous antibiotics, and c) coefficients of variation. These four were matched against the remaining 142 patients, and a further seven patients excluded in whom none of these short listed biomarkers was abnormal. 89 patients (3 or 4 biomarkers abnormal) have been definitely included to progress into the Multidose Trial, and a further 46 (1 or 2 biomarkers abnormal) are awaiting the final primary outcome selection. The Run-in study has, therefore, been able to a) select a cohort of ‘optimal’ patients in which to assess gene therapy and b) provide an indication of which may be the more useful biomarkers to use in phase IIB clinical trials of novel therapeutic agents.

P106 Inflammatory markers: data from the UK CF Gene Therapy Consortium Run-In Study

Inflammatory markers in sputum and serum have been used with variable success as outcome measures in interventional studies. Limited data are available on reproducibility of such assays in cystic fibrosis (CF) particularly over a long time period. This study was designed to address this; stable patients (FEV1>40, >10 years age) were recruited into the study which ran over an 18-month period with up to four hospital visits. Patients provided a 24 h sputum collection, for weighing at each visit. Spontaneous sputum was collected at the beginning of each visit; if insufficient sample was obtained, sputum was induced with hypertonic saline. Inflammatory markers were measured in dithiothreitol-processed sputum (total and differential cell counts, IL8 (and other cytokines), calprotectin, neutrophil elastase, myeloperoxidase and extracellular DNA). Blood was collected at each visit for cytokines (IL1β, IL6, IL8, IL10, IL12 (p40) and TNFα) and calprotectin. Data are available from 189 patients at 655 visits. Adequate sputum for analysis was obtained at only 60% of visits. Sputum induction accounted for only 16% of adequate samples. Median and range for each detectable assay are shown below. Serum cytokines IL1β, IL10, IL12 (p40) and TNFα were undetectable at each visit, and IL6 was only detectable in 17% of samples. To assess intra-individual variability the coefficient of variation of results across each visit for each patient is presented. Both sputum and serum assays showed a large range of results at each visit, but the variation for each individual was much higher than the ideal 10%. Serum assays were not able to discriminate between CF and non-CF, apart from calprotectin (CF 10.1(0.8–72.5) vs non-CF 0.40 (0.2–1.12) p<0.001). Due to the difficulty in obtaining sputum samples reliably and the large variability of results between visits in these stable patients, we consider it unlikely that a change due to a new therapy would be detectable. As such, we are not considering sputum inflammatory markers as primary or secondary efficacy endpoints in our multidose gene therapy trial. Serum markers also appear to be of limited use is assessing efficacy but will still be useful for toxicology and safety studies.Abstract P106 Table 1