1. ¹Department of Gene Therapy, Imperial College, London, United Kingdom
2. ²Medical Genetics Section, University of Edinburgh, Edinburgh, United Kingdom
3. ³Gene Medicine Group, University of Oxford, Oxford, United Kingdom
4. ⁴UK Cystic Fibrosis Gene Therapy Consortium

Abstract

Cystic fibrosis (CF) has long been a model disease to study lung gene therapy and we and others have carried out a number of phase I/II clinical trials using viral or non-viral vectors carrying the cystic fibrosis transmembrane conductance regulator (CFTR) gene. In addition to safety, measurements of potential difference (PD) across the airway epithelium, which is altered in CF due to the altered ion transport, have often been included as a primary endpoint. However, although important, the information obtained from PD measurements in pre-clinical and clinical gene therapy research has to be interpreted carefully, because changes in PD after transfection with the CFTR gene do not provide information about the number of cells transfected. In addition, it is unclear, how a change in PD after gene therapy correlates with more clinically relevant endpoints. The UK CF Gene Therapy Consortium has, therefore, devoted a significant amount of time and resources to refine further existing assays and set-up and validate a panel of new pre-clinical and clinical assays to screen non-viral gene transfer agents.The CF knockout mouse is the only available CF animal model and although the CF mouse does not develop traditional CF lung disease, it has long been demonstrated that the CF ion transport abnormalities are recapitulated in the murine nasal epithelium. Importantly, the cell composition of the murine nasal epithelium is also similar to the human lower airways. We have chosen the FABp-CF knockout mouse (Zhou 1994) as the standard model in our core facility. Fatty acid binding promoter (FABp)-mediated expression of CFTR in the gut, but not the airways, prevents the intestinal disease of the CF knockout mouse, thus easing work in a core facility setting. When choosing assays we aimed to cover several aspects of defective CFTR level and function [1. CFTR protein (vector- and epithelial cell-specific detection of CFTR mRNA and protein). 2. Ion transport (PD and short circuit current measurements). 3. Airway surface liquid (ASL) (ASL height), 4. Host bacterial interaction (Bacterial adherence to airway epithelial cells). During assay validation we compared FABP-CF mice and wild-type littermates and used the results to carry out power calculations that informed cohort size when assessing efficiency of gene transfer agents. Importantly, we learnt that n numbers have to be large to detect small, but potentially clinically relevant, changes in these assays and that care has to be taken not to underpower assays for assumed treatment effects.