Cystic Fibrosis, a recessive genetic disease. Mutations within the cystic fibrosis transmembrane regulator (CFTR) gene can impair the function of the CFTR anion channel and disrupt the mucociliary escalator; the first line of defense against lung infection. Due to the monogenetic nature of the disease and the accessibility of the airways to nebulizers, CF lung disease is an obvious candidate for gene therapy. It is unclear how many cells expressing functional CFTR are required for restoration of ion channel activity in CF models whilst considering the downregulatory effect of the abnormal sputum produced in the CF lung (including upregulated cytokines, bacterial toxins, altered pH). With gene editing technology continually improving, we want to identify how much functional CFTR is required to restore activity of CF cultures in the presence of CF sputum. To answer this question we used two models: human bronchiolar epithelial cell cultures of mixed CF (CFBE):normal (NHBE; a model for gene editing) from alternate sexes and CFBE transduced with CFTR under the expression of a high activity promoter (a model for gene therapy). Cells were differentiated at airway-liquid interface. Cells were incubated with apically applied normal or CF induced sputum pooled from 9 donors before functional analysis. Digital droplet PCR (ddPCR) and immunohistochemistry were performed for characterisation. Short circuit current (Isc) and airway surface liquid (ASL) height were measured as functional read-outs. We demonstrated a 41% decrease (n=5, p<0.001) in CFTR-mediated Isc and a loss of vasoactive intestinal peptide (VIP, activator of CFTR) stimulated ASL secretion (n=5, p<0.001) after incubation with CF sputum compared to normal sputum. Using ddPCR analysis of AMEL-X/Y to accurately determine the number of CFBE vs NHBE in the mixed cell cultures, we observed that CFTR-mediated Isc increased in a linear fashion with the percentage NHBE cells (R2=0.91, n=28). The VIP-stimulated increase in ASL height followed a similar trend. In comparison, ~16% transduction of CF cultures with CFTR was sufficient to fully restore Cl- secretion and increase ASL height (n=6) in the presence of CF sputum. Our data implies that overexpression rather than endogenous expression of CFTR is more effective at restoring function in a CF culture. However, this technology is limited by its transient and immunoreactive nature. Currently, permanent restoration of function in the CF lung would require an unachievable level of gene editing events. Thus, we propose the use of an enhanced CFTR gene we have developed for gene editing. Codon optimization and a substitution mutation in cytosolic loop 3 produced ~7 fold greater processing in to protein and dramatically increased anion transport activity as shown by a YFP quenching assay in Hek293T cells (n=3). It is our hope that this enhanced gene could be used to reduce the editing events necessary to restore function in the CF lung.