Background: Cystic fibrosis (CF) is a genetic disease caused by mutations in the CF transmembrane conductance regulator (CFTR) protein. CF results in airway surface dehydration and the inability to clear mucus, which leads to chronic infections, inflammation, and premature death most commonly due to respiratory failure. Gene therapy can be used to insert a correct copy of the CFTR gene into the genome to restore organ function by fixing the underlying genetic condition independent of CFTR mutation type. Lentiviruses (LV) are a promising gene therapy vehicle as they stably integrate the therapeutic gene into the genome. To achieve permanent genetic correction, we are focusing on developing vectors to improve cell targeting and specificity to overcome the current challenges of CF gene therapy. Methods: LV vectors containing reporter genes (LacZ, Luc-GFP) or epitope-tagged CFTR were developed and tested in vitro. Subsequently, each vector was administered to the nasal epithelia or lungs of CF mice or rats. Transduction efficiency, duration, localisation and CFTR function were assessed using histology, electrophysiological measurements, and in vivo bioluminescence imaging. Results: LV vectors containing reporter genes or CFTR have been shown to successfully transduce the lung and express transgenes long-term. Non-specific alveoli cells and macrophages are transduced in conjunction with a low proportion of therapeutically-relevant basal cells. Importantly, LV vector containing tagged or non-tagged CFTR produced functional correction of the nasal potential difference with tagging of CFTR having no effect on CFTR function. Conclusions: We have shown that our vector has the potential for long-term expression, and can correct CF mutation-induced electrophysiological defects. Using vector-surface engineering strategies, we now aim to overcome the current challenges of basal cell targeting, immune responses, and off-target transduction of non-therapeutic cell types. Combatting these critical obstacles will enable full and rapid progress in gene therapy treatment for this life-limiting disease.