Background: A limitation to efficient lentivirus‐mediated airway gene transfer is the lack of receptors to commonly used viral envelopes on the luminal surface of airway epithelia. The use of viral envelopes with natural tropism to the airway could be useful for overcoming this limitation. Methods: We investigated influenza hemagglutinin (HA) pseudotyped equine infectious anemia virus‐derived lentiviral vector‐mediated gene transfer to the airway epithelium of adult and newborn mice. For these studies, high‐titer vectors were delivered by intranasal administration. In addition, we tested the feasibility of vector re‐dosing to the nasal airway. Results: Delivery of high‐titer HA pseudotyped lentiviral vectors by nasal administration to newborn mouse pups or adult mice results in the efficient transduction of airway epithelial cells in the nose, trachea, and lungs. In the nose, vector expression was predominant in the respiratory epithelium and was not observed in the olfactory epithelium. In the trachea and large airways of the lung, approximately 46% and 40%, respectively, of surface epithelial cells could be transduced. The efficiency of re‐dosing to the nasal airway of mice was found to be dependent of the age of the animal when the first dose is administered, as well as the length of time between doses. Conclusions: A single intranasal dose of concentrated influenza HA‐pseudotyped lentiviral vector is sufficient for efficient gene transfer to the airways of mice. This is a promising result that could lead to the development of effective gene transfer reagents for the treatment of cystic fibrosis and other human lung diseases. Copyright © 2013 John Wiley & Sons, Ltd.

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