A Concentrated and Stable Aerosol Formulation of Cationic Lipid:DNA Complexes Giving High-Level Gene Expression in Mouse Lung

Abstract

Advances in gene therapy vectors and techniques hold promise for treatment of many inherited and acquired diseases. For lung indications, especially those involving the epithelium, delivery of the gene therapy vehicle ideally will involve the use of an aerosol. Aerosol delivery of transgenes using cationic lipids is currently limited by the ability to generate highly concentrated formulations of lipid:DNA complexes that are stable and retain their activity following aerosolization. We have examined many of the variables inherent in aerosolizing cationic lipid gene delivery vehicles and have devised a new formulation that incorporates small amounts of a polyethylene glycol-containing lipid. This formulation has allowed the preparation of concentrated dispersions of cationic lipid:plasmid DNA (pDNA) complexes (>20 mM pDNA) at approximately 10-fold higher concentrations than previously reported. Most of the pDNA in these formulations was bound to the lipid component and thereby protected from nebulizer-induced shearing; the pDNA also maintained full biological activity both in vitro and in vivo. This new formulation thus represents a significant improvement over current methods to prepare concentrated, active cationic lipid gene delivery vectors, and provides a new tool with which to test gene transfer to the lung. A novel cationic lipid formulation has been developed that overcomes many of the difficulties associated with the aerosol delivery of cationic lipid:DNA complexes. The formulation is stable at high concentrations of lipid and DNA over a wide range of cationic lipid:DNA ratios. Complexes prepared with this lipid formulation in the absence of ions in the suspending media were transferred efficiently and the lipid:DNA ratio was maintained through aerosolization. This formulation solves many of the problems previously identified in aerosolizing cationic lipid:DNA complexes. Aerosol delivery of this complex to the lungs of BALB/c mice resulted in high and dose-dependent levels of expression of the reporter gene product chloramphenicol acetyl transferase (CAT). Transgene expression was transient, however, with CAT levels at day 14 being less than 10% of the peak levels observed at day 2.