Efficiency of Cationic Lipid-Mediated Transfection of Polarized and Differentiated Airway Epithelial CellsIn VitroandIn Vivo

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Abstract
Systematic analysis of a large number of different cationic lipids has led to the identification of novel structures (GL-67) and formulations of cationic lipid:plasmid DNA (pDNA) complexes that facilitate high levels of gene expression in lungs of mice. However, despite significant improvement in gene transfer activity, we show here that the efficiency of GL-67-mediated gene transduction of intact airway epithelia is still relatively low. Administration of GL-67:pCF1-CFTR (encoding the cystic fibrosis transmembrane conductance regulator) complexes into the nasal epithelium of cystic fibrosis (CF) transgenic mice resulted only in marginal correction of the ion transport defects. Measurements of nasal potential differences (PD) showed no correction of the sodium (Na+) transport defect, and only partial restitution of the chloride (Cl¯) transport defect was achieved in a small proportion of the animals after perfusion of the nasal epithelium with the complexes. Furthermore, in contrast to results obtained following instillation of GL-67:pDNA complexes into the lungs of mice, perfusion of GL-67:pDNA into the nasal epithelium resulted only in a moderate enhancement of gene transduction activity relative to that attained with naked pDNA alone. To determine the basis for this low efficiency of transfection, a series of studies was conducted to identify some of the barriers governing cationic lipid-mediated gene transfer to the airway epithelium. We show here that the transfection activity of GL-67 was affected by the polarization, differentiation, and proliferative state of the cells. Diminished transfection activity was observed with nonmitotic, highly polarized and differentiated airway epithelial cells. This observed reduction in gene expression with nonmitotic cells was determined to be due in part to inefficient nuclear translocation of the pDNA from the cytoplasm. Together these data indicate that much improvement in the ability of cationic lipids to transfect polarized and differentiated airway epithelial cells is a necessary prerequisite for effective cationic lipid-mediated gene therapy of airway diseases such as CF. Progress continues to be made in the design and identification of new cationic lipid entities with enhanced gene transfer activity in vivo. However, despite these improvements, the efficiency of cationic lipid-mediated transfection, particularly of differentiated airway epithelium, is still relatively low when compared with that of adenovirus-based vectors. In this study, we assessed the relative ability of the cationic lipid GL-67 to mediate the transduction of genes into the nasal epithelium and lungs of transgenic CF mice. We showed that although GL-67 was capable of effecting partial restoration of the Cl¯ transport defect associated with CF mice, the effects were marginal and were associated with some toxicity. To understand the basis for the low efficiency of cationic lipid-mediated transfection, studies were undertaken to determine the factors regulating the gene transduction activity of this vector system. We showed that the transfection activity of cationic lipids was influenced by the mitotic, polarization, and differentiation state of the airway epithelial cells. Inefficient nuclear translocation of cytoplamic pDNA was partially responsible for the decreased gene transfer activity observed in postmitotic cells. These studies highlight the need for increased understanding of the mechanisms governing cationic lipid-mediated gene transfection and of strategies to improve their transduction efficiency.