180. Transfection Efficiency of Albumin-Containing DNA/Polyethylenimine Complexes in Primary Human Respiratory Epithelial Cells and In Vivo in the Murine Airways

Abstract

We have previously shown that human serum albumin (HSA) enhances PEI-mediated gene transfer into immortalized cell lines derived from various anatomical regions of the airways (1). Therefore, we sought to confirm these results on ex-vivo cultures of primary respiratory cells: nasal polyps, nasal brushings and bronchial cells obtained from lung biopsies. The complexes were prepared by adding different amounts of HSA (0.2–20 μg) to preformed PEI/DNA polyplexes. HSA increased PEI-mediated gene transfer in nasal outgrowths (n=5) and nasal cells obtained from brushings (n=3) by 2.6 and 4.8 times, respectively. Transfection of primary bronchial epithelial cells by HSA/PEI/DNA complexes was increased 3.2 times, relative to values obtained with PEI. It has been shown that non-viral vectors are more efficient in vivo if they are prepared in water (2, 3). Therefore, albumin/PEI/DNA (luciferase) complexes were prepared in water and their transfection efficiency was evaluated in the lungs of C57Bl/6 mice. We found that polyplexes with an N/P ratio of 15 produced the highest levels of luciferase expression. We also observed that the presence of albumin, both HSA and murine serum albumin (MSA), increased transfection mediated by PEI/DNA complexes. Immunohistochemical studies, employing an antibody against green fluorescent protein (GFP), further revealed that PEI mediated transfection with the GFP gene in the lung is restricted to bronchial epithelium. The same pattern of transfection was observed for PEI/ DNA/MSA complexes. To evaluate whether in vivo gene transfer is influenced by acute respiratory infection, C57Bl/6 mice were injected in the lung with the Pseudomonas aeruginosa PAO1 strain. Forty-eight hours after infection mice were transfected with PEI polyplexes containing the luciferase gene and prepared in the presence or absence of MSA. We observed that the levels of luciferase expression were higher in infected animals than in healthy animals when transfection was performed with PEI/DNA complexes, while no difference between infected and healthy animals was observed when the animals were transfected with PEI/DNA/MSA complexes. Overall, these results show that albumin increases gene transfer to the respiratory epithelium in ex-vivo and in-vivo models and suggest that the transfection efficiency of albumin-containing polyplexes is not likely to be hampered by opportunistic pathogen airway infections in CF patients. 1) Carrabino S et al. Serum albumin enhances polyethylenimine- mediated gene transfer to human respiratory epithelial cells. J Gene Med 2005; 7: 1555–1564, 2005. 2) Jenkins RG et al. Formation of LID vector complexes in water alters physicochemical properties and enhances pulmonary gene expression in vivo. Gene Ther 2003; 10: 1026–1034. 3) Rudolph C et al. Aerosolized nanogram quantities of plasmid DNA mediate highly efficient gene delivery to mouse airway epithelium. Mol Ther 2005; 12: 493–501. Supported by Lega Italiana Fibrosi Cistica, Italian Cystic Fibrosis Research Foundation and Associazione Lombarda Fibrosi Cistica-ONLUS.