Intracellular rate-limiting steps of gene transfer using glycosylated polylysines in cystic fibrosis airway epithelial cells.

To identify the intracellular barriers to efficient gene transfer, we studied the intracellular trafficking of biotinylated plasmid DNA complexed with either fluorescein-conjugated lactosylated or mannosylated polylysine by confocal microscopy. Both are known to be taken up by cystic fibrosis airway epithelial cells (SigmaCFTE29o- cells), but their gene transfer efficiencies differ markedly: lactosylated polylysine is the most efficient glycosylated polylysine while mannosylated polylysine is quite inefficient for gene transfer. Mannosylated complexes appeared to stay longer in endosomes labeled by anti-transferrin receptor antibody than lactosylated complexes (from 30 min to 3 h and from 10 min to 30 min, respectively). At 24 h, higher percentages of mannosylated than lactosylated complexes were localized inside lysosomes labeled by anti-LAMP-1 antibody (41.8 +/- 6.6% versus 19.8 +/- 5.2%, respectively, P < 0.05). Between 30 min and 8 h, complexes reached the nuclei labeled by anti-lamin A/C antibody and no difference was observed between the nuclear amounts of mannosylated and lactosylated complexes. However, as analyzed by a nuclease S1 transcription assay, initiation of transcription was prevented when plasmid DNA was complexed to mannosylated polylysine. Our results indicate that the major limiting steps for mannosylated versus lactosylated polylysine transfer of plasmid DNA are delayed exit from endosomes, high accumulation in lysosomes and limited transcription of the complexed plasmid DNA.