Cell delivery, intracellular trafficking and expression of an integrin-mediated gene transfer vector in tracheal epithelial cells

The mechanism of cell entry and intracellular fate of a gene transfer vecto r composed of a receptor-targeting, DNA-condensing peptide, RGD-oligolysine , a luciferase encoding plasmid DNA (pDNA) and a cationic liposome was exam ined. We demonstrate by confocal microscopy, electron microscopy and subcel lular fractionation that the major mechanism of entry of the Vector is endo cytic. The vector complex rapidly (5 min) internalizes into early endosomes , then late endosomes and lysosomes. Entry involves, at least in part, clat hrin-coated pit-mediated endocytosis since different conditions or drugs kn own to influence this pathway modify both uptake of pDNA and its expression . The observed increase in expression with addition of a lip some correlate d with an increase in the rate of transfer of the pDNA to lysosomes, a decr ease in intracellular recycling and exocytosis of the pDNA and an increase in the amount of pDNA in the nuclear fraction. Trafficking within the cell involved endosome fusion and the acid environment of the endosomes-lysosome s was beneficial for expression. After 30 min both the peptide and pDNA loc alized to the nucleus and the amount of intact pDNA in the nuclear fraction was highest with liposome and peptide. A better understanding of the cellu lar mechanisms by which vectors transfer to and traffic in cells should hel p design improved vectors.