Specific gene transfer mediated by lactosylated poly-L-lysine into hepatoma cells

Abstract

Plasmid DNA/glycosylated polylysine complexes were used to transfer in vitro a luciferase reporter gene into human hepatoma cells by a receptor-mediated endocytosis process. HepG2 cells which express a galactose specific membrane lectin were efficiently and selectively transfected with pSV2Luc/lactosylated polylysine complexes in a sugar dependent manner: i) HepG2 cells which do not express membrane lectin specific for mannose were quite poorly transfected with pSV2Luc/mannosylated polylysine complexes, ii) HeLa cells which do not express membrane lectin specific for galactose were not transfected with pSV2Luc/lactosylated polylysine complexes. The transfection efficiency of HepG2 cells with pSV2Luc/lactosylated polylysine complexes was greatly enhanced either in the presence of chloroquine or in the presence of a fusogenic peptide. A 22-residue peptide derived from the influenza virus hemagglutinin HA2 N-terminal polypeptide that mimics the fusogenic activity of the virus, was selected. In the presence of the fusogenic peptide, the luciferase activity in HepG2 cells was 10 fold larger than that of cells transfected with pSV2Luc/lactosylated polylysine complexes in the presence of chloroquine.

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

“…3,[11][12][13] Indeed, such a mechanism is likely at work for glycosylated polylysines. Our previous findings 7 and those reported herein suggest such a mechanism: a mannose-specific membrane lectin is strongly expressed at the surface of ΣCFTE29oϪ cells and mannosylated complexes are the glycosylated complexes most efficiently incorporated into the cells, as shown by flow cytometry 7 and confocal microscopy (this study).…”

“…14,15 To partly overcome this barrier, we used chloroquine, a endosomolytic agent which is thought to protect the internalized plasmid from intracellular degradation by neutralizing acidic compartments and delaying the transfer of material from endosomes to lysosomes. 3,16,17 It is pertinent to note that, although both mannosylated and lactosylated complexes were tested in the presence of chloroquine, their transfer kinetics from endosomes to lysosomes differed. Indeed, mannosylated complexes remained in endosomes longer and more were present in lysosomes, as compared with lactosylated complexes.…”

“…28 The average number of lactosyl or mannosyl residues bound per poly-L-lysine molecule was calculated from the sugar content determined using the resorcinol sulfuric acid micromethod 29 and was found to be Gene Therapy 66 ± 5 and 76 ± 4, respectively. Fluorescein-conjugated glycosylated polylysines were prepared as previously described 3 and contained 4 ± 2 fluorescein residues per poly-L-lysine molecule.…”

“…We and others have previously shown that polylysine substituted with sugar residues can efficiently transfect various cell types. [3][4][5][6][7][8] Indeed, membrane lectins, ie cell-surface sugar-specific receptors, are thought to mediate the uptake of plasmid DNA complexed to glycosylated polylysine ('glycoplex') and to carry it into endosomes. The subsequent intracellular trafficking of glycoplexes is largely unknown, but appears to vary according to the sugar moiety substituting the polylysine.…”

Non-viral approaches to gene therapy

“…The ligand triggers binding to the target cell and the DNA is internalized upon endocytosis of the ligand-DNA complex bound to the target receptor [21]. A wide variety of conjugates for receptor-mediated gene delivery are now available ( [7,8,19,20,[22][23][24][25][26]; see Table 1). …”