Gene transfer into hepatocytes using asialoglycoprotein receptor mediated endocytosis of DNA complexed with an artificial tetra-antennary galactose ligand.
We have constructed an artificial ligand for the hepatocyte-specific asialoglycoprotein receptor for the purpose of generating a synthetic delivery system for DNA. This ligand has a tetra-antennary structure, containing four terminal galactose residues on a branched carrier peptide. The carbohydrate residues of this glycopeptide were introduced by reductive coupling of lactose to the alpha- and epsilon-amino groups of the two N-terminal lysines on the carrier peptide. The C-terminus of the peptide, containing a cysteine separated from the branched N-terminus by a 10 amino acid spacer sequence, was used for conjugation to 3-(2-pyridyldithio)propionate-modified polylysine via disulfide bond formation. Complexes containing plasmid DNA bound to these galactose-polylysine conjugates have been used for asialoglycoprotein receptor-mediated transfer of a luciferase gene into human (HepG2) and murine (BNL CL.2) hepatocyte cell lines. Gene transfer was strongly promoted when amphipathic peptides with pH-controlled membrane-disruption activity, derived from the N-terminal sequence of influenza virus hemagglutinin HA-2, were also present in these DNA complexes. Thus, we have essentially borrowed the small functional domains of two large proteins, asialoglycoprotein and hemagglutinin, and assembled them into a supramolecular complex to generate an efficient gene-transfer system.
Plank
C
C
Research Institute of Molecular Pathology, Vienna, Austria.
Zatloukal
K
K
Cotten
M
M
Mechtler
K
K
Wagner
E
E
D000595:Amino Acid Sequence; D000818:Animals; D037263:Asialoglycoprotein Receptor; D001212:Asialoglycoproteins / Q000378:metabolism; D002478:Cells, Cultured; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D004705:Endocytosis; D005690:Galactose / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D008024:Ligands; D008099:Liver / Q000166:cytology / Q000378:metabolism*; D008156:Luciferases / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D010957:Plasmids; D011971:Receptors, Immunologic / Q000378:metabolism*; D014162:Transfection / Q000379:methods*
D037263:Asialoglycoprotein Receptor; D001212:Asialoglycoproteins; D008024:Ligands; D011971:Receptors, Immunologic; D004247:DNA; D008156:Luciferases; D005690:Galactose