Abstract
In order to develop the non-viral Bioplex vector system for targeted delivery of genes to hepatocytes, we have evaluated the structure-function relationship for a number of synthetic ligands designed for specific interaction with the hepatic lectin ASGPr. Biotinylated ligand derivatives containing two, three or six beta-linked N-acetylgalactosamine (GalNAc) residues were synthesized, bound to fluorescent-labeled streptavidin and tested for binding and uptake to HepG2 cells using flow cytometry analysis (FACS). Uptake efficiency increased with number of displayed GalNAc units per ligand, in a receptor dependent manner. Thus, a derivative displaying six GalNAc units showed the highest uptake efficacy both in terms of number of internalizing cells and increased amount of material taken up per each cell. However, this higher efficiency was shown to be due not so much to higher number of sugar units, but to higher accessibility of the sugar units for interaction with the receptor (longer spacer). Improving the flexibility and accessibility of a trimeric GalNAc ligand through use of a longer spacer markedly influenced the uptake efficiency, while increasing the number of GalNAc units per ligand above three only provided a minor contribution to the overall affinity. We hereby report the details of the chemical synthesis of the ligands and the structure-function studies in vitro. Published in 2003.
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References
Wu J, Nantz MH, Zern MA, Targeting hepatocytes for drug and gene delivery: emerging novel approaches and applications, Front Biosci 7, D717-25 (2002).
Lee YC, Biochemistry of carbohydrate-protein interaction, Faseb J 6, 3193-200 (1992).
Kawasaki T, Ashwell G, Chemical and physical properties of an hepatic membrane protein that specifically binds asialoglycoproteins, J Biol Chem 251, 1296-302 (1976).
Stockert RJ, The asialoglycoprotein receptor: relationships between structure, function, and expression, Physiol Rev 75, 591-609 (1995).
Zhang X, Simmons CG, Corey DR, Liver cell specific targeting of peptide nucleic acid oligomers, Bioorg Med Chem Lett 11, 1269-72 (2001).
Wu GY, Wu CH, Receptor-mediated gene delivery and expression in vivo, J Biol Chem 263, 14621-4 (1988).
Sliedregt LA, Rensen PC, Rump ET, van Santbrink PJ, Bijsterbosch MK, Valentijn AR, van der Marel GA, van Boom JH, van Berkel TJ, Biessen EA, Design and synthesis of novel amphiphilic dendritic galactosides for selective targeting of lipo-somes to the hepatic asialoglycoprotein receptor, J Med Chem 42, 609-18 (1999).
Remy JS, Kichler A, Mordvinov V, Schuber F, Behr JP, Targeted gene transfer into hepatoma cells with lipopolyamine-condensed DNA particles presenting galactose ligands: a stage toward artifi-cial viruses, Proc Natl Acad Sci USA 92, 1744-8 (1995).
Maier MA, Yannopoulos CG, Mohamed N, Roland A, Fritz H, Mohan V, Just G, Manoharan M, Synthesis of antisense oligonu-cleotides conjugated to a multivalent carbohydrate cluster for cellular targeting, Bioconjug Chem 14, 18-29 (2003).
Lee RT, Lee YC, Facile synthesis of a high-affinity lig-and for mammalian hepatic lectin containing three termi-nal N-acetylgalactosamine residues, Bioconjug Chem 8, 762-5 (1997).
Kichler A, Schuber F, Versatile synthesis of bi-and tri-antennary galactose ligands: Interaction with the Gal/GalNAc receptor of human hepatoma cells, Glycoconj J 12, 275-81 (1995).
Ren T, Zhang G, Liu D, Synthesis of galactosyl compounds for targeted gene delivery, Bioorg Med Chem 9, 2969-78 (2001).
Biessen EA, Beuting DM, Roelen HC, van der Marel GA, van Boom JH, van Berkel TJ, Synthesis of cluster galactosides with high affinity for the hepatic asialoglycoprotein receptor, J Med Chem 38, 1538-46. (1995).
Valentijn AR, van der Marel GA, Sliedregt LA, van Berkel TJ, Biessen EA, van Boom JH, Solid-phase synthesis of lysine-based cluster galactosides with high affinity for the asialoglycoprotein receptor, Tetrahedron 53, 759-70 (1997).
Svahn MG, Lundin KE, Ge R, Tornquist E, Simonson EO, Oscarsson S, Leijon M, Branden LJ, Smith CI, Adding functional entities to plasmids, J Gene Med 6 Suppl1, S36-44 (2004).
Lundin KE, Ge R, Svahn MG, Tornquist E, Leijon M, Branden LJ, Smith CI, Cooperative strand invasion of supercoiled plasmid DNA by mixed linear PNA and PNA-peptide chimeras, Biomol Eng 21, 51-9 (2004).
Branden LJ, Smith CI, Bioplex technology: novel synthetic gene delivery system based on peptides anchored to nucleic acids, Methods Enzymol 346, 106-24 (2002).
Branden LJ, Christensson B, Smith CI, In vivonuclear delivery of oligonucleotides via hybridizing bifunctional peptides, Gene Ther 8, 84-7 (2001).
Branden LJ, Mohamed AJ, Smith CI, A peptide nucleic acid-nuclear localization signal fusion that mediates nuclear transport of DNA, Nat Biotechnol 17, 784-7 (1999).
van Rossenberg SM, Sliedregt-Bol KM, Prince P, van Berkel TJ, van Boom JH, van der Marel GA, Biessen EA, A targeted peptide nucleic acid to down-regulate mouse microsomal triglyceride transfer protein expression in hepatocytes, Bioconjug Chem 14, 1077-82 (2003).
Lee YC, Binding modes of mammalian hepatic Gal/GalNAc re-ceptors, Ciba Found Symp 145, 80-93 (1989).
Joziasse DH, Lee RT, Lee YC, Biessen EA, Schiphorst WE, Koeleman CA, van den Eijnden DH, alpha3-galactosylated glyco-proteins can bind to the hepatic asialoglycoprotein receptor, Eur J Biochem 267, 6501-8 (2000).
Rice KG, Weisz OA, Barthel T, Lee RT, Lee YC, Defined geometry of binding between triantennary glycopeptide and the asialogly-coprotein receptor of rat heptocytes, J Biol Chem 265, 18429-34 (1990).
Biessen EA, Vietsch H, Van Berkel TJ, Cholesterol derivative of a new triantennary cluster galactoside directs low-and high-density lipoproteins to the parenchymal liver cell, Biochem J 302(Pt 1), 283-9 (1994).
van Berkel TJ, Kruijt JK, Spanjer HH, Nagelkerke JF, Harkes L, Kempen HJ, The effect of a water-soluble tris-galactoside-terminated cholesterol derivative on the fate of lowdensity lipopro-teins and liposomes, J Biol Chem 260, 2694-9 (1985).
Singh M, Ariatti M, Targeted gene delivery into HepG2 cells using complexes containing DNA, cationized asialoorosomucoid and activated cationic liposomes, J Control Release 92, 383-94 (2003).
Kolatkar AR, Leung AK, Isecke R, Brossmer R, Drickamer K, Weis WI, Mechanism of N-acetylgalactosamine binding to a C-type animal lectin carbohydrate-recognition domain, J Biol Chem 273, 19502-8 (1998).
Iobst ST, Drickamer K, Selective sugar binding to the carbohydrate recognition domains of the rat hepatic and macrophage asialogly-coprotein receptors, J Biol Chem 271, 6686-93 (1996).
Fadden AJ, Holt OJ, Drickamer K, Molecular characterization of the rat Kupffer cell glycoprotein receptor, Glycobiology 13, 529-37 (2003).
Schwartz AL, Fridovich SE, Knowles BB, Lodish HF, Characterization of the asialoglycoprotein receptor in a continuous hepatoma line, J Biol Chem 256, 8878-81 (1981).
Andersson M, Oscarson S, Öberg S, Synthesis of oligosaccharides with oligoethylene glycol spacers and their conversion into gly-coconjugates using N,N,NN-tetramethyl(succinimido)uronium tetrafluoroborate as coupling reagent, Glycoconjugate J, 10, 197-201 (1993).
Bailen MA, Chinchilla R, Dodsworth DJ, Najera C, O-Succinimidyl-1,3-dimethyl-1,3-trimethyleneuronium salts as efficient reagents in active ester synthesis, Tetrahedron Lett 43, 1661-4 (2002).
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Westerlind, U., Westman, J., Törnquist, E. et al. Ligands of the asialoglycoprotein receptor for targeted gene delivery, part 1: Synthesis of and binding studies with biotinylated cluster glycosides containing N-acetylgalactosamine. Glycoconj J 21, 227–241 (2004). https://doi.org/10.1023/B:GLYC.0000045095.86867.c0
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DOI: https://doi.org/10.1023/B:GLYC.0000045095.86867.c0