Ligands of the asialoglycoprotein receptor for targeted gene delivery, part 1: Synthesis of and binding studies with biotinylated cluster glycosides containing N-acetylgalactosamine

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.

Bioplex Technology: Novel Synthetic Gene Delivery Pharmaceutical Based on Peptides Anchored to Nucleic Acids

“…(5). These ligands, tested in vivo, differ greatly from the ones described above by Westerlind et al [61], and are less efficient in receptor binding due to steric hindrance, suboptimal orientation and geometry of the terminal sugars (data not shown). In this experiment no difference in liver distribution was seen when comparing the GalNAc and Gal FEs.…”

“…Westerlind et al [61] showed that synthetic ligands containing trimetric units of GalNAc bound to fluorescentlabelled streptavidin exhibit high binding affinity and specific uptake in the HepG2 hepatocyte cell line. Furthermore, they demonstrated that the uptake could be blocked with EDTA, which chelates the divalent calcium ions, thus inhibiting binding of the calcium dependent ASGPr.…”

“…(3). The conclusion of this report was that although increase in binding and uptake was seen when numbers of GalNAc units increased (up to six), uptake efficiency was mainly influenced by increasing linker spacing and flexibility between biotin and FE [61]. We have investigated the liver targeting ability of PNA-DNA hybrids, where the PNA is covalently linked to branched multimeric FEs, in vivo using the ASGPr.…”

“…Uptake was abolished only when the cells where preincubated with 1000 times excess of asialofetuin. Reprinted from Glycoconjugate Journal, 21, Westerlind et al, Ligands of the asialoglycoprotein receptor for targeted gene delivery, part 1: Synthesis of and binding studies with biotinylated cluster glycosides containing Nacetylgalactosamine, 227-41, ©2004, with permission from Kluwer Academic Publisher[61].…”

Design of intrahepatocyte copper(I) chelators as drug candidates for Wilson's disease

“…Interestingly, among the structures proposed for optimal ASGP‐R recognition are tripods bearing, on each arm, a β‐linked GalNAc moiety kept about 20 Å away from the branching point of the tripod by an ethylene glycol spacer . The tripodal architecture of L 2 is thus perfectly suitable for the design of the triantennary glycoside cluster Chel2 (Fig.…”