title: Efficient Synthesis and Biological Evaluation of 5'-GalNAc Conjugated Antisense Oligonucleotides.
abstract: Conjugation of triantennary N-acetyl galactosamine (GalNAc) to oligonucleotide therapeutics results in marked improvement in potency for reducing gene targets expressed in hepatocytes. In this report we describe a robust and efficient solution-phase conjugation strategy to attach triantennary GalNAc clusters (mol. wt. ∼2000) activated as PFP (pentafluorophenyl) esters onto 5'-hexylamino modified antisense oligonucleotides (5'-HA ASOs, mol. wt. ∼8000 Da). The conjugation reaction is efficient and was used to prepare GalNAc conjugated ASOs from milligram to multigram scale. The solution phase method avoids loading of GalNAc clusters onto solid-support for automated synthesis and will facilitate evaluation of GalNAc clusters for structure activity relationship (SAR) studies. Furthermore, we show that transfer of the GalNAc cluster from the 3'-end of an ASO to the 5'-end results in improved potency in cells and animals.
authors.affiliation: Isis Pharmaceuticals, Inc., 2855 Gazelle Court, Carlsbad, California 92010, United States.
authors.forename: Michael E
authors.initials: ME
authors.lastname: Østergaard
authors.affiliation: Isis Pharmaceuticals, Inc., 2855 Gazelle Court, Carlsbad, California 92010, United States.
authors.forename: Jinghua
authors.initials: J
authors.lastname: Yu
authors.affiliation: Isis Pharmaceuticals, Inc., 2855 Gazelle Court, Carlsbad, California 92010, United States.
authors.forename: Garth A
authors.initials: GA
authors.lastname: Kinberger
authors.affiliation: Isis Pharmaceuticals, Inc., 2855 Gazelle Court, Carlsbad, California 92010, United States.
authors.forename: W Brad
authors.initials: WB
authors.lastname: Wan
authors.affiliation: Isis Pharmaceuticals, Inc., 2855 Gazelle Court, Carlsbad, California 92010, United States.
authors.forename: Michael T
authors.initials: MT
authors.lastname: Migawa
authors.affiliation: Isis Pharmaceuticals, Inc., 2855 Gazelle Court, Carlsbad, California 92010, United States.
authors.forename: Guillermo
authors.initials: G
authors.lastname: Vasquez
authors.affiliation: Isis Pharmaceuticals, Inc., 2855 Gazelle Court, Carlsbad, California 92010, United States.
authors.forename: Karsten
authors.initials: K
authors.lastname: Schmidt
authors.affiliation: Isis Pharmaceuticals, Inc., 2855 Gazelle Court, Carlsbad, California 92010, United States.
authors.forename: Hans J
authors.initials: HJ
authors.lastname: Gaus
authors.affiliation: Isis Pharmaceuticals, Inc., 2855 Gazelle Court, Carlsbad, California 92010, United States.
authors.forename: Heather M
authors.initials: HM
authors.lastname: Murray
authors.affiliation: Isis Pharmaceuticals, Inc., 2855 Gazelle Court, Carlsbad, California 92010, United States.
authors.forename: Audrey
authors.initials: A
authors.lastname: Low
authors.affiliation: Isis Pharmaceuticals, Inc., 2855 Gazelle Court, Carlsbad, California 92010, United States.
authors.forename: Eric E
authors.initials: EE
authors.lastname: Swayze
authors.affiliation: Isis Pharmaceuticals, Inc., 2855 Gazelle Court, Carlsbad, California 92010, United States.
authors.forename: Thazha P
authors.initials: TP
authors.lastname: Prakash
authors.affiliation: Isis Pharmaceuticals, Inc., 2855 Gazelle Court, Carlsbad, California 92010, United States.
authors.forename: Punit P
authors.initials: PP
authors.lastname: Seth
mesh_terms: D000116:Acetylgalactosamine / Q000737:chemistry*; D000818:Animals; D002478:Cells, Cultured; D022781:Hepatocytes / Q000166:cytology / Q000187:drug effects*; D008099:Liver / Q000166:cytology / Q000187:drug effects*; D051379:Mice; D008810:Mice, Inbred C57BL; D016376:Oligonucleotides, Antisense / Q000138:chemical synthesis* / Q000494:pharmacology*
chemical_list: D016376:Oligonucleotides, Antisense; D000116:Acetylgalactosamine