title: Intra-endosomal trafficking mediated by lysobisphosphatidic acid contributes to intracellular release of phosphorothioate-modified antisense oligonucleotides.
abstract: Antisense oligonucleotides (ASOs) with phosphorothioate (PS) linkages are broadly used as research tools and therapeutic agents. Chemically modified PS-ASOs can mediate efficient target reduction by site-specific cleavage of RNA through RNase H1. PS-ASOs are known to be internalized via a number of endocytotic pathways and are released from membrane-enclosed endocytotic organelles, mainly late endosomes (LEs). This study was focused on the details of PS-ASO trafficking through endocytic pathways. It was found that lysobisphosphatidic acid (LBPA) is required for release of PS-ASOs from LEs. PS-ASOs exited early endosomes (EEs) rapidly after internalization and became co-localized with LBPA by 2 hours in LEs. Inside LEs, PS-ASOs and LBPA were co-localized in punctate, dot-like structures, likely intraluminal vesicles (ILVs). Deactivation of LBPA using anti-LBPA antibody significantly decreased PS-ASO activities without affecting total PS-ASO uptake. Reduction of Alix also substantially decreased PS-ASO activities without affecting total PS-ASO uptake. Furthermore, Alix reduction decreased LBPA levels and limited co-localization of LBPA with PS-ASOs at ILVs inside LEs. Thus, the fusion properties of ILVs, which are supported by LBPA, contribute to PS-ASO intracellular release from LEs.
authors.affiliation: Department of Core Antisense Research, Ionis Pharmaceuticals, Inc. 2855 Gazelle Court, Carlsbad, CA 92010, USA.
authors.forename: Shiyu
authors.initials: S
authors.lastname: Wang
authors.affiliation: Department of Core Antisense Research, Ionis Pharmaceuticals, Inc. 2855 Gazelle Court, Carlsbad, CA 92010, USA.
authors.forename: Hong
authors.initials: H
authors.lastname: Sun
authors.affiliation: Department of Medicinal Chemistry, Ionis Pharmaceuticals, Inc. 2855 Gazelle Court, Carlsbad, CA 92010, USA.
authors.forename: Michael
authors.initials: M
authors.lastname: Tanowitz
authors.affiliation: Department of Core Antisense Research, Ionis Pharmaceuticals, Inc. 2855 Gazelle Court, Carlsbad, CA 92010, USA.
authors.forename: Xue-Hai
authors.initials: XH
authors.lastname: Liang
authors.affiliation: Department of Core Antisense Research, Ionis Pharmaceuticals, Inc. 2855 Gazelle Court, Carlsbad, CA 92010, USA.
authors.forename: Stanley T
authors.initials: ST
authors.lastname: Crooke
mesh_terms: D001692:Biological Transport; D002135:Calcium-Binding Proteins / Q000378:metabolism; D002352:Carrier Proteins / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000378:metabolism; D045744:Cell Line, Tumor; D004705:Endocytosis; D056827:Endosomal Sorting Complexes Required for Transport / Q000378:metabolism; D011992:Endosomes / Q000378:metabolism*; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D042541:Intracellular Space / Q000378:metabolism*; D007700:Kinetics; D008246:Lysophospholipids / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000378:metabolism; D008954:Models, Biological; D050178:Monoglycerides / Q000378:metabolism*; D000077296:Niemann-Pick C1 Protein; D016376:Oligonucleotides, Antisense / Q000737:chemistry / Q000378:metabolism*; D054735:Phosphorothioate Oligonucleotides / Q000378:metabolism*
chemical_list: D002135:Calcium-Binding Proteins; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D056827:Endosomal Sorting Complexes Required for Transport; D047908:Intracellular Signaling Peptides and Proteins; D008246:Lysophospholipids; D008562:Membrane Glycoproteins; D050178:Monoglycerides; C106881:NPC1 protein, human; D000077296:Niemann-Pick C1 Protein; D016376:Oligonucleotides, Antisense; C117020:PDCD6IP protein, human; D054735:Phosphorothioate Oligonucleotides; C012786:bis(monoacylglyceryl)phosphate