title: Stabilin-1 and Stabilin-2 are specific receptors for the cellular internalization of phosphorothioate-modified antisense oligonucleotides (ASOs) in the liver.
abstract: Phosphorothioate (PS)-modified antisense oligonucleotides (ASOs) have been extensively investigated over the past three decades as pharmacological and therapeutic agents. One second generation ASO, Kynamro™, was recently approved by the FDA for the treatment of homozygous familial hypercholesterolemia and over 35 second generation PS ASOs are at various stages of clinical development. In this report, we show that the Stabilin class of scavenger receptors, which were not previously thought to bind DNA, do bind and internalize PS ASOs. With the use of primary cells from mouse and rat livers and recombinant cell lines each expressing Stabilin-1 and each isoform of Stabilin-2 (315-HARE and 190-HARE), we have determined that PS ASOs bind with high affinity and these receptors are responsible for bulk, clathrin-mediated endocytosis within the cell. Binding is primarily dependent on salt-bridge formation and correct folding of the intact protein receptor. Increased internalization rates also enhanced ASO potency for reducing expression of the non-coding RNA Malat-1, in Stabilin-expressing cell lines. A more thorough understanding of mechanisms by which ASOs are internalized in cells and their intracellular trafficking pathways will aid in the design of next generation antisense agents with improved therapeutic properties.
authors.affiliation: University of Nebraska-Lincoln, Dept. of Biochemistry, 1901 Vine Street Lincoln NE 68588, USA.
authors.forename: Colton M
authors.initials: CM
authors.lastname: Miller
authors.affiliation: Ionis Pharmaceuticals, 2855 Gazelle Ct, Carlsbad, CA 92010, USA.
authors.forename: Aaron J
authors.initials: AJ
authors.lastname: Donner
authors.affiliation: University of Nebraska-Lincoln, Dept. of Biochemistry, 1901 Vine Street Lincoln NE 68588, USA.
authors.forename: Emma E
authors.initials: EE
authors.lastname: Blank
authors.affiliation: University of Nebraska-Lincoln, Dept. of Biochemistry, 1901 Vine Street Lincoln NE 68588, USA.
authors.forename: Andrew W
authors.initials: AW
authors.lastname: Egger
authors.affiliation: University of Nebraska-Lincoln, Dept. of Biochemistry, 1901 Vine Street Lincoln NE 68588, USA.
authors.forename: Brianna M
authors.initials: BM
authors.lastname: Kellar
authors.affiliation: Ionis Pharmaceuticals, 2855 Gazelle Ct, Carlsbad, CA 92010, USA.
authors.forename: Michael E
authors.initials: ME
authors.lastname: Østergaard
authors.affiliation: Ionis Pharmaceuticals, 2855 Gazelle Ct, Carlsbad, CA 92010, USA pseth@ionisph.com.
authors.forename: Punit P
authors.initials: PP
authors.lastname: Seth
authors.affiliation: University of Nebraska-Lincoln, Dept. of Biochemistry, 1901 Vine Street Lincoln NE 68588, USA eharris5@unl.edu.
authors.forename: Edward N
authors.initials: EN
authors.lastname: Harris
mesh_terms: D000818:Animals; D015816:Cell Adhesion Molecules, Neuronal / Q000235:genetics / Q000378:metabolism*; D022163:Clathrin-Coated Vesicles / Q000378:metabolism; D004705:Endocytosis; D042783:Endothelial Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D015870:Gene Expression; D057809:HEK293 Cells; D006801:Humans; D007700:Kinetics; D008099:Liver / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D051379:Mice; D016376:Oligonucleotides, Antisense / Q000138:chemical synthesis / Q000378:metabolism* / Q000493:pharmacokinetics; D054735:Phosphorothioate Oligonucleotides / Q000138:chemical synthesis / Q000378:metabolism* / Q000493:pharmacokinetics; D061251:Primary Cell Culture; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D062085:RNA, Long Noncoding / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley
chemical_list: D015816:Cell Adhesion Molecules, Neuronal; C557040:Malat1 long non-coding RNA, mouse; D016376:Oligonucleotides, Antisense; D054735:Phosphorothioate Oligonucleotides; D020033:Protein Isoforms; D062085:RNA, Long Noncoding; C486732:Stab1 protein, mouse; C486733:Stab2 protein, mouse