Stabilin-1 and Stabilin-2 are specific receptors for the cellular internalization of phosphorothioate-modified antisense oligonucleotides (ASOs) in the liver

Authors

Colton M. Miller, University of Nebraska- LincolnFollow
Aaron J. Donnerr, Ionis Pharmaceuticals, Carlsbad, CA
Emma E. Blank, University of Nebraska- Lincoln
Andrew W. Egger, University of Nebraska- Lincoln
Brianna M. Kellar, University of Nebraska- Lincoln
Michael E. Østergaard, Ionis Pharmaceuticals, Carlsbad, CA
Punit P. Seth, Ionis Pharmaceuticals, Carlsbad, CAFollow
Edward N. Harris, University of Nebraska-LincolnFollow

Document Type

Article

Date of this Version

2-2016

Citation

Nucleic Acids Research, 2016, Vol. 44, No. 6, pp. 2782–2794

doi: 10.1093/nar/gkw112

Comments

Copyright (c) The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution License

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^TM, 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. Amore 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.