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Review
, 28 (3), 119-127

Receptor-Mediated Uptake of Phosphorothioate Antisense Oligonucleotides in Different Cell Types of the Liver

Affiliations
Review

Receptor-Mediated Uptake of Phosphorothioate Antisense Oligonucleotides in Different Cell Types of the Liver

Colton M Miller et al. Nucleic Acid Ther.

Abstract

Oligonucleotide therapeutics have emerged as a third distinct platform for drug discovery within the pharmaceutical industry. Five oligonucleotide-based drugs have been approved by the US FDA and over 100 oligonucleotides drugs are currently at different stages of human trials. Several of these oligonucleotide drugs are modified using the phosphorothioate (PS) backbone modification where one of the nonbridging oxygen atoms of the phosphodiester linkage is replaced with sulfur. In this review, we summarize our knowledge on receptor-mediated uptake of PS antisense oligonucleotides (ASOs) within different cell types of the liver-a privileged organ for the discovery of oligonucleotide-based therapeutics.

Keywords: Stabilins; asialoglycoprotein receptor; hepatocytes; sinusoidal endothelial cells.

Conflict of interest statement

The authors declare no financial conflicts of interest. M.T., A.J.D., T.P.P., E.E.S., and P.P.S. are employees of Ionis Pharmaceuticals, Inc.

Figures

<b>FIG. 1.</b>
FIG. 1.
Stabilin interactions with PS ASOs. (A) Flp-In HEK293 cells stably expressing Stabilin-1 (gray), Stabilin-2 (black), or null plasmid/ EV (white) were incubated with 0.1 micromolar 125I-PS ASO for 9 h, washed, and radioactivity was measured against total cellular protein to determine overall endocytosis rates. (B) Stabilin-2-expressing cells were incubated with 0.05 micromolar 125I-PS ASO for 90 min also (black) or with the indicated competing ASOs 353382 (unlabeled parent ASO) and 771994 (mixed PO/PS unlabeled parent ASO). (C) Purified ectodomain (1 microgram/mL) of the small Stabilin-2 isoform (190-HARE) was plated in polysorb wells and incubated with increasing amounts of 125I-PS ASO for 2 h. The wells were washed and eluted with 1% SDS and radioactivity was assessed for binding. (D) Quantification of MALAT-1 lncRNA in the presence of increasing concentrations of anti-MALAT-1 PS antisense oligonucleotide in both EV and 190-HARE cells as evaluated by qPCR. ASO; HARE, hyaluronic acid receptor for endocytosis; EV, empty vector; PO, phosphodiester; PS, phosphorothioate; SDS, sodium dodecyl sulfate.
<b>FIG. 2.</b>
FIG. 2.
ASGR1 contributes to uptake and activity of parent PS ASOs both in vitro and in vivo. (A) Doxycycline-inducible ASGR1 HEK cells were incubated with parent and GalNAc-conjugated cy3-labeled ASOs with or without induction of ASGR1 expression and subjected to flow cytometry to measure relative uptake. (B) Doxycycline-inducible ASGR1 HEK cells were incubated with parent and GalNAc-conjugated gapmer ASOs with or without induction of ASGR1 expression and subjected to RT-qPCR analysis to measure knockdown of MALAT1 RNA. (C) Mice were dosed with ASO targeting mouse liver ASGR1 and subsequently dosed with parent or GalNAc-conjugated ASOs targeting FXI. Shown is relative knockdown of liver ASGR1 and FXI RNA. Doses are expressed as mg ASO/kg body weight. ASGR1, asialoglycoprotein receptor. GalNAc, galactose and N-acetyl galactosamine.
<b>FIG. 3.</b>
FIG. 3.
Uptake of parent and GalNAc-conjugated ASOs at their respective IC50s is similar. Interpolated values from flow cytometry uptake experiments with ASGR1 HEK cells were used to compare relative uptake of parent and GalNAc-conjugated ASOs at their respective IC50s.
<b>FIG. 4.</b>
FIG. 4.
The PS backbone promotes binding to plasma proteins and facilitates ASO distribution to the liver and other tissues from the site of injection. ASOs are efficiently internalized into LSECs by the stabilin receptors by interactions with the PS backbone. In contrast, ASOs are internalized less efficiently into hepatocytes by interactions with the PS backbone. Targeted delivery of ASOs into hepatocytes by the ASGR using GalNAc-ASO conjugates improves potency for inhibition of gene targets expressed in hepatocytes. LSECs, liver sinusoidal endothelial cells.

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