Endocytosis of Phosphorothioate Antisense Oligonucleotides (ASO) by the Stabilin Receptors
Authors: Egger Andrew , Keller Brianna , Miller Colton M. , Harris Edward N. 
Published: November 2023

Many clinically relevant drugs are prescribed in high doses to compensate for degradation and removal by liver and kidney‐mediated mechanisms. It is necessary to understand the molecular mechanisms of these clearance systems for the rational design of pharmaceuticals In the liver, the hepatocytes and sinusoidal endothelial cells (SECs) are responsible for the removal and neutralization of many pharmaceuticals The Stabilin receptors (Stabilin‐1 and Stabilin‐2) expressed by SECs and undergo rapid and constituitive clathrin‐mediated endocytosis. Here, we characterized the Stabilin‐2 receptor (large isoform – 315‐HARE and small isoform – 190‐HARE) binding and internalization of phosphorothioate‐modified antisense oligonucleotides (ASOs). Endocystosis assays were preformed over a 9 hour period to compare relative rates of oligo uptake by empty vector (EV) parental control cells, Stabilin‐1, and both isoforms of Stabilin‐2. To investigate how much ASO is internalized via clathrin‐dependent mechanisms, cells were subjected to 0.4M Sucrose (hyperosmolarity) and two clathrin‐inhibiting drugs Dynasore and Pit Stop 2. Results suggested that over half of all internalization was clathrin mediated. Since ASOs are charged and tend to stick to membranes, we investigated how they bound to cells with and without expressing receptor. Cells were placed on ice with 125I‐ ASO for one hour, washed and assessed for radioactivity. There was no difference in binding between the EV, Stab1 and 315‐HARE cells and a slight increase in the 190‐HARE cells suggesting that cellular binding is not receptor dependent, but internalization was largely facilitated by each receptor. Next, we assessed whether the cells were degrading the internalized ASO or if the ASO was being secreted intact through exocytosis. To do this, approximately 6×106 cells were incubated in 100 mm dishes, incubated with 0.1 μM 125I‐ASO for 2 hrs, washed with Hank's Buffered Salt Solution (HBSS) 3x, and incubated with fresh media for 6 hrs. At the indicated time point, the media was harvested and passed through a 1 mL bed volume of DEAE affinity resin to capture intact ASO. The column was washed with 0.2 M NaCl to elute fragmented ASO, and eluted with 2.0 M NaCl to wash out the intact ASO. Degradation rates of 125I‐ASO were much higher in the Stab2 and Stab1‐expressing cells in contrast to the EV cells. These data in addition to the fluorescent microscopy data showing colocalization of Cy3‐ASO with lysosomes suggests that the ASO is trafficked to lysosomes and degraded before efflux from the cells. From these data, we conclude that SECs are a major site of ASO clearance from the blood and internalization is primarily mediated by both Stab1 and Stab2 receptors.Support or Funding InformationThis work was supported by the National Institutes of Health [5R01HL094463 to ENH], The University of Nebraska Center for Integrated Biomolecular Communication (CIBC) Research Cluster Development grant, and the University of Nebraska Undergraduate Creative Activities and Research Experience (UCARE) program.