Antisense oligonucleotides (ASOs) are short (~20 bp) oligonucleotides that have chemically-modified backbones to resist endonuclease activity in cells and biological fluids. ASOs bind to targeted mRNAs within cells and act as catalysts for RNAse H to destroy the mRNA, thus decreasing gene expression. The most common ASO modification is the substitution of the unbridging oxygen atom of the phosphodiester group with a sulfur atom to create the phosphorothioate (PS) moiety which is most often used in clinical ASOs to date. Further stabilization and nuclease resistance may be conferred through the modification of the 2’ position of ribose of the ASO. It is widely known that the liver is the natural sink for PS-ASOs, however, the mechanism for this activity is not clear. We have discovered that the Stabilin class (SR-H) scavenger receptors (Stabilin-1 and Stabilin-2) are the primary mechanism for systemic PS-ASO clearance. Stabilins are expressed in a number of tissues including the sinusoids of liver, lymph nodes, spleen, Type II macrophages, bone marrow, etc which may have implications for PS-ASO delivery to many tissues. The gap in knowledge is how interactions of PS-ASOs with the Stabilins increases PS-ASO knock-down of targeted mRNAs. Our central hypothesis is that Stabilin-mediated endocytosis of PS-ASO proceeds along 2 pathways; one in which the PS- ASO is shuttled to the lysosome (destruction) and the other in which the PS-ASO is allowed to escape the endosome to interact with mRNAs (efficacy). Our primary objectives for this project are first, to determine the biological interactions of Stabilin-PS-ASO binding complexes with the use of biolayer interferometry with competing ligands in both known solutions and in plasma. Second, to determine the kinetics of PS-ASO endocytosis in both recombinant stable cells lines expressing the Stabilins and in primary sinusoidal endothelial cells of liver. We will also dissect the endocytosis mechanisms of Stabilin-2 in which we will elucidate interacting molecules that are necessary for PS-ASO activity (endosomal escape). Third, to determine the systemic clearance and bioactivity of PS-ASOs in WT and Stabilin knock-out mice to assess Stabilin-dependent biodistribution and activation in tissues other than just the liver. We will use global and tissue-specific Stabilin knockout mice for these studies. This will further delineate the two possible pathways (destruction vs activation) in multiple tissues and how the Stabilins contribute to each pathway. The expected outcomes of this project will lend greater understanding for the structure-activity relationship, efficacy, and overall metabolism of clinical-grade PS-ASOs and Stabilin biology/biochemistry.