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Identification of Antisense Oligonucleotide Internalization and Transport by Scavenging Receptor Proteins in Liver Sinusoidal Endothelial Cells
Antisense Oligonucleotides (ASOs) are an emerging field of gene therapeutics. These drugs are small DNA/RNA hybrid nucleoside polymers that modulate mRNA levels of targets identified in the progression of the specific disease. ASOs have multiple mechanisms and chemistries that may be utilized, with the most common being a 5-10-5 gapmer, which is traditionally designed with a 10 base pair region of DNA that codes for the specific mRNA target, flanked on both ends by modified RNA regions. Traditionally, these RNA molecules are modified with a phosphorothioate (PS) substitution for the non-bridging oxygen within the backbone, which assists in retaining stability of these molecules. Although many discoveries surrounding these drugs have come about, many mechanisms of internalization and degradation of these targets are left undiscovered, prompting further evaluation.^ Our lab has previously identified a class of scavenging receptors, Stabilin-1 and Stabilin-2, which is also referred to as the hyaluronic acid receptor for endocytosis (HARE). These Class I receptors have been shown to facilitate scavenging roles in the liver sinusoidal endothelial cells for a variety of negatively charged molecules.^ We hypothesized that these scavenging receptor proteins are involved in the uptake of ASOs with the second generation (Gen-2) PS modification, including 2’ RNA modifications, based on the molecules negative charge. We have identified that the Stabilin receptors were functioning as scavengers for ASO drugs that proceeded through a clathrin mediated endocytosis.^ We further evaluated post-endocytosis activity to determine the nature of ASO escape from endosomal vesicles. Surprisingly, we found that Rab5C and EEA1 in the early endosome were involved in the transport for ASOs to the late endosome where they were escaping before transport to the lysosome for degradation.^ Collectively, we were able to determine one of the first receptors involved in the internalization of ASOs into the cell. We then were able to examine this further to determine the pathway by which these drugs were able to escape the endosomal pathway before degradation to reach their mRNA targets within the cell. This work provide the groundwork for other pathways and a detailed understanding of how this novel class of drugs function.^
Miller, Colton McKenzie, "Identification of Antisense Oligonucleotide Internalization and Transport by Scavenging Receptor Proteins in Liver Sinusoidal Endothelial Cells" (2018). ETD collection for University of Nebraska - Lincoln. AAI10840179.