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Investigating the Intracellular Trafficking of Antisense Oligonucleotide After Stabilin Receptor Mediated Endocytosis and Finding Novel Strategies to Enhance Its Endosomal Escape
Phosphorothioate antisense oligonucleotides (PS-ASOs) are internalized inside the cells via various receptor mediated endocytic pathways. Previous studies have reported that non-DNA binding Stabilin2/HARE receptor present on endothelial cells specifically binds to and internalize several classes of ASOs. However, once internalized by Stabilin receptor, ASOs are trafficked within endosomes (>99%), ultimately resulting in destruction in the lysosome. The ASO entrapment in endosomes is considered a bottleneck in the oligonucleotide therapeutic advancement. As a first step towards solving endosomal entrapment problem, we first characterized the intracellular route transversed by ASOs after Stabilin receptor mediated uptake. We found that PS-ASO as well as Stabilin-2 receptor follow the classical path: Clathrin coated vesicle > early endosome > late endosome > lysosome. However, Stabilin-1 traffick the PS-ASO via different route after clathrin mediated endocytosis which needs further investigation. Next, we investigated different strategies to enhance endosomal escape of PS-ASO after Stabilin-2 receptor mediated endocytosis. First, we evaluated a small molecule, Chloroquine, as an effective strategy for promoting the release of PS-ASO from endosomes. Chloroquine effectively enhanced the activity of PS-ASO in Stabilin-2 overexpressing cell lines. We also established its dominant role in enhancing PS-ASO escape as compared to other important endosome associated proteins. Next, we identified a novel role of galectin’s in enhancing endosomal escape of PS-ASO in Stabilin-2 cell line. Till date, galectins have been used as an imaging sensor for visualizing endosomal escape of siRNA. In our study, we showed that galectin knockdown efficiently enhances activity of PS-ASO by 50-70%. Galectin-1 modulated the expression of endosomal associated gene. This observation led us to hypothesize that galectin-1 affect endosomal vesicle maturation which could be a possible mechanism for how galectins are enhancing PS-ASO activity. This study brings us one step closer towards solving endosomal entrapment problem of PS-ASOs. Enhancing endosomal escape will result in lower drug administration, which will reduce the toxicity, thereby making PS-ASO drugs more cost-effective.
Biochemistry|Cellular biology|Molecular biology
Pandey, Ekta, "Investigating the Intracellular Trafficking of Antisense Oligonucleotide After Stabilin Receptor Mediated Endocytosis and Finding Novel Strategies to Enhance Its Endosomal Escape" (2023). ETD collection for University of Nebraska - Lincoln. AAI30573580.