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Second Generation PS‐ASO Internalization and Endosomal Escape
Author(s) -
Miller Colton,
Egger Andrew,
Kellar Bri,
Hass Blake,
Seth Punit P.,
Harris Edward N.
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.766.23
Subject(s) - internalization , endosome , endocytosis , microbiology and biotechnology , receptor , gene knockdown , small interfering rna , transfection , chemistry , biology , cell culture , biochemistry , genetics
Phosphorothioate (PS)‐modified antisense oligonucleotides (ASOs) have been extensively investigated over the past few decades as therapeutic agents. Clearance of ASOs by liver and kidney greatly affect their efficacy for gene knockdown or modifications on the RNA level. The liver sinusoidal endothelial cells (LSECs) are highly endocytically active cells involved with the clearance of many ECM‐derived molecules as well as synthetic macromolecules such as heparan sulfates, hyaluronan, and ASOs. LSECS express the class H receptors known as the Stabilin‐1 and Stabilin‐2 receptors. Stabilin‐2 is also known as the Hyaluronic Acid Receptor for Endocytosis (HARE) which is found as two isoforms; 315‐kDa (315‐HARE) and 190‐kDa (190‐HARE). The Stabilin receptors are also found in the endothelium of many other tissues such as the bone marrow, spleen, and lymph node. Our previous work identified the Stabilin receptors as a primary mechanism for the internalization of ASOs in LSECs. ASO internalization occurs by clathrin‐mediated endocytosis, and are trafficked to the lysosome. Here we analyze how ASOs are able to escape the endosomal pathway to inhibit RN A expression in the cell. Methods We use recombinant cell lines stably expressing Stabilin‐1, 315‐HARE and 190‐HARE to analyze ASO vesicular escape. Using siRNAs for proteins involved in the early and late endosomal pathway, we will determine the levels of the non‐coding RNA, malat‐1, which is targeted by the ASO. Malat‐1 expression is directly correlated with escape efficiency of the ASO which targets it. The siRNA targeted gene that prevents the least amount of ASO functionality is likely the best candidate involved with ASO escape in the cytoplasm. We also analyzed vesicular transport by encapsulating ASOs in liposomes, and analyze ASO escape based on modifications resembling events of endosomal trafficking such as changes in pH, and lipid reshuffling, which will model vesicular maturation. Future Aims By determining the precise component of the vesicular endosomal pathway by which the ASO escape mechanism occurs, we will be able to gain a better understanding of how these therapeutics function. This will help identify how these molecules, once internalized within the cell are able to functionally bind to mRNA in the cytosol and nucleus for RNase H recruitment and degradation. Understanding this mechanism will greatly assist in rational design of therapeutic ASOs.