Inhibition of productive/competitive endocytic pathways enhances siRNA delivery and cell specific targeting
July 14-18, 2019
While the use of short interfering RNAs (siRNAs) for laboratory studies is now common practice, development of siRNAs for therapeutic applications has slowed, due in part to a still limited understanding of the endocytosis and intracellular trafficking of siRNA-containing complexes. As a result, it is difficult to design delivery vehicles for specific cell types, resulting in inefficient delivery, cytotoxicity, or immunogenicity when used in vivo. Our aim is to identify which endocytosis and intracellular trafficking pathways lead to active silencing by siRNA-containing complexes. Our work explores the preferential mechanism of endocytosis (whether by clathrin, caveolin, Arf6, Graf1, flotillin, or macropinocytosis) across multiple cell types (HeLa (cervical), H1299 (lung), HEK293 (kidney), and HepG2 (liver)). Using Lipofectamine 2000 (LF2K), fluorescently-labeled siRNAs were delivered to cells stably expressing green fluorescent protein (GFP). Chemical inhibitors (Filipin, Dynasore, Cytochalasin D, Chlorpromazine, Amiloride, and Methyl-β-cyclodextrin) were used to identify the specific endocytic pathway internalizing the complexes. By measuring the effect of inhibitors on both intracellular levels of siRNA and GFP silencing, we were able to categorize pathways as being productive/competitive according to their functional role in facilitating gene silencing. In productive pathways, siRNAs are actively delivered to a cell and silence a target protein, whereas in competitive pathways, siRNAs are endocytosed but do not lead to silencing.
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S. Patrick Walton, Daniel Vocelle, and Christina Chan, "Inhibition of productive/competitive endocytic pathways enhances siRNA delivery and cell specific targeting" in "Biochemical and Molecular Engineering XXI", Christina Chan, Michigan State University, USA Mattheos Koffas, RPI, USA Steffen Schaffer, Evonik Industries, Germany Rashmi Kshirsagar, Biogen, USA Eds, ECI Symposium Series, (2019). https://dc.engconfintl.org/biochem_xxi/65