(175ao) Endoplasmic Reticulum (ER) Stress-Induced Autophagy Alters the Efficacy of Cationic-Lipid Delivered Sirnas

The ER is part of the endomembrane system, linking it to the Golgi apparatus, cell membrane, and lysosomes via trafficking of membrane encapsulated vesicles. Endocytosis of siRNA-containing complexes begins when an initiation event causes the cell membrane to envelop the complex forming an endosome. These vesicles are then trafficked to the sorting endosome that determines to which endomembrane organelle the cargo will be distributed. To achieve a therapeutic effect, siRNAs must escape the endomembrane system during vesicle processing by either delivery vehicle fusion with the vesicle membrane or forming pores in the vesicle membrane.

One change to endomembrane processing that occurs during ER stress is autophagy. Stress causing agents near the ER initiate a membrane formation event that forms a phagophore, engulfing the problematic compounds. Phagophores mature into autophagosomes before merging with lysosomes for cargo degradation. Endomembrane transport vesicles like maturing endosomes can be trafficked to autophagosomes to reduce the amount of material within the membrane system. Large, charged molecules including siRNAs encapsulated in autophagosomes have reduced access to the cytoplasm due to the highly polar, rigid membranes of autophagosomes.

In this work, we sought to examine how autophagosome formation associated with ER stress influenced endocytosis, endosomal escape, and function of siRNAs delivered by cationic lipids. HeLa cells constitutively expressing EGFP were treated with increasing levels of tunicamycin (TM), a common ER stress inducer, followed by transfection with Lipofectamine 2000/siRNA complexes targeting EGFP. 3-methyladenine was used to inhibit autophagy during TM treatment, which alleviated siRNA accumulation levels. Confocal laser scanning microscopy was used to confirm the overlap of siRNA and LC3B stained autophagosomes. Finally, the rate of siRNA decay and silencing over 5 days was measured. siRNA accumulation was increased and persisted longer with delayed and reduced maximal silencing. Taken together our results inform clinical studies of cationic delivery of siRNA, suggesting that conditions and comorbidities that cause increased autophagy may increase toxicity due to delayed siRNA degradation and cytoplasmic access.