(556f) Endoplasmic Reticulum (ER) Stress Decreases the Efficacy of Delivered siRNA Via Lysosomal Sequestration

RNA-based therapeutics are increasingly important tools for the treatment and prevention of diseases. Therapeutics are routinely developed and tested under idealized conditions; however, clinical comorbidities can impact the effectiveness of therapeutics, including therapeutic RNAs. For example, COVID-19 mRNA vaccines showed lower antibody titers in obese vaccine recipients. The physiological impacts of comorbidities vary, but many induce endoplasmic reticulum (ER) stress on affected cells. In response to ER stress, cells increase chaperone protein expression, increase protein degradation pathways, decrease overall protein production, and, when all else fails, initiate apoptosis.

The ER is part of the “endomembrane system,” linking it to the Golgi apparatus, cell membrane, and lysosomes via trafficking of membrane encapsulated vesicles. ER stress alters vesicle trafficking due to chaperone-mediated autophagy (CMA). In CMA, misfolded proteins are tagged by chaperones for degradation in lysosomes. The ER then forms a vesicle containing the tagged proteins and exports it to the lysosome. When the ER vesicle fuses with a lysosome, the lysosome membrane becomes more rigid due to the increased proportion of cholesterol and decreased fraction of free negatively charged phospholipids. The more rigid lysosomes sequester the misfolded proteins, leading to significant accumulation of material in lysosomes.

In this work, we sought to examine how lysosomal accumulation 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, a common ER stress inducer, followed by transfection with Lipofectamine 2000/siRNA complexes targeting EGFP. Fillipin III staining for cholesterol, LysoTracker Red staining, flow cytometry, and confocal laser scanning microscopy were used to show that cells under ER stress have increased cholesterol accumulation in larger more abundant lysosomes that contain siRNA. In addition, thioperamide maleate, a histone 3 receptor agonist that increases negatively charged phospholipids, was shown to reverse this effect. These results will inform RNA delivery vehicle design for treating patients with ER stress inducing comorbidities.