(315a) Endoplasmic Reticulum (ER) Stress Decreases the Efficacy of Delivered siRNA Via Cholesterol Dysregulation

RNA-based therapeutics are increasingly important tools for the treatment and prevention of diseases. Because therapeutics are routinely developed and tested under idealized conditions, it is important to understand how clinical comorbidities can impact the effectiveness of delivered RNA. Obesity, diabetes, altered immune function, bacterial infections, and nutrient deprivation are examples of comorbidities that could be present in recipients of RNA-based therapeutics and reduce their efficacy. For example, the COVID-19 mRNA vaccines developed by Pfizer and Moderna showed lower antibody titers in obese vaccine recipients. The physiological impacts of these comorbidities vary, but all result in stress on the endoplasmic reticulum (ER). ER stress develops when there is a buildup of unfolded or misfolded proteins in the ER. In human cells, there are three proteins that sense ER stress: ATF6, IRE1, and PERK. Upon activation, these proteins begin cascades that lead to either prosurvival or apoptotic changes in cell function.

In response to ER stress, ATF6 is translocated to the Golgi, where it is activated. Membrane-bound transcription site-2 protease (S2P), the same protein that activates ATF6, activates sterol regulatory element binding proteins (SREBPs), which are the global regulators of cholesterol biosynthesis. When IRE1 is activated, it splices X-box binding protein 1 (XBP1) mRNA. The protein coded by this spliced mRNA (XBP1s) is translated into a transcription factor that controls transcription of genes that alleviate ER stress by increasing cholesterol biosynthesis. Overall, ER stress causes an increase in the amount of cellular cholesterol. Increased cholesterol subsequently reduces the fluidity of the cell and ER membranes. This results in increased rigidity in vesicles trafficking among the cell membrane, ER, and Golgi. Cholesterol rich vesicles are less permeable and quickly convert into lysosomes for degradation of the contents, preventing further ER stress but also potentially sequestering/destroying delivered RNAs. In this work, we demonstrate that sequestering of RNA inside membrane bound vesicles due to increased cholesterol levels from the ER stress response results in decreased effectiveness of delivered siRNAs.

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, flow cytometry, and confocal laser scanning microscopy were used to show that cells under ER stress have increased membrane cholesterol and reduced EGFP silencing. In addition, transfected siRNAs are colocalized with cholesterol. Addition of AEBSF, an inhibitor of cholesterol biosynthesis, restored silencing.

Our results demonstrate that when designing RNA based therapeutics, ER stress must be considered. Therapies that must precisely control the level of protein produced in a cell will have altered efficacy in patients with high levels of ER stress.