(742e) Environmental and Chemical Stress Alters siRNA-Mediated Silencing

RNA-based therapeutics achieved widespread adoption in 2020 with the FDA authorization of mRNA vaccines produced by Moderna and Pfizer. This follows the approval of two siRNA therapeutics, Onpattro (approved in 2018) and Givlaari (approved in 2019), targeting diseases in the liver. RNA-based therapeutics are developed and tested under idealized conditions, but patients often have comorbidities that cause additional environmental and chemical stress on diseased cells; therefore, it is important to understand how comorbidities alter cellular processing and function of RNA-based therapeutics.

A growing number of Americans are obese. Obesity and many diseases activate ER stress and the unfolded protein response, which alters cell membrane maintenance pathways. Additionally, patients with diseases that are proposed as targets of RNA-based therapeutics (e.g., carcinomas, leukemias, and amyloidoses) often also have compromised immune function, increasing their risk of infection. Toxins from E. coli, Shigella, and C. difficile inhibit actin cytoskeleton rearrangement by Rho-GTPases, which are crucial for many endocytic and exocytic pathways. Because RNAs are large and electrostatically excluded from entering cells without the use of active transport, cell membrane maintenance, endocytosis, and exocytosis are important steps along the therapeutic pathway for RNA-based therapies. Tunicamycin, an ER glycosylation inhibitor, and C. difficile toxin A were used as models for ER stress and bacterial infection, respectively, in cultured human cells. We hypothesized siRNA delivered to tunicamycin treated HeLa and NCI-H1299 cells would achieve less silencing than untreated cells. Further, we expected that nanoparticle delivery systems that utilize endocytosis pathways other than Flotillin-mediated endocytosis would achieve less silencing in C. difficile toxin A treated cells. Using flow cytometry, laser scanning confocal microscopy, and an established suite of chemical inhibitors of endocytosis, our results will show alterations in silencing efficacy and identify the endocytic mechanisms that are impacted by the environmental and chemical stressors.