Flow Cytometric Profiling of Leukocyte Responses to Nanocarrier-Delivered RyR2-Targeted siRNA

A major challenge in RNA-based therapeutics is the potential induction of unintended immune responses, either by the RNA molecules themselves or their delivery systems. Within the framework of the PNRR–National Center for Gene Therapy and RNA-Based Drugs, we developed and optimized a spectral flow cytometry-based in vitro assay to evaluate both biological activity and off-target effects of nucleic acid (NA)-based compounds on human immune cells. Using this platform, we investigated the immunological impact of siRNA U10 targeting the Ryanodine receptor 2 (Ryr2) gene. This siRNA is designed to treat patients with dominant catecholaminergic polymorphic ventricular tachycardia (CPVT) caused by a specific Ryr2 mutation. To enhance cellular uptake, siRNA U10 was encapsulated in calcium phosphate nanoparticles (CaP). Human peripheral blood mononuclear cells (PBMCs) were cultured for 24–72 hours with increasing concentrations of CaP-loaded U10 (12.5–200 pmol/ml). CaP alone and CaP-loaded scrambled siRNA served as controls. Uptake efficiency was assessed using Cy5-labeled siRNA. Our results demonstrated efficient, time-dependent uptake of CaP-siRNA by PBMCs across all tested concentrations, without compromising cell viability. Monocytes were identified as the primary leukocyte subset internalizing CaP-siRNA (~30%), as also confirmed by confocal microscopy. We next investigated the impact of CaP-siRNAs on leukocyte distribution. Notably, CaP-U10, but not CaP-siRNA scramble and CaP alone, significantly increased the relative frequency of lymphocytes while reducing the monocyte proportions. Collectively, these findings the utility of our assays for preclinical safety evaluation of RNA-based therapeutics. Moreover, our results suggest that CaP-U10 is non-toxic to human leukocytes and may modulate immune cell composition. Ongoing studies aim to further elucidate the immunomodulatory potential of CaP-U10 and determine whether this RNA-based therapeutic could elicit inappropriate immune activation.