Imaging and safety profiling of inhaled siRNA RyR2 in human respiratory models.

RNA therapeutics hold strong potential for treating genetic disease, yet progress is often limited by delivery, stability, and safety concerns. Here, we profile respiratory safety and cellular uptake of an inhaled siRNA targeting the cardiac RyR2 gene, which is responsible for catecholaminergic polymorphic ventricular tachycardia (CPVT), delivered via inhalation with calcium phosphate nanoparticles (CaP NPs). Barrier integrity, cytotoxicity, siRNA uptake, and immune activation, were assessed using a human 3D normal bronchial epithelial model (NHBE) under Air-Liquid Interphase (ALI) and dendritic cells (DCs), tested in monoculture and in co-culture. Barrier function, measured by transepithelial electrical resistance (TEER), remained stable after 48 h of exposure to 400 nM siRNA RyR2, scramble control, or CaP NPs, indicating preserved epithelial performance. Confocal imaging showed efficient internalization of Cy5-labeled siRNA in both mono- and co-cultures. Cytokine profiling revealed IL-8 release across all conditions in NHBE and NHBE + DC models, with IL-6 and TNF-α limited to immune-competent co-cultures; IFN-γ was below the limit of detection. No cytotoxicity was observed. Together, these data demonstrate that CaP NP-mediated delivery achieves robust siRNA uptake without compromising airway barrier integrity, while eliciting only modest, context-dependent immune responses (primarily IL-8 and TNF-α in the presence of DCs). This work supports the respiratory safety and translational potential of inhaled RyR2-targeting siRNA for CPVT.