Mechanistic intracellular PK/PD modeling to inform development strategies for small interfering RNA therapeutics.

Small interfering RNA (siRNA) therapeutics provide a targeted approach to silence disease-related genes, with notable success in liver-targeting applications. However, the quantitative effects of siRNA properties, such as stability and affinity, as well as biological factors like cell proliferation, mRNA turnover, and abundance, on gene silencing, particularly for extrahepatic targets, remain poorly understood. To identify determinants influencing gene knockdown extent and duration, we developed a mechanistic intracellular pharmacokinetic/pharmacodynamic (PK/PD) model for RNAiMAX-delivered siRNA, based on cytoplasmic siRNA disposition, RISC-loaded siRNA exposure, and mRNA knockdown across different targets in MCF7 and BT474 cells. The model highlighted the critical roles of cell proliferation in silencing duration and mRNA turnover rates on knockdown extent. In rapid-dividing cells, mRNA half-life drives knockdown profiles, whereas chemical siRNA stabilization extends silencing in slow-dividing cells. Targets with extremely low or high mRNA abundance pose silencing challenges. While sufficient RISC occupancy is essential, increasing RISC exposure has minimal impact on silencing extent; enhancing siRNA-mRNA target engagement is more effective. The model also defined a quantitative relationship for maximal mRNA knockdown, governed by cell proliferation, mRNA half-life, and RISC-mediated cleavage rates. This mechanistic PK/PD modeling provides insights into optimizing siRNA design and target selection in therapeutic development.