Quantitative Pharmacology of Internalization, Intracellular Trafficking and Gene Silencing of siRNA
Authors: Abbiati Roberto Andrea , Yeung Bertrand Z. , Lu Ze , Wientjes Guill M. , Au Jessie L‐S 
Published: November 2023

RNA interference (RNAi) represents a promising approach to correct genetic lesions, and several RNAi candidates including short interfering RNA (siRNA) are in clinical evaluation. In cells, siRNA is loaded on RISC (RNA Induced Silencing Complex) and causes degradation of the targeted mRNA. Due to instability and immunogenicity, siRNA is delivered as a complex with cationic liposomes (Lipoplex, 100–200 nm diameter). Hence, siRNA pharmacodynamics is determined by multiple kinetic steps including cell binding, endocytosis, endosomal transport and recycling, cytoplasmic release from endosomes and Lipoplex, loading in cytoplasmic RISC, and degradation of mRNA and protein. The goal was to develop quantitative pharmacology models of the concentration/time dependent mRNA and protein knockdowns by Lipoplex.For model development, Lipoplex and siRNA transfer between extracellular and intracellular compartments or organelles, and degradation of mRNA/protein were described as first order processes, and the mathematical formulation was based on a system of ordinary differential equations solved numerically. Model parameters were identified by first assigning initial estimates, then fitting the model to the experimental data to yield the best‐fit parameters (i.e., lowest sum of squared errors and within experimental variations).For experimental results, human breast MCF7 cancer cells were incubated with fluorescence‐labeled Lipoplex, and Lipoplex cellular binding and endocytosis were monitored using live cell confocal microscopy or fluorescence microscopy together with markers for endosomal organelles (Rab5 for early endosomes and macropinosomes, Rab‐7 for late endosomes, LAMP1 for lysosomes). Images were acquired every 10 min for up to 12 h, and the fluorescence intensity (e.g., signals colocalizing with the markers) were converted to siRNA molecules using quantitative image analysis (ImageJ and MatLab software). The siRNA molecules on RISC (siRNARISC) were quantified using RISC immunoprecipitation followed by siRNA extraction and quantification with RT‐qPCR (quantitative reverse transcription polymerase chain reaction). The mRNA level was measured using RT‐qPCR and the protein level using Western blotting. The experimental results and computational modeling allowed the definition of two models: (i) the kinetic model described the steps from extracellular Lipoplex to siRNARISC and comprised 16 compartments and 18 rate constants. (ii) The pharmacodynamic model described the mRNA and protein knockdown by siRNARISC and comprised 3 compartments and 7 rate constants. The two models jointly yielded the concentration/time dependent gene silencing by Lipoplex. In conclusion, this study demonstrates the successful application of quantitative pharmacology to capture the kinetics of siRNA embedded in Lipoplex deliver to its target RISC and the resulting pharmacodynamics of gene silencing.Support or Funding InformationSupported in part by research grants RO1 CA163015 (GW, JA) from National Cancer Institute and RO1EB015253 (JA, GW) from National Institute of Biomedical Imaging and Bioengineering, DHHS.