Multiplexed siRNA Immunoassay Unveils Spatial and Quantitative Dimensions of siRNA Function, Abundance, and Localization In Vitro and In Vivo

Small interfering RNAs (siRNAs) have been successfully used as therapeutics to silence disease-causing genes when conjugated to ligands or formulated in lipid nanoparticles to target relevant cell types for efficacy while sparing other cells for safety. To support the development of new methods for delivery of siRNA therapeutics, we developed and characterized a panel of antibodies generated against chemically modified nucleotides used in therapeutic siRNA molecules, identifying a monoclonal antibody that detects a broad range of siRNA representing distinct sequences and modification patterns. By integrating this anti-siRNA antibody with additional reagents, we created a multiplex siRNA immunoassay that simultaneously quantifies siRNA uptake, trafficking, and silencing activity. Using immunohistochemistry (IHC), we applied our method on tissues from mice treated with unconjugated, GalNAc-conjugated, or cholesterol-conjugated siRNAs and quantitatively assessed the biodistribution and activity of siRNAs in various organs. In addition, we used high-content imaging (HCI) and applied our multiplex siRNA immunoassay in tissue culture to enable simultaneous quantification of siRNA uptake, activity, and intracellular colocalization with endosome markers. These methods provide a robust platform for testing nucleic acid delivery methods in vitro and in vivo , allowing precise analysis and visualization of the pharmacokinetics and pharmacodynamics of siRNA therapeutics with cellular and subcellular resolution.