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Multiplexed siRNA Immunoassay Unveils Spatial and Quantitative Dimensions of siRNA Function, Abundance, and
Michael Ly1, Sandra Diaz-Garcia1, Nathaniel Roscoe1
1Johnson & Johnson Innovative Medicine, La Jolla, California, USA.
Toxicologic Pathology
|January 20, 2025
Summary
Researchers developed a novel antibody and assay to measure small interfering RNA (siRNA) delivery, uptake, and activity in cells and tissues. This tool aids in developing safer and more effective siRNA therapeutics.
Area of Science:
- Biotechnology
- Molecular Biology
- Pharmacology
Background:
- Small interfering RNAs (siRNAs) are promising therapeutics for gene silencing.
- Effective delivery and cellular uptake are critical for siRNA therapeutic efficacy and safety.
- Current methods for assessing siRNA pharmacokinetics and pharmacodynamics are limited.
Purpose of the Study:
- To develop and characterize a novel antibody for detecting chemically modified siRNA molecules.
- To create a multiplex immunoassay for simultaneous quantification of siRNA uptake, trafficking, and activity.
- To establish a robust platform for evaluating siRNA delivery methods in vitro and in vivo.
Main Methods:
- Generation and characterization of a monoclonal antibody against chemically modified siRNA nucleotides.
- Development of a multiplex siRNA immunoassay integrating the antibody with other reagents.
- Application of the immunoassay using immunohistochemistry (IHC) on mouse tissues and high-content imaging (HCI) in cell culture.
- Quantitative assessment of siRNA biodistribution, activity, and intracellular localization.
Main Results:
- Identification of a monoclonal antibody with broad detection of diverse siRNA sequences and modifications.
- Successful development of a multiplex immunoassay enabling simultaneous measurement of siRNA uptake, trafficking, and silencing.
- Quantitative analysis of siRNA biodistribution and activity in various organs of treated mice using IHC.
- In vitro validation of the assay for simultaneous quantification of siRNA uptake, activity, and endosomal colocalization using HCI.
Conclusions:
- The developed anti-siRNA antibody and multiplex immunoassay provide a powerful tool for siRNA drug development.
- This platform enables precise analysis and visualization of siRNA pharmacokinetics and pharmacodynamics.
- The methods facilitate robust testing of nucleic acid delivery strategies with cellular and subcellular resolution.
Keywords:
2’ O-Me: 2’ O-Methyl2’-F: 2’-FluoroCTNNB1: β-cateninDAPI: 4′,6-Diamidino-2-phenylindoleGalNAc: N-acetylgalactosamineHCI: high-content imagingIF: ImmunofluorescenceIHC: ImmunohistochemistryPK/PD: Pharmacokinetics and PharmacodynamicsPNPDB3 and PNPDB1: siRNA antibodiesRT-qPCR: Reverse Transcription Quantitative PCRTMA: tissue microarraysiRNA: small interfering RNA
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