Abstract 5479: Development of novel luminescent assays for sensitive and specific quantitation of double-stranded RNA in mRNA vaccines

Abstract Personalized mRNA vaccines, designed with patient-specific tumor mutations, are now advancing in clinical trials and showing promising results. Additionally, the delivery of therapeutic proteins (e.g., antibodies) encoded by mRNA is under active development. The large-scale production of mRNA is through the process of in vitro transcription (IVT). A significant byproduct and contaminant of IVT is double-stranded RNA (dsRNA), which is highly immunogenic. dsRNA can trigger inflammation, induce cell death, and inhibit protein translation. Therefore, accurate quantification of dsRNA is essential to ensure the safety and efficacy of mRNA therapeutics. However, current technologies, such as dot blot and ELISA, lack the sensitivity and quantitation needed for robust dsRNA detection. In this work, we describe the development of novel luminescent assays for the sensitive and specific quantitation of dsRNA in IVT products. First, we applied NanoBiT® split luciferase technology to detect dsRNA in mRNA drug substance. In this assay, dsRNA-binding domains are fused to the SmBiT and LgBiT components of NanoBiT®. In the presence of dsRNA, these domains dimerize, reconstituting a functional NanoBiT® luciferase enzyme that generates light proportional to the dsRNA concentration. The assay detects dsRNA longer than 30 base pairs and is agnostic to sequence and size. It exhibits high specificity, with no cross-reactivity to single-stranded RNA or DNA, and a limit of quantitation below 10 pg/ml. As a complementary approach, we developed a cell-based bioassay to detect encapsulated dsRNA within LNPs. Upon endocytosis, LNP-encapsulated dsRNA is recognized by Toll-like receptor 3 (TLR3). This activation is captured via a TLR3-specific promoter driving luciferase expression, offering a physiologically relevant readout of dsRNA bioactivity. Both assays are streamlined in an “add-mix-read” format, requiring no wash steps or media transfers, and deliver robust, sensitive, and reproducible performance. Together, these tools enable precise detection of dsRNA contaminants in IVT products, ensuring the quality and safety of mRNA therapeutics. Citation Format: Rich Moravec, Jun Wang, Jim Hartnett, Mei Cong, Jamison Grailer. Development of novel luminescent assays for sensitive and specific quantitation of double-stranded RNA in mRNA vaccines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5479.