Development of Novel Bioluminescent Assays for Sensitive and Specific Quantitation of Double-Stranded RNA in mRNA Therapeutics

mRNA-based therapeutics have shown clinical efficacy, evidenced by the success of the mRNA vaccines for SARS-CoV-2. Personalized mRNA vaccines containing patient-derived tumor mutations are now entering the clinic and have shown promise. Delivery of mRNA-encoded therapeutic proteins (e.g., antibodies) is also under development. mRNA drug substance is typically generated by in vitro transcription (IVT). One byproduct and contaminant of IVT is the formation of double-stranded RNA (dsRNA). dsRNA is highly immunogenic, can induce inflammation and cell death, and inhibit protein translation, including the protein encoded by the therapeutic mRNA. As such, dsRNA must be measured for all IVT products to ensure safety and efficacy. However, existing dot bot and ELISA technology to detect dsRNA is neither sensitive nor quantitative. Herein, we describe the development of novel bioassays for sensitive and specific quantitation of dsRNA in IVT products and following encapsulation of IVT products in lipid nanoparticles (LNPs)