RNA ImmunoGenic Assay: Simple method for detecting immunogenicity of in vitro transcribed mRNA. Issue 2 (6th February 2020)

• Record Type: Journal Article
• Authors: Haque, AKM Ashiqul; Weinmann, Petra; Biswas, Sumit; Handgretinger, Rupert; Mezger, Markus; Kormann, Michael S. D.; Antony, Justin S.
• Is Part Of: Advances in cell and gene therapy. Volume 3:Issue 2(2020)
• Page Start: n/a
• Page End: n/a
• Publication Date: 2020-02-06
• Subjects: cmRNA -- cytokines -- immunogenicity -- IVT -- whole blood assay; Cellular therapy -- Periodicals; Gene therapy -- Periodicals; Immunotherapy -- Periodicals; Cancer -- Treatment -- Periodicals; Bone marrow -- Diseases -- Treatment -- Periodicals; Blood -- Diseases -- Treatment -- Periodicals; 615.5
• DOI: 10.1002/acg2.79
• Languages: English
• ISSNs: 2573-8461

Abstract

Abstract: In vitro transcribed (IVT) mRNA therapies is a promising approach for the effective and safe treatment of various diseases. However, IVT ‐ mRNA triggers immune responses due to recognition by human RNA sensors, but incorporation of chemically modified nucleosides have been shown to reduce such responses. Nonetheless, an assay reflecting the complexity of the human immune system is still needed. Here, we present a simple and fast ex vivo method called "RNA Immunogenic Assay" for measuring the immunogenicity of IVT‐mRNA in human whole blood. Chemically modified and unmodified mRNA were complexed with a transfection reagent (TransIT), and co‐incubated in human whole blood and specific cytokines were quantified (TNF‐α, INF‐α, IL‐6, and IL‐12p70) using ELISAs. The qPCR analysis was conducted to unveil the activation of specific immune pathway. Our findings demonstrated that the complete replacement of uridine with pseudouridine reduced TNF‐α, IL‐6, and IL‐12p70 levels and did not elevate the expression of genes involved in immune response against RNA including IRF7/3, EIF2A, & RNASEL. In addition, we observed that the transcript length was not found to be a confounding factor in RNA immunogenicity generation and our assay provide the feasibility to dissect donor specific immune response against mRNA therapeutics.