Abstract
Studies conducted using murine arthritis models have indicated that the use of in vitro-transcribed messenger RNA (IVT mRNA) is an effective therapeutic approach for joint diseases. However, the use of IVT mRNA in human synovial cells has not been widely studied. Recently, the outbreak of the novel coronavirus disease has accelerated the development of innovative mRNA vaccines, such as those containing a modified nucleic acid, N1-methylpseudouridine-5′-triphosphate (m1ψ). IVT mRNA is an attractive tool for biological experiments and drug discovery. To verify the protein expression from IVT mRNA in vitro, primary cultured fibroblast-like synoviocytes (FLS) and MH7A human synovial fibroblast cells were transfected with enhanced green fluorescent protein (EGFP) mRNA with or without m1ψ incorporation. EGFP was detected using western blotting and fluorescence microscopy. A multiplex assay was performed to comprehensively understand IVT mRNA-induced immunogenicity. Gene expression levels were measured using reverse transcription polymerase chain reaction. In both MH7A cells and FLS, cells transfected with EGFP mRNA containing m1ψ generated higher levels of EGFP than those transfected with unmodified EGFP or control mRNAs. The multiplex assay of the FLS culture supernatant and reverse transcription polymerase chain reaction for FLS revealed that both concentration and expression of IL-6, TNF-α, and CXCL10 were upregulated by unmodified EGFP mRNA, whereas they were suppressed by EGFP mRNA with m1ψ. Overall, m1ψ incorporation enhanced protein expression and decreased the expression of cytokines. These findings may contribute to arthritis research.
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Acknowledgements
We used MH7A cells with the MTA from KISSEI Pharmaceutical Co., Ltd.. We would like to thank Shigeru Miyaki (Hiroshima University) for the preparation of experimental devices.
Funding
This research was funded by JSPS KAKENHI (Grant Number 19K18499), Mitsubishi Foundation, Takeda Science Foundation, Mochida Memorial Foundation for Medical and Pharmaceutical Research, Japanese Respiratory Foundation Grant, Japan Rheumatism Foundation, The Japan College of Rheumatology Grant for Promoting Research for Early RA, and The Nakatomi Foundation to S.M.
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SM, HW, HK, MI, and KA performed the experiments and analyzed the data. SM, HW, SH, and ES planned the experiments and wrote the manuscript.
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Mokuda, S., Watanabe, H., Kohno, H. et al. N1-methylpseudouridine-incorporated mRNA enhances exogenous protein expression and suppresses immunogenicity in primary human fibroblast-like synoviocytes. Cytotechnology 74, 503–514 (2022). https://doi.org/10.1007/s10616-022-00540-4
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DOI: https://doi.org/10.1007/s10616-022-00540-4