key: cord-0859498-arpq5bet
authors: Mokuda, Sho; Watanabe, Hirofumi; Kohno, Hiroki; Ishitoku, Michinori; Araki, Kei; Hirata, Shintaro; Sugiyama, Eiji
title: N1-methylpseudouridine-incorporated mRNA enhances exogenous protein expression and suppresses immunogenicity in primary human fibroblast-like synoviocytes
date: 2022-03-23
journal: bioRxiv
DOI: 10.1101/2022.03.22.485393
sha: 10ef030c5b4a884ab772019db60eb8ccf9d17bc3
doc_id: 859498
cord_uid: arpq5bet

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 of IVT mRNA in vitro, primary cultured human fibroblast-like synoviocytes (FLS) 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. FLS transfected EGFP mRNA containing m1ψ generated higher levels of EGFP than unmodified EGFP mRNA or control RNAs. The multiplex assay of the FLS culture supernatant revealed that concentrations 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 cytokine expressions in primary cultured FLS. The findings may contribute to arthritis research.

Studies conducted using murine arthritis models have indicated that the use of in vitro-57 transcribed messenger RNA (IVT mRNA) is an effective therapeutic approach for joint diseases 58 1,2 . IVT mRNA is synthesized in vitro from template DNA. When IVT mRNA penetrates the cell 59 membrane and enters the cytoplasm, it instantly initiates the production of foreign proteins 60 using intracellular ribosomes. Unlike plasmid DNA and viral vectors, IVT mRNA can function 61 without entering the nucleus and is degraded through the endogenous metabolic pathway after 62 protein production 3 . These therapeutic strategies using IVT mRNA are attractive and have 63 been examined in vivo. However, the in vitro-use of IVT mRNA in human synoviocytes has 64 been poorly studied 4 . 65

IVT mRNA introduced into the cytoplasm stimulates intracellular innate immune responses 66 through pattern-recognition receptors (PRRs) 3 . Exogenous single-stranded RNA (ssRNA) is 67 known to react with Toll-like receptor 7 (TLR7), TLR8, protein kinase R (PKR) and retinoic acid-68 inducible gene-I (RIG-I) [5] [6] [7] [8] [9] [10] . In addition, when IVT mRNA forms a hairpin or partially forms 69 double-stranded RNA (dsRNA), it may also be recognized by PRRs such as TLR3, RIG-I, PKR, 70 2′-5′-oligoadenylate synthetase (OAS)/ribonuclease L and melanoma differentiation-71 associated protein 5 (MDA5) [11] [12] [13] [14] . It has been reported that the incorporation of modified 72 nucleic acids, such as pseudouridin-5′-triphosphate (ψ), into IVT mRNA is an effective method 73 to reduce immunogenicity 10,15,16 . The advantages of ψ and 5-methylcytidine-5′-triphosphate 74 have also been reported 1,17 . 75

Recently, the novel coronavirus disease 2019 pandemic, which has been ongoing since 76

December 2019, has led to the development of innovative mRNA vaccines such as BNT162b2 77 and mRNA-1273, containing N 1 -methylpseudouridine-5′-triphosphate (m1ψ) 18,19 . These 78 vaccines seldom have only a few negative effects on the human body, at least in the short term 79

(within approximately 1 year), indicating the safety of m1ψ. The incorporation of m1ψ into IVT 80 mRNA has been highlighted as a useful method for enhancing protein expression 20-27 ; m1ψ 81 has a unique benefit for therapeutic methods using IVT mRNA. 82

In this study, we aimed to evaluate the efficiency of exogenous gene expression and 83 To prepare templates for IVT mRNA with a cap structure and poly A sequence, the following 112 plasmids were previously constructed in our laboratory: pcDNA3-A124 and pcDNA3-enhanced Results 189

We prepared the following four types of IVT mRNA: mock mRNA with or without m1ψ- efficiency. The efficiency of EGFP mRNA (m1ψ)-transfected FLS was more than 80% (Fig 1c) . 201

And, the viability of these cells was more than 90% (Fig 1d) . 202

These findings indicate that IVT mRNA containing m1ψ can induce adequate foreign 203 protein expression with acceptable cell viability in primary cultured FLS. 204 205

Introduced IVT mRNA is considered to be recognized by some PRRs and induce post-viral 207 infection-like responses, which can activate two main cascades, namely, nuclear factor-kappa 208 B (NF-κB)-mediated signaling and interferon-regulatory factor (IRF)-mediated signaling 3,27 . To 209 clarify these responses against IVT mRNA, we used a multiplex assay to measure 13 different 210 proteins in the culture supernatants from 0 to 24 h after transfection: IL-1β, IL-6, IL-8, IL-10, 211 IL-12p70, IFN-α2, IFN-β, IFN-λ1, IFN-λ2/3, IFN-γ, TNF-α, CXCL10, and GM-CSF. IL-6, TNF-212 α, CXCL10, and IL-8 were detected, whereas the other nine proteins were below the detection 213 limits. Unmodified mock mRNA increased the protein levels of IL-6 and CXCL10. Moreover, 214 unmodified EGFP mRNA increased the protein levels of IL-6, TNF-α, and CXCL10 (Fig 2a-c) . 215

Both mock and EGFP mRNAs incorporated with m1ψ downregulated this elevation. IL-8 216 showed high endogenous excretion in the no RNA control group, and it did not increase in the 217 presence of unmodified IVT mRNA (Fig 2d) . 218

In summary, unmodified IVT mRNA simultaneously elevated both NF-κB-mediated pro-219 inflammatory cytokines, IL-6 and TNF-α, and an IRF-mediated chemokine, CXCL10. In 220 addition, m1ψ-incorporation into IVT mRNA suppressed this elevation. 221 Inflammation exacerbates the pathophysiology of joint diseases, and human RA-derived 231 FLS reportedly produce high levels of IL-6 and CXCL10 37-39 . Therefore, it is desirable that IVT 232 mRNA has low immunogenicity when developing a therapeutic strategy involving gene 233 replacement for arthritis. To verify the usefulness of m1ψ-incorporated IVT mRNA for cultured 234 cells, we utilized primary cultured human FLS. To date, transfection of both unmodified IVT 235 mRNA and plasmid DNA using lipofection in FLS is technically difficult, and these molecules 236 must be introduced using electroporation or viral vectors. Our results support that m1ψ-237 modified IVT mRNA-lipofection method is convenient, facilitates high protein production, and 238 as noted below, has low immunogenicity. 239

To avoid immune responses induced by IVT mRNA, it is necessary to block the interaction 240 between these single-stranded RNAs, which partially form double strands, and PRRs such as 

Wwp2 maintains cartilage homeostasis through regulation of 278

Messenger RNA delivery of a cartilage-anabolic transcription factor as a 280 disease-modifying strategy for osteoarthritis treatment

MRNA-based therapeutics-developing a new class 282 of drugs

The proto-oncogene survivin splice variant 2B is induced by PDGF 284 and leads to cell proliferation in rheumatoid arthritis fibroblast-like synoviocytes

Species-specific recognition of single-stranded RNA via toll-like receptor 287 7 and 8

Innate Antiviral 289 Responses by Means of TLR7-Mediated Recognition of Single-Stranded RNA

Recognition of single-stranded RNA viruses by Toll-like receptor 7

Differential roles of MDA5 and RIG-I helicases in the recognition of RNA 294 viruses

RIG-I-mediated antiviral responses to single-stranded RNA bearing 296

Incorporation of pseudouridine into mRNA enhances translation 298 by diminishing PKR activation

The RNA helicase RIG-I has an essential function in double-300 stranded RNA-induced innate antiviral responses

Length-dependent recognition of double-stranded ribonucleic acids by 302 retinoic acid-inducible gene-I and melanoma differentiation-associated gene 5

Structural basis of double-stranded RNA recognition by the RIG-I like 305 receptor MDA5

Nucleoside modifications in RNA limit activation of

oligoadenylate synthetase and increase resistance to cleavage by RNase L

Suppression of RNA recognition by 310

Toll-like receptors: the impact of nucleoside modification and the evolutionary origin of 311 RNA

Incorporation of pseudouridine into mRNA yields superior 313 nonimmunogenic vector with increased translational capacity and biological stability

From the RNA world to the clinic

Safety and Efficacy of the BNT162b2 mRNA Covid-19 Vaccine

Efficacy and Safety of the mRNA-1273 SARS-CoV-2 Vaccine

N1-methylpseudouridine-incorporated mRNA outperforms 322 pseudouridine-incorporated mRNA by providing enhanced protein expression and 323 reduced immunogenicity in mammalian cell lines and mice

N1-methyl-pseudouridine in mRNA enhances translation through 326 eIF2α-dependent and independent mechanisms by increasing ribosome density

Dendritic Cell Targeting mRNA Lipopolyplexes Combine 329

Strong Antitumor T-Cell Immunity with Improved Inflammatory Safety

Purification of mRNA Encoding Chimeric Antigen Receptor Is 332

Critical for Generation of a Robust T-Cell Response

Optimizing Modified mRNA In Vitro Synthesis Protocol for

M1ψ efficiency in fibroblasts 14

N 1-Methylpseudouridine substitution enhances the performance of 337 synthetic mRNA switches in cells

Impact of mRNA chemistry and manufacturing process on innate 339 immune activation

Modifications in an Emergency: The Role of

Methylpseudouridine in COVID-19 Vaccines

The American Rheumatism Association 1987 revised criteria for the 343 classification of rheumatoid arthritis

Modification of antigen-encoding RNA increases stability, 345 translational efficacy, and T-cell stimulatory capacity of dendritic cells

Induction of virus-specific cytotoxic T lymphocytes in vivo by 348 liposome-entrapped mRNA

Characterization of a Messenger RNA Polynucleotide Vaccine 350

Dendritic cells pulsed with RNA 352 are potent antigen-presenting cells in vitro and in vivo

In vitro-synthesized infectious RNA as an attenuated live vaccine in 355 a flavivirus model

Induction of Antitumor Immunity by Vaccination of Dendritic Cells 357

Transfected with MUC1 RNA

Intra-pinna anti-tumor vaccination with self-replicating infectious 359 RNA or with DNA encoding a model tumor antigen and a cytokine

Detection of prokaryotic mRNA signifies microbial viability and 362 promotes immunity

Fibroblast-like synoviocytes: key effector cells in 364 rheumatoid arthritis

Serum CXCL10 levels are associated with better responses to 366 abatacept treatment of rheumatoid arthritis

Activin A Expressed in Rheumatoid Synovial Cells Downregulates 369

TNFα-Induced CXCL10 Expression and Osteoclastogenesis

Recognition of double-372 stranded RNA and activation of NF-kappaB by Toll-like receptor 3

expression of therapeutic proteins after delivery of chemically 375 modified mrNA in mice