Efficient reduction of synthetic mRNA induced immune activation by simultaneous delivery of B18R encoding mRNA

Ana Velic,Meltem Avci-Adali,Heidrun Steinle,Stefanie Krajewski,Boris Macek,Tatjana Michel,Ludmilla Hann,Christian Schlensak,Hans Peter Wendel,Sonia Golombek

doi:10.1186/s13036-019-0172-5

Ana Velic, Meltem Avci-Adali + Show 8 more

Open Access

https://doi.org/10.1186/s13036-019-0172-5

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Abstract

The application of synthetic modified messenger RNA (mRNA) is a promising approach for the treatment of a variety of diseases and vaccination. In the past few years, different modifications of synthetic mRNA were applied to render the mRNA more stable and less immunogenic. However, the repeated application of synthetic mRNA still requires the suppression of immune activation to avoid cell death and to allow a sufficient production of exogenous proteins. Thus, the addition of type I interferon (IFN) inhibiting recombinant protein B18R is often required to avoid IFN response. In this study, the ability of B18R encoding mRNA to prevent the immune response of cells to the delivered synthetic mRNA was analyzed. The co-transfection of enhanced green fluorescent protein (eGFP) mRNA transfected fibroblasts with B18R encoding mRNA over 7-days resulted in comparable cell viability and eGFP protein expression as in the cells transfected with eGFP mRNA and incubated with B18R protein. Using qRT-PCR, significantly reduced expression of interferon-stimulated gene Mx1 was detected in the cells transfected with B18R mRNA and stimulated with IFNβ compared to the cells without B18R mRNA transfection. Thereby, it was demonstrated that the co-transfection of synthetic mRNA transfected cells with B18R encoding mRNA can reduce the IFN response-related cell death and thus, improve the protein expression.

Highlights

During the last few years, synthetic messenger RNA has gained great interest as a therapeutic agent
Proteome analysis for the detection of translated B18R protein in the cells Fibroblasts were transfected with 1.5 μg enhanced green fluorescent protein (eGFP) messenger RNA (mRNA)
The cells treated with B18R mRNA demonstrated a similar cell viability as the cells treated with B18R. These results demonstrated that the cells transfected with synthetic mRNA could be simultaneously transfected with B18R encoding mRNA to reduce immune reactions and maintain the cell viability and protein expression

Summary

Introduction

During the last few years, synthetic messenger RNA (mRNA) has gained great interest as a therapeutic agent. The synthetic mRNA-based therapies promise new opportunities for the treatment of different diseases by the induction of functional protein expression in desired cells [1,2,3,4]. To increase the translation and stability of synthetic mRNAs, different types of modifications can be introduced during the in vitro transcription (IVT) [10, 11]. A poly-(A)-tail is attached to the 3′-end to enhance the stability and translation of synthetic mRNA [12]. A synthetic cap analog, such as the anti-reverse cap analog (ARCA, 3′-O-Me-m7G(5′)ppp(5′)G RNA cap structure analog), can be used to further increase the mRNA stability and translation efficiency. Thereby, the mRNA degradation is prevented, the translation efficiency is improved, and the immune

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