Chemical modification of uridine modulates mRNA-mediated proinflammatory and antiviral response in primary human macrophages

Hanieh Moradian,Toralf Roch,Larissa Anthofer,Andreas Lendlein,Manfred Gossen

doi:10.1016/j.omtn.2022.01.004

Abstract

In vitro transcribed (IVT)-mRNA has been accepted as a promising therapeutic modality. Advances in facile and rapid production technologies make IVT-mRNA an appealing alternative to protein- or virus-based medicines. Robust expression levels, lack of genotoxicity, and their manageable immunogenicity benefit its clinical applicability. We postulated that innate immune responses of therapeutically relevant human cells can be tailored or abrogated by combinations of 5′-end and internal IVT-mRNA modifications. Using primary human macrophages as targets, our data show the particular importance of uridine modifications for IVT-mRNA performance. Among five nucleotide modification schemes tested, 5-methoxy-uridine outperformed other modifications up to 4-fold increased transgene expression, triggering moderate proinflammatory and non-detectable antiviral responses. Macrophage responses against IVT-mRNAs exhibiting high immunogenicity (e.g., pseudouridine) could be minimized upon HPLC purification. Conversely, 5′-end modifications had only modest effects on mRNA expression and immune responses. Our results revealed how the uptake of chemically modified IVT-mRNA impacts human macrophages, responding with distinct patterns of innate immune responses concomitant with increased transient transgene expression. We anticipate our findings are instrumental to predictively address specific cell responses required for a wide range of therapeutic applications from eliciting controlled immunogenicity in mRNA vaccines to, e.g., completely abrogating cell activation in protein replacement therapies.

Highlights

IntroductionGrowing demands for rapid, robust, and scalable production of therapeutics for disease prevention or treatment lead to remarkable advances in mRNA-based medicines over the past few years.[1–3] Lack of genotoxicity and facile production, as well as efficient intracellular delivery are advantages of mRNA therapeutics, when compared with preceding non-cellular, nucleic acids-basedAdvanced Therapy Medicinal Products such as recombinant viruses of DNA or recombinant protein-based medicines.[4,5] Clinical applications of mRNA include both, protein replacement therapies[6] and mRNA vaccines,[7,8] deployed for treatment of inherited and non-infectious acquired diseases such as cancer,[9] and viral diseases, such as recently the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).[10,11] The latter is a showcase example for the power of mRNA technology in tackling disease, outpacing other types of vaccines, with rather fast development from bench to market.[12]Despite progress in mRNA production technology by in vitro transcription (IVT) via bacteriophage enzymes such as SP6, T3, and T7 RNA polymerases, potential immunogenicity of transcripts remains a major issue for some mRNA-based medicines.[5,13] The exogenous in vitro transcribed mRNAs (IVT-mRNA) can be recognized by various endosomal and cytosolic pattern recognition receptors (PRRs).[8]
Primary human macrophages were generated from blood-derived CD14+ monocytes
In the present study, we investigated the effects of different cap and nucleotide modifications of In vitro transcribed (IVT)-mRNA upon macrophage transfection

Summary

Introduction

Growing demands for rapid, robust, and scalable production of therapeutics for disease prevention or treatment lead to remarkable advances in mRNA-based medicines over the past few years.[1–3] Lack of genotoxicity and facile production, as well as efficient intracellular delivery are advantages of mRNA therapeutics, when compared with preceding non-cellular, nucleic acids-basedAdvanced Therapy Medicinal Products such as recombinant viruses of DNA or recombinant protein-based medicines.[4,5] Clinical applications of mRNA include both, protein replacement therapies[6] and mRNA vaccines,[7,8] deployed for treatment of inherited and non-infectious acquired diseases such as cancer,[9] and viral diseases, such as recently the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).[10,11] The latter is a showcase example for the power of mRNA technology in tackling disease, outpacing other types of vaccines, with rather fast development from bench to market.[12]Despite progress in mRNA production technology by in vitro transcription (IVT) via bacteriophage enzymes such as SP6, T3, and T7 RNA polymerases, potential immunogenicity of transcripts remains a major issue for some mRNA-based medicines.[5,13] The exogenous in vitro transcribed mRNAs (IVT-mRNA) can be recognized by various endosomal and cytosolic pattern recognition receptors (PRRs).[8]. Clinical applications of mRNA include both, protein replacement therapies[6] and mRNA vaccines,[7,8] deployed for treatment of inherited and non-infectious acquired diseases such as cancer,[9] and viral diseases, such as recently the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).[10,11]. The latter is a showcase example for the power of mRNA technology in tackling disease, outpacing other types of vaccines, with rather fast development from bench to market.[12].

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