Optimizing the Functionality of in vitro-Transcribed mRNA

Marina Tusup^1,2, Lars E French^1,2, Emmanuella Guenova^1,2, Thomas Kundig^1,2, and Steve Pascolo^1,2*

Received: July 22, 2018;   Published: July 27, 2018

DOI: 10.26717/BJSTR.2018.07.001487

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Abstract

Background: In vitro-transcribed messenger RNA (ivt mRNA) is a safe genetic vector that can be used for vaccination and gene therapy. We investigated the impact of the 3’ untranslated region (UTR) and of modified nucleotides on the functionality of ivt mRNA in vitro as well as in vivo. We confirmed that a 3’ UTR consisting of a tandem repeat of beta-globin 3’ UTR enhances the expression of ivt mRNA in non-transformed cells in vitro and in liver in vivo. In addition, we compared ivt mRNA made with the four canonical bases (“ACGU”), with uridine replaced by N1-methyl pseudouridine (“ACGPseudo”) or with both uridine and cytidine replaced by N1-methyl pseudouridine and 5-methylcytidine, respectively (“A5mCGPseudo”). We report that the “ACGU” and “ACGPseudo” ivt mRNA have superior functionality in tumour cells. However, in immune cells and in vivo, the “ACGPseudo” composition allows better expression of the synthetic mRNA. The additional substitution of cytidine with 5-methylcytidine in “A5mCGPseudo” ivt mRNA is deleterious for expression. The “ACGU” ivt mRNA induces cytokines in immune cells, while both “ACGPseudo” and “A5mCGPseudo” do not. We conclude that an ivt mRNA containing A, C, G and N1-methyl pseudouridine residues and having a 3’ tandem repeat of the beta-globin UTR is the optimal design of this vector for gene therapy.

Keywords: in vitro-transcribed mRNA; Pseudouridine; 5-methylcytidine, Globin UTR

Abstract | Introduction | Materials and Methods | Results and Discussion | Conclusion | Acknowledgement | References |