Optimizing the Functionality of in vitro-Transcribed mRNA
Journal Title: Biomedical Journal of Scientific & Technical Research (BJSTR) - Year 2018, Vol 7, Issue 2
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. The utilization of in vitro-transcribed messenger RNA (ivt mRNA) for vaccination (induction of an immune response against the encoded protein) and gene therapy (expression of a protein without triggering innate and adaptive immune response) is now intensively developed [1,2]. Since 2006, the production of ivt mRNA at a large scale and under good manufacturing practice (GMP) is well established and has made possible the clinical testing of ivt mRNA in patients with melanomas or carcinomas [3-6]. Features that affect the efficacy of ivt mRNA include the capacity of the 5’ untranslated region (UTR) to recruit initiation factors and ribosome subunits, of the coding region to be efficiently translated and of the 3’ UTR to stabilize the mRNA. In addition to its primary function of serving as a matrix for protein production, ivt mRNA is a danger signal [7]. It can trigger different types of immune responses, depending, for example, on the size of the particles with which it is associated [8]. Although the induction of innate immunity by ivt mRNA can be a good adjuvant in the context of vaccines, it can also limit expression (type I interferon blunts the translation machinery) and prevent the utilization of this format for gene therapy. Substituting canonical unmodified nucleotides such as uridine and cytidine with modified nucleotides such as pseudouridine () and 5-methylcytidine (5mC), respectively, was shown to reduce the immunogenicity of synthetic RNA [9]. It was also reported that those two substitutions can increase the translation of mRNA in immune (e.g., dendritic cells) and cancer cell lines (HEK293T) [10,11]. Synthetic mRNA containing N1-methyl pseudouridine (1) was found to outperform in a broad range of cells (the tumour cell lines A549 and HeLa, human fibroblast BJ cells and human primary keratinocytes) [12].
Authors and Affiliations
Marina Tusup, Lars E French, Emmanuella Guenova, Thomas Kundig, Steve Pascolo
Keywords
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- EP ID EP592756
- DOI 10.26717/BJSTR.2018.07.001487
- Views 185
- Downloads 0
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