An engineered T7 RNA polymerase that produces mRNA free of immunostimulatory byproducts

Abstract

In vitro transcription (IVT) is a DNA-templated process for synthesizing long RNA transcripts, including messenger RNA (mRNA). For many research and commercial applications, IVT of mRNA is typically performed using bacteriophage T7 RNA polymerase (T7 RNAP) owing to its ability to produce full-length RNA transcripts with high fidelity; however, T7 RNAP can also produce immunostimulatory byproducts such as double-stranded RNA that can affect protein expression. Such byproducts require complex purification processes, using methods such as reversed-phase high-performance liquid chromatography, to yield safe and effective mRNA-based medicines. To minimize the need for downstream purification processes, we rationally and computationally engineered a double mutant of T7 RNAP that produces substantially less immunostimulatory RNA during IVT compared with wild-type T7 RNAP. The resulting mutant allows for a simplified production process with similar mRNA potency, lower immunostimulatory content and quicker manufacturing time compared with wild-type T7 RNAP. Herein, we describe the computational design and development of this improved T7 RNAP variant.

Self-Amplifying RNA Vaccine Candidates: Alternative Platforms for mRNA Vaccine Development

“…dsRNA can be removed by purification of the RNA, e.g., by high-performance liquid chromatography or by cellulose-based purification [ 53 , 54 ]. Alternatively, dsRNA generation during in vitro transcription can be reduced by optimizing the nucleoside triphosphate ratios or constructing mutant polymerases for RNA synthesis [ 55 , 56 , 57 ]. Additionally, incorporation of modified nucleosides can reduce the immunogenicity of the single-stranded mRNA [ 58 ].…”

Trendbericht Organische Chemie 2023

“…Ein Durchbruch in der Verbesserung von RNA-Polymerasen zur Herstellung von mRNA-Therapeutika kommt von Moore und Rabideau: Sie berichten über eine T7-RNA-Polymerase-Variante, mit der sich mRNA-Transkripte nahezu frei von immunstimulierenden Nebenprodukten wie doppelsträngiger RNA herstellen lassen. 88) Diese verbesserte T7-RNA-Polymerase könnte sich zur Anwendung in der Synthese von mRNA-Therapeutika eignen. In einem ähnlichen Ansatz veränderten die Arbeitsgruppen um Herdewijn und Holliger eine RNA-Polymerase derart, dass diese die effiziente enzymatische Synthese von 2'-O-Methyl-RNA (2'-OMe-RNA) und 2'-O-2'-Methoxyethyl-RNA (MOE-RNA) erlaubt (Abbildung 23E).…”

Towards the controlled enzymatic synthesis of LNA containing oligonucleotides

“…For instance, antisense oligonucleotides consist of short, fully-modified sequences and the de novo genome synthesis requires the error-free assembly of massive amounts of shorter stretches of unmodified DNA ( Masaki et al, 2022 ; Matthey-Doret et al, 2022 ). On the other end of the spectrum, mRNA vaccines require the production of long (several thousands of nucleotides) oligonucleotides containing modified residues such as N 1-methyl-pseudouridine ( Nance and Meier, 2021 ; Dousis et al, 2022 ) while studies aiming at understanding the mechanisms and functions of larger RNAs such as long non-coding RNAs or mRNA call in for the synthesis of long, heavily modified sequences ( Zuckerman et al, 2020 ; Statello et al, 2021 ; Liu and Wang, 2022 ).…”