1. Introduction
Synthetic mRNA has potential use in a wide range of applications,
including cancer immunotherapy, protein replacement therapy, genome
editing, pluripotent stem cell generation, and vaccines against
infectious diseases (Baden et al., 2021; Breda et al., 2023; Gan et al.,
2019; Qin et al., 2022; Vavilis et al., 2023). In all cases, mRNA
molecules are currently produced in standardised in vitrotranscription (IVT) systems, comprising an RNA Polymerase biocatalyst,
DNA template, modified nucleosides, magnesium-containing buffer and a
capping enzyme/analog (Ouranidis et al., 2022). These simple, modular,
cell-free production platforms embed flexibility and predictability in
mRNA manufacture, while substantially reducing process-related
impurities (Whitley et al., 2022). However, the requirement for purified
input components is associated with relatively high costs, and critical
reagent shortages (Kis et al., 2021). Moreover, downstream purification
processes are complicated by complex product-related impurity profiles,
that include immunostimulatory double-stranded RNA and abortive
transcripts (Gholamalipour et al., 2018; Rosa et al., 2021). However,
despite these drawbacks, expanding product diversification (particularly
with respect to size), highly variable intended applications (with
associated variability in required production scale, purity, cost, etc),
and the increasing pressure placed on reagent/equipment supplies by
growing demand for mRNA synthesis, there are currently no alternative
technology platforms available for mRNA manufacture.
Cell-based production systems are the dominant choice for manufacture of
other bioproducts, such as AAV vectors, recombinant proteins and
recombinant DNA plasmids (Agostinetto et al., 2022; Jiang and Dalby,
2023; McElwain et al., 2022). Although they are associated with
relatively complex and costly downstream processing steps to remove
host-cell impurities, this is somewhat mitigated by the availability of
well-characterised chromatographic and membrane-based unit operations
(Fan et al., 2023; Sripada et al., 2022). As a relatively simple
macromolecule, synthetic mRNA could theoretically be produced in
virtually any microbial cell factory. E. coli is a particularly
attractive expression host given that decades of use in recombinant
plasmid DNA production has led to development of very low-cost,
standardised, easy to scale (up to 100,000L) flexible manufacturing
platforms (Pontrelli et al., 2018; Yang et al., 2021). Indeed, these
benefits have seen E. coli utilised as a biocatalyst for
production of RNA aptamers and double stranded RNA (dsRNA) molecules
(Delgado-Martín and Velasco, 2021; Ma et al., 2020; Ponchon and Dardel,
2011; Ponchon et al., 2009, 2013)
The primary limitation of mRNA production in microbial expression hosts
is endogenous pathways that encode rapid RNA turnover, where the average
mRNA half-life in E.coli is ~5 mins (Esquerré et al.,
2015; Mohanty and Kushner, 2022). For dsRNA manufacture, multigram per
liter yields have been achieved in E. coli bioprocesses by
deleting RNase III, a non-essential dsRNA-targeting endonuclease
(Pertzev and Nicholson, 2006). However, single stranded mRNA decay is
mediated by RNAseE, an essential enzyme required for global RNA
metabolism. Although RNAseE has broad substrate specificity, various
sequence features have been shown to increase its relative specific
activity on individual mRNA species, including unstructured AU rich
regions, and, most critically, the presence of a 5’-monophosphate (Bae
et al., 2023; Callaghan et al., 2005; Richards and Belasco, 2023).
However, other molecule-specific features, such as RNA-binding protein
binding sites, codon usage, and secondary structure profiles can reduce
RNAse E mediated mRNA turnover (Börner et al., 2023; Roux et al., 2022).
More generically, global mRNA half-life is affected by both the relative
abundance and activity level of RNAse E (Mohanty and Kushner, 2022).
Accordingly, the half-life of a specific mRNA molecule within anE. coli cell chassis is determined by a complex interplay between
the mRNA sequence/structure and the host cell’s complement of RNA
degradation machinery components.
Herein we report coordinated mRNA, DNA, media and host cell engineering
to dramatically increase synthetic mRNA accumulation and maintenance inE. coli cell factories. Achieving mRNA yields
>40-fold greater than standard ‘unengineered’ E.
coli expression systems, we demonstrate rapid production and
purification of a range of functional mRNA products. In doing so, we
introduce a new technology platform for mRNA manufacturing solution
spaces. This may be particularly useful in contexts where IVT systems
are unavailable (e.g., due to reagent shortages), product formats
necessitate process optimisation (e.g., production of very large RNA
molecules), or manufacturing costs need to be significantly reduced.