4. Concluding remarks
The in vivo mRNA manufacturing system we have presented here can
be utilised to produce synthetic mRNA molecules for a wide range of
research and commercial applications. Although we have demonstrated for
the first time that functional mammalian mRNA can be produced at
high-yields in E. coli cell factories, widespread adoption of
this platform requires further optimisation of both i) downstream
processing steps to increase product purities and ii) upstream processes
to maximise product titres by enhancing cell specific productivity and
cell biomass accumulation/maintenance. While we anticipate that the
developed system will be particularly suitable for production of
circular mRNA products, it’s utility for manufacturing linear mRNA
molecules would be significantly enhanced by optimising co-expression of
a T7-Capping enzyme fusion protein (Qin et al., 2023) to enable
synthesis of capped mRNA species in E. coli . Similarly,
co-expression of nucleotide-modifying enzymes, such as Psuedouridine
synthetase (Carlile et al., 2019), would permit production of linear
mRNA products with required immunostimulatory properties for therapeutic
applications.
The potential utility of microbial cell factories for large-scale mRNA
manufacturing is being increasingly recognised. Indeed, earlier this
year saw announcements of plans to develop commercial cell-based mRNA
production processes using Eukaryotic cell-hosts. The availability of
such platforms will become increasingly critical in coming years as
product lines begin to diversify (e.g., adoption of more complex
molecular formats) and the scale of global mRNA manufacturing continues
to increase. By engineering the core components of an E.
coli -based mRNA production system this study has added a novel
technology to the mRNA manufacturing solution space, providing
flexibility to achieve context- and/or application-specific design
criteria.