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\begin{document}

\title{Engineering an E. coli -based in vivo mRNA manufacturing platform}



\author[1]{Edward Curry}%
\author[1]{George Muir}%
\author[1]{Jixin Qu}%
\author[1]{Zoltán Kis}%
\author[2]{Martyn Hulley}%
\author[1]{Adam Brown}%
\affil[1]{The University of Sheffield Department of Chemical and Biological Engineering}%
\affil[2]{AstraZeneca UK Limited}%


\vspace{-1em}



  
  \date{May 27, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
Synthetic mRNA is currently produced in standardised \emph{in vitro}
transcription systems. However, this one-size-fits-all approach has
associated drawbacks in supply chain shortages, high reagent costs,
complex product-related impurity profiles and limited design options for
molecule-specific optimisation of product yield and quality. Herein, we
describe for the first time development of an \emph{in vivo} mRNA
manufacturing platform, utilising an \emph{E. coli} cell chassis.
Coordinated mRNA, DNA, cell and media engineering, primarily focussed on
disrupting interactions between synthetic mRNA molecules and host cell
RNA degradation machinery, increased product yields
\textgreater{}40-fold compared to standard `unengineered' \emph{E. coli}
expression systems. Mechanistic dissection of cell factory performance
showed that product mRNA accumulation levels approached theoretical
limits, accounting for \textasciitilde{}30\% of intracellular total RNA
mass, and that this was achieved via host-cell's reallocating
biosynthetic capacity away from endogenous RNA and cell biomass
generation activities. We demonstrate that varying sized functional mRNA
molecules can be produced in this system and subsequently purified in
large- or small-scale processes. Accordingly, this study introduces a
new mRNA production technology, expanding the solution space available
for mRNA manufacturing.%
\end{abstract}%



\sloppy


\textbf{Engineering an \emph{E. coli}-based \emph{in vivo} mRNA
manufacturing platform}

Edward Curry\textsuperscript{1*}, George Muir\textsuperscript{1}, Jixin
Qu\textsuperscript{1}, Zolt\selectlanguage{ngerman}án Kis\textsuperscript{1}, Martin
Hulley\textsuperscript{2}, Adam Brown\textsuperscript{1*1}Department of
Chemical and Biological Engineering, University of Sheffield, Sheffield,
UK.

\textsuperscript{2}Bioprocess Development, AstraZeneca, Cambridge, UK.
*Corresponding Authors:

Adam J. Brown, Department of Chemical and Biological Engineering,
University of Sheffield,

Mappin St., Sheffield, S1 3JD

Telephone: +44-(0)114-222-7608, Email: adam.brown@sheffield.ac.uk

Edward Curry, Department of Chemical and Biological Engineering,
University of Sheffield,

Mappin St., Sheffield, S1 3JD

Email: eajcurry1@sheffield.ac.uk

ORCID ID: 0000-0002-5123-8866

Co-author contact details

George Muir: gwmuir1@sheffield.ac.uk

Jixin Qu: jqu8@sheffield.ac.uk

Zoltán Kis: z.kis@sheffield.ac.uk

Martyn Hulley: martyn.hulley@astrazeneca.com

\textbf{Abstract}

Synthetic mRNA is currently produced in standardised \emph{in
vitro}transcription systems. However, this one-size-fits-all approach
has associated drawbacks in supply chain shortages, high reagent costs,
complex product-related impurity profiles and limited design options for
molecule-specific optimisation of product yield and quality. Herein, we
describe for the first time development of an \emph{in vivo} mRNA
manufacturing platform, utilising an \emph{E. coli} cell chassis.
Coordinated mRNA, DNA, cell and media engineering, primarily focussed on
disrupting interactions between synthetic mRNA molecules and host cell
RNA degradation machinery, increased product yields
\textgreater{}40-fold compared to standard `unengineered' \emph{E.
coli}expression systems. Mechanistic dissection of cell factory
performance showed that product mRNA accumulation levels approached
theoretical limits, accounting for \textasciitilde{}30\% of
intracellular total RNA mass, and that this was achieved via host-cell's
reallocating biosynthetic capacity away from endogenous RNA and cell
biomass generation activities. We demonstrate that varying sized
functional mRNA molecules can be produced in this system and
subsequently purified in large- or small-scale processes. Accordingly,
this study introduces a new mRNA production technology, expanding the
solution space available for mRNA manufacturing.

\textbf{Keywords:} Synthetic mRNA, mRNA manufacture, \emph{E. coli} ,
synthetic biology, biopharmaceutical engineering

\emph{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 \emph{in
vitro}transcription (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. \emph{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 \emph{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 \textasciitilde{}5 mins (Esquerré et al.,
2015; Mohanty and Kushner, 2022). For dsRNA manufacture, multigram per
liter yields have been achieved in \emph{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 an\emph{E.
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
in\emph{E. coli} cell factories. Achieving mRNA yields
\textgreater{}40-fold greater than standard `unengineered' \emph{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.

2. Materials and Methods

\emph{Plasmid Design and Construction}

The baseline mRNA production construct was designed with 5' and 3' UTR
sequences from Xenopus beta-globin, directly flanking a human codon
optimised eGFP sequence. All DNA sequences containing stabilisation
features of interest flanking the UTRs and coding sequence were
synthesized and cloned into pET-24b (Novagen) by Twist Bioscience.
Sequences were inserted using the BamHI and XhoI restriction sites,
placing the GOI downstream of the T7 promoter and lac operon, and
removing the ribosome binding site from the original plasmid. High copy
number plasmids were constructed by amplifying the mRNA encoding region
from the original pET29b vector by PCR, and inserting it into pRNA128A,
a vector with the ColE1 origin of replication. Synthetic triple
terminator constructs were assembled using DNA fragments purchased from
Genewiz. The fragments consisted of the T3 and \emph{E. coli} endogenous
rrnB T1 terminators, and were ligated into vectors using the XhoI and
BlpI restriction sites, directly upstream of the T7 terminator.
Sequences of all DNA parts are available in Supplementary table 1.

\emph{Synthetic mRNA Expression}

mRNA encoding plasmids were transformed into BL21 (DE3) or BL21 Star
(DE3) (Invitrogen) \emph{E. coli} strains, typically used for
recombinant protein production. 5ml starter cultures were inoculated
with a single colony, and grown overnight in Luria-Bertani (LB) broth
(Thermo Fisher), containing 50 ug/ml kanamycin (Thermo Fisher) at 37C,
200 rpm. For small scale expression, 100 ul of starter culture was used
to inoculate 10 ml of LB medium containing 50 ug/ml kanamycin, and cells
were grown at 37C, 200rpm, until the OD600nm reached 0.4-0.6. For large
scale expression, 200 ml of LB was inoculated with 5 ml of overnight
culture. IPTG was then added to a final concentration of 1 mM. Where
RNase E inhibitor was used, it was added from a 100x stock at the point
of IPTG addition 500 ul of culture was pelleted at 30 minute intervals
post induction, and pellets stored at -80C. Total cellular capacity
(integral of viable cell concentration), cell-specific growth rate and
cell\selectlanguage{english}-specific productivity were calculated as previously described
(Brown et al., 2019).

\emph{Small Scale RNA Extraction}

Total RNA was extracted using the GenElute Total RNA purification kit
(Sigma-Aldrich) according to the manufacturer's instructions. Briefly,
cell pellets were resuspended in 100 ul of TE buffer containing 1 mg/ml
Lysozyme, and incubated at room temperature for 5 minutes. 300 ul of
buffer RL and 200 ul of 96-100\% ethanol were added to the lysate before
vortexing. Lysate was then applied to the spin column resin, before
washing with ethanol solution. RNA was eluted in 50 ul of elution
solution. Residual DNA in the RNA sample was then removed through
addition of 2 units of RNase free DNase I, and incubation at 37C for 30
minutes. RNA was purified from the DNase reaction using the Monarch RNA
Cleanup kit (50 ug) (New England Biolabs), following the manufacturer's
instructions. Briefly, 2 volumes of RNA binding buffer and 3 volumes of
96-100\% ethanol were added to the RNA sample. The RNA was then bound to
the spin column resin, before washing with ethanol solution. RNA was
eluted from the column in nuclease free water, and stored at -80C. The
concentration of samples was determined using a Nanodrop
spectrophotometer (Thermo Fisher). Integrity of RNA samples were
assessed using denaturing agarose gel electrophoresis. An equal volume
of 2X RNA loading dye was added to 200 ng of total RNA, before heating
to 65C for 5 minutes, and loading on a 1\% agarose gel, which was run at
80 V for 40 minutes.

\emph{Large Scale RNA Extraction}

200 ml of \emph{E. coli} culture was pelleted at 10000 xg for 10
minutes, before extraction by a protocol based upon RNASwift (Nwokeoji
et al., 2016). Cells were suspended in 5 ml of 5 mg/ml lysozyme solution
(Thermo Fisher), and allowed to lyse for 10 minutes at room temperature.
12ml of lysis solution (4\% SDS, 0.5 M NaCl (Thermo Fisher)) was added,
before incubation for 5 minutes at 65 oC. 6.8ml of 5M NaCl of was added,
and suspensions were placed on ice for 5 minutes to promote
precipitation of SDS. Suspensions were centrifuged at 10000 xg for 20
minutes at 4 oC, and supernatant was transferred to a separate tube. RNA
was precipitated from the supernatant by isopropanol or ethanol, and
stored at -20 oC.

\emph{Digital Droplet PCR}

mRNA copy number per nanogram of Total RNA was determined by one-step
reverse transcription digital droplet PCR (RT-ddPCR). Primers were
designed to amplify a 150 bp region in the middle of eGFP, universal to
every construct under investigation. ddPCR reaction mixtures of a final
volume of 20 ul were comprised of 5 ul one step RT-ddPCR supermix
(Bio-Rad), 2 ul reverse transcriptase (Bio-Rad), 1 ul 300 mM DTT
(Bio-Rad), 1 ul 900 nM forward/reverse primer mix (Integrated DNA
Technologies), 1 ul 250 nM 5'-FAM probe (Integrated DNA Technologies),
9ul H20 and 1 ul Total RNA at a concentration of 1 ng/ul. The 20ul
reaction mixture and 70ul of droplet generation oil (Bio--Rad) were
loaded into a DG8 Cartridge, and 40ul of droplets were generated with
the Bio-Rad QX200 droplet generator. Droplets were transferred to a
96-well PCR plate (Bio-Rad), sealed with foil, and placed in a Bio-rad
C1000 thermal cycler. Reverse transcription was performed at 50C for 1
hour. Polymerase activation was carried out for 10 minutes at 95C,
before 40 cycles of denaturation for 1 minute at 95C, before a combined
annealing and extension phase for 1 minute at 60C. Enzymes were then
deactivated at 98C for 10 minutes, before a final hold phase at 12C for
30 minutes. Positive droplets were detected by the QX200 droplet reader
(Bio-Rad), using automatically assigned amplitude thresholds determined
by QuantaSoft software (Bio-Rad). Samples were only used in analysis if
the number of measured droplets exceeded 12000.

\emph{Small Scale mRNA Purification}

mRNA was purified from total RNA extraction samples by oligo (dT)
enrichment, using the Dynabeads mRNA purification kit (Invitrogen).
Approx. 30 ug of Total RNA was adjusted to a volume of 100 ul with
nuclease free water. Samples were heated to 65C for 2 minutes to disrupt
secondary structure, before placing on ice. Magnetic beads were
equilibrated in 100 ul of binding buffer (10 mM Tris-HCl, 1M LiCl),
before addition of the 100 ul total RNA samples. Beads were incubated
with RNA samples for 5 minutes at room temperature with constant
rotation, before removal of the supernatant. Beads were washed twice
with 200 ul of washing buffer (10 mM Tris-HCl, 1M LiCl). mRNA was eluted
from the magnetic beads by addition of 10 mM Tris-HCl, pH 7.5, and its
concentration determined by Nanodrop spectrophotometer.

\emph{Large Scale mRNA Purification}

mRNA from large scale extractions was purified by oligo-dT enrichment
utilising an AKTA pcc chromatography system (Cytiva). 10ml total RNA was
bound to a 1 ml volume Oligo-dt(18) column (Sartorius) in binding buffer
(50 mM Sodium Phosphate pH 7, 500 mM NaCl). The column was washed in 20
column volumes of wash buffer (50 mM Sodium Phosphate, 150 mM NaCl).
Bound mRNA was eluted in 5ml of water, before quantification by Nanodrop
spectrophotometer.

\emph{Capillary Gel Electrophoresis}

Capillary Gel Electrophoresis (CGE) analysis of RNA integrity and purity
was performed with a 5200 Fragment Analyzer System (Agilent, CA, USA),
using the DNF-471 RNA Kit (15 nt) (Agilent, CA, USA). The capillary
cassette used was FA 12-Capillary Array Short, 33 cm (Agilent, CA, USA).
Samples were diluted to \textless{}100 ng/ul in nuclease free water.
Before each separation, a pre-run voltage was applied (8 kV for 30
seconds), the capillaries were conditioned with the conditioning
solution and the capillaries were dipped twice in the rinse buffer.
Following this, the capillaries were filled with RNA separation gel (by
pressure) and then the sample was introduced using a voltage injection
(5 kV for 4 seconds). The separation was then conducted by applying a
voltage of 8 kV for 45 minutes. Detection was carried out using Laser
Induced Fluorescence (LIF), by fluorescent dye tagging of the RNA.

\emph{Transfection of mRNA into HEK293 cells}

Suspension adapted HEK293 cells (Thermo Fisher) were routinely cultured
in serum-free medium (Thermo Fisher). Cells were maintained in 30 ml
volume in 125 ml Erlenmeyer flasks (Corning), at 37C, 85\% humidity, and
5\% CO2, with agitation at 140rpm. Cell density and viability was
determined by the Countess 3 automated cell counter system (Thermo
Fisher). mRNA that required capping prior to transfection was capped
using the NEB Vaccinia virus capping system (New England Biolabs),
following the manufacturers protocol Cells for transfection were
cultured in 24-shallow well plates (Corning), containing 500 ul culture
volume, with agitation at 240 rpm. For mRNA transfection, cells were
seeded at a density of 0.3 x 10\textsuperscript{6} cells per ml in
24-shallow well plates, and incubated for 24 hours. \emph{Trans} IT-mRNA
transfection reagent (Mirus) was used to transfect 500 ng of mRNA per
well as per manufacturer's instructions. GFP transfection efficiency was
determined by the Countess 3 system using a GFP filter. For fluorescence
measurements, cells were by centrifugation at 200 xg for 5 minutes,
before resuspension in DPBS (Sigma-Aldrich), and determination of
fluorescence by plate reader at 488 nm/507 nm.

\emph{3. Results and Discussion3.1) mRNA engineering to increase product
stability in E. coli}

\emph{} The specific activity of RNAse E on a discrete mRNA species
directly determines the half-life of that molecule in an \emph{E. coli}
cell chassis (Mauger et al., 2019; Mohanty and Kushner, 2022; Viegas et
al., 2018). While coding sequence design could theoretically be employed
to enhance accumulation of a particular mRNA molecule in \emph{E.
coli}(E.g., by optimising codon-usage), the efficacy of such methods
would be highly product-specific, dependent on the available
design-space for each primary amino acid sequence. Algorithms are
available for enhancing mRNA sequence through codon usage, however the
design rules applied are unlikely to produce sequences optimal for
stability in both an \emph{E. coli} and mammalian context (Leppek et
al., 2022; Zhang et al., 2023). Accordingly, to achieve product-agnostic
increases in mRNA stability, we focussed on engineering elements that
are located outside the protein coding sequence. Firstly, we introduced
`scaffold' tRNA-Lysine motifs at both the 5' and 3' termini
(TermtRNA-mRNA, Fig 1A), based on previous findings that i) stable
secondary structures significantly reduced the activity of RNAse E on
mRNA molecules (Richards and Belasco, 2023; Zhang et al., 2021), and ii)
incorporation of tRNA motifs increased production of short RNA species
in \emph{E. coli} (Nelissen et al., 2012; Ponchon et al., 2009).
Secondly, given that RNaseE is preferentially active on RNA species that
have a 5' monophosphate (Callaghan et al., 2005), we incorporated
ribozyme sequences either side of the untranslated regions (UTRs) to
promote self-circularisation of product mRNA (SelfCirc-mRNA, Fig 1B).
This design step was aided by recent work describing elements which
efficiently catalyze mRNA self-circulation via the `Permuted Intron
Exon' method (Rostain et al., 2020; Wesselhoeft et al., 2018). Finally,
we designed a hybrid approach, where sections of the tRNA-Lys motif were
inserted at the 5' and 3' termini. Hybridisation of these complementary
sequences creates a pseudo-circular molecule, where the gene expression
cassette is contained in a single stranded loop attached to the tRNA-Lys
structural element (CirctRNA-mRNA, Fig 1C).

TermtRNA-, SelfCirc- and CirctRNA- features were incorporated into a
widely used bacterial GFP-expression vector, where transcription of
product mRNA was driven by an inducible T7 promoter. These expression
plasmids lacked a bacterial ribosome binding site (i.e., to prevent
translation of GFP-mRNA in the host-cell) but contained commonly used
mammalian 5' and 3' untranslated regions, including an encoded polyA
tail, to permit translation of purified mRNA in human cells. GFP-vectors
were transformed into the \emph{E. coli} protein production strain BL21
(DE3) and small-scale mRNA production processes were carried out. Total
RNA was extracted 150 min after induction of expression and GFP mRNA
yields were quantified by digital droplet PCR. As shown in Fig 1D, each
of the engineered constructs facilitated substantial increases in mRNA
product yield, as compared to the standard unengineered control. The
best performing construct, SelfCirc-mRNA, enhanced GFP mRNA yield by
\textgreater{}11-fold, indicating that these circularised molecules were
efficiently shielded from RNAse E-mediated product decay. CirctRNA-
(6-fold increase in product yield compared to control) had a
significantly reduced stabilising effect compared to TermtRNA- (10-fold
increase), which may be due to relatively inefficient formation of the
tRNA motif in this molecular context. Unlike the tRNA-based elements,
SelfCirc-mRNA is also protected from degradation by 3'-5' exonucleases,
which play a relatively minor role in global mRNA decay in \emph{E.
coli} but are particularly active on polyadenylated transcripts (Mohanty
and Kushner, 2022). Moreover, circular mRNA molecules can be directly
utilised in downstream applications without requiring the addition of a
cap structure or incorporation of modified nucleotides. Indeed, given
its low immunogenicity and high molecular stability, both in mammalian
cells and during product storage (Deviatkin et al., 2023), circular mRNA
is considered a promising molecular format for a variety of applications
(Bai et al., 2023; Liu et al., 2022; Qu et al., 2022). As circularised
mRNA facilitated the highest production yields in \emph{E. coli} , while
also being associated with simplified downstream processing
requirements, we concluded that this emerging product class was
particularly well-suited for an \emph{in vivo} biomanufacturing system.
Accordingly, we focussed further optimisation of our platform on
enhancing production of SelfCirc-mRNA.

\emph{3.2 Cell, DNA and media engineering to maximise mRNA product
yield}

We previously described a whole pathway engineering approach that
maximised production of recombinant proteins in Chinese Hamster Ovary
cells (Brown et al., 2019). We hypothesized that a similar strategy
could be applied to mRNA manufacturing in \emph{E. Coli} by sequentially
improving the host cell chassis, DNA expression vector and cell culture
media. Commercially available \emph{E. coli} cell lines have been
engineered to reduce RNase E activity to levels that enhance recombinant
mRNA stability without impacting global mRNA homeostasis (Heyde and
Norholm, 2021; Miroux and Walker, 1996). Although these strains were
originally designed to increase production of `easy to express'
recombinant proteins, they theoretically provide a highly permissive
cell background for synthetic mRNA manufacture. To directly test this,
we compared SelfCirc-mRNA production in previously utilised standard
BL21(DE3) cells and engineered BL21 Star\selectlanguage{english} (DE3) ( (DE3)) cells. As shown
in Fig 2A, cells expressing a mutated RNAse E produced
\textasciitilde{}1.8-fold more GFP mRNA than the unengineered strain.
Although circular mRNA is efficiently protected from RNase E mediated
degradation, covalent circularisation requires synthesis of the
full-length transcript. A reduction in RNase E activity may therefore
enhance synthetic mRNA yields by preventing turnover of nascent product
mRNA, increasing the pool of mRNA molecules available for
circularisation. Product yields may be further enhanced by cell
engineering strategies that increase the host cell's mRNA biosynthesis
(E.g., T7 expression level) and/or cell biomass accumulation capacities.

We rationalised that promoter engineering was unlikely to increase
product yields, as the expression plasmid already contained a T7
promoter optimised to maximise recombinant mRNA transcription rates.
However, enhancing the number of plasmid copies per cell has previously
been shown to enhance manufacture of short dsRNA molecules (Ponchon et
al., 2013). Accordingly, we tested the effect of using a pUC origin of
replication (Ori), which permits very high plasmid copy numbers per cell
(\textasciitilde{}500-700; (Lee et al., 2006; Lin\selectlanguage{english}-Chao et al., 1992)).
As shown in Fig 2B, the use of this element did not increase GFP mRNA
yields in Star BL21 cells, as compared to the use of the original
Rop-ColE1 Ori, despite that construct only encoding maintenance of
\textasciitilde{}15-20 copies per cell (Bolivar et al., 1977; Lee et
al., 2006). This may be caused by the intrinsic metabolic burden
associated with replicating and transcribing very high DNA plasmid
loads. It is likely that testing a range of synthetic Oris (Joshi et
al., 2022; Rouches et al., 2022) will identify a plasmid copy number
`sweet spot' that optimises the quantity of DNA templates available for
product biosynthesis without negatively impacting other desirable
cellular bioproduction phenotypes.

Beyond the promoter and the Ori, the final DNA plasmid element that can
be engineered is the transcriptional terminator. The original expression
plasmid utilised a standard class I intrinsic late T7 terminator, T\selectlanguage{greek}Φ,
\selectlanguage{english}however this is known to encode a termination efficiency of only
\textasciitilde{}74\% (Carter et al., 1981). Replacing T\selectlanguage{greek}Φ \selectlanguage{english}with a
previously described novel triple terminator, comprising a combination
of T7 T\selectlanguage{greek}Φ, \selectlanguage{english}T3 and \emph{E. coli} rrnBT1 endogenous terminators, enhanced
GFP mRNA yields by \textasciitilde{}40\% (Fig 2B). This triple
terminator has been shown to effectively eliminate read-through
transcription by T7 RNA Polymerase (Mairhofer et al., 2015).
Accordingly, this terminator facilitates enhanced RNA Polymerase
recycling efficiency and increases the total biocatalyst time available
for productive synthetic mRNA biosynthesis.

Producing high levels of synthetic mRNA may create product
titer-limiting burden/bottlenecks in host cell metabolic pathways. We
tested the effect of replacing the commonly utilised protein and plasmid
production cell culture media Luria-Bertani broth with other
commercially available formulations. Terrific Broth and Bacto CD Supreme
Fermentation media were investigated as their use of glycerol, as
opposed to oligopeptides, as a carbon source has been reported to
increase maximum cell culture densities (Kram and Finkel, 2015).
However, both media formulations significantly reduced mRNA product
titers (Fig 2C), likely due to the lower cell growth rates achieved
(data not shown). We also tested supplementation with L-Glutamine, based
on the hypothesis that an additional nitrogen source would enhance mRNA
biosynthetic capacity by increasing nucleoside biogenesis, however this
did not significantly impact product yields (Fig 2C). Finally, we
evaluated the chemical effector design space to identify small molecules
that could specifically enhance mRNA production in \emph{E. coli} . The
most promising chemicals identified were a range of RNAse E inhibitors
that reduce enzyme activity via interactions with the N-terminal domain.
However, only one of these inhibitors was commercially available, and
accordingly we tested the effect of supplementing LB media with
3-(4-Hydroxy-5-isopropyl-6-oxo-1,6-dihydro-pyrimidin-2-ylsulfanyl)-propionic
acid (AS2). It was determined that 2 mM AS2 was the optimal
concentration for maximising mRNA maintenance in the cell chassis
(Supplementary data, Fig S1), which has previously been shown to reduce
RNase E activity in \emph{E. coli} by \textgreater{} 80\% (Kime et al.,
2015; Mardle et al., 2020) . Utilising AS2 at this concentration
increased mRNA yield by \textasciitilde{}50\% (Fig 2C), where higher
concentrations reduced cellular productivity. While a similar increase
in titer may be possible via BL21 STAR cell engineering to further
attenuate RNAse E activity, AS2 supplementation offers a robust
mechanism to precisely optimise the synthetic mRNA-RNAse E interactome
in a product-specific manner. Similarly, the use of AS2 in combination
with a mutated RNAse E permits use of inhibitor concentrations with
reduced off-target effects on the host cell.

The optimal combination of engineered mRNA construct (SelfCirc-mRNA),
DNA expression plasmid (Triple terminator), cell host (BL21 STAR) and
media formulation (LB + AS2), facilitated a 44x increase in mRNA product
yield, compared to the standard control system (Fig 2D). Capillary gel
electrophoresis analysis confirmed that product mRNA was full-length and
constituted a substantial proportion of total cellular RNA
(\textgreater{}20\%, compared to \textless{}1\% for the standard control
system; Fig3A). Moreover, high yields of full-length synthetic mRNA were
maintained when the relatively small GFP coding sequence (720 nt) was
substituted for Cypridina Luciferase (cLuc) (1662 nt) or SARS-COV-2
Spike Protein (3783 nt) (Fig 3B), demonstrating that the
engineered\emph{in vivo} biomanufacturing system can produce larger,
more complex molecules. Finally, using oligo-dT magnetic beads, we
validated that achieved increases in product yield were maintained
following small-scale purification processes (Fig 3C). This also
demonstrates that mRNA manufactured in an \emph{E. coli} cell-host can
be purified using simple low-tech methodologies, facilitated by the
absence of abundant endogenous mRNAs with PolyA tails \textgreater{} 5
nucleotides in length (Laalami et al., 2014; Mohanty and Kushner, 2019).

\emph{3.3 Mechanistic dissection of synthetic mRNA production in E. coli
host cell chassis.}

\emph{} In order to understand how \emph{E. coli} host cells utilise
available biosynthetic capacity for mRNA production, we profiled cell
biomass, total RNA and product mRNA accumulation/maintenance during a
6hr manufacturing time course. We utilised the previously optimised
cell-DNA-media composition (see Fig. 2) to manufacture SARS-COV-2 Spike
Protein mRNA. To evaluate mechanistic differences between biosynthesis
of circular and linear molecules, we separately manufactured SelfCirc-
and TermtRNA-mRNA products. With respect to the latter, we confirmed
that the optimal BL21 STAR-Triple terminator-LB(AS2) system combination
permitted a 36-fold increase in TermtRNA-GFP yields as compared to the
standard control system (see Supplementary data, Fig. S2), similar to
the 44-fold increase achieved for SelfCirc-GFP.

As shown in Fig 4A, manufacture of circular and linear synthetic mRNA
products induced a significant metabolic burden on the host cell.
Producer cells reached maximum cell density 2-3 h post induction of mRNA
expression, whereas uninduced cells continued to accumulate biomass up
to the 6 h harvest timepoint. Indeed, these cells exhibited a 25\%
reduction in cell specific growth rate during the first 2 h post
expression induction (Fig 4B). Moreover, the final maximum cell density
achieved was \textasciitilde{}50\% lower for producer cells, as compared
to non-producers, associated with a \textasciitilde{}35\% reduction in
the integral of cell concentration (cumulative cell hours; Fig. 4B).
This indicates that producing substantial amounts of synthetic mRNA
forces the cell to reallocate biosynthetic capacity away from cell
biomass generation activities. Accordingly, approaches to overcome
product biosynthesis-associated burden represent a potentially effective
way to enhance total biocatalyst activity and further increase product
yields. The simplest method to achieve this may be optimisation of
expression induction kinetics, although genetic engineering and/or
directed evolution strategies will likely deliver the most significant
impact on maximum achievable cell densities (Al'abri et al., 2022;
Badran and Liu, 2015; Esvelt et al., 2011). Either way, optimising the
biocatalytic capacity available for mRNA product synthesis is critically
required to take full-advantage of the ability to scale \emph{E.
coli}production processes up to 100,000 L.

As shown in Figure 4C, total RNA synthesised per cell was stable
throughout the production process, at \textasciitilde{}60 fg/cell,
despite Spike Protein-mRNA accumulating over time (Fig. 4D). This is
likely due to feedback mechanisms that act to maintain intracellular
concentrations of key macromolecules within relatively narrow
concentration ranges (Rado\selectlanguage{polish}š \selectlanguage{english}et al., 2022). Accordingly, accumulation of
highly-stable product-mRNA forces the cell to reduce biosynthesis and/or
induce degradation of endogenous RNA species, with potential associated
off-target effects on desirable bioproduction phenotypes such as cell
growth rate. These RNA homeostasis mechanisms place a theoretical limit
on the total quantity of product-mRNA that can be maintained per cell,
above which concentrations of key endogenous RNA molecules will become
critically limiting leading to cell death and/or downregulation of
product expression. Indeed, as shown in Fig 4D, intracellular
concentrations of Spike Protein-mRNA peaked at 4 h, before decreasing
slightly at 6 h. At 4 h, SelfCirc-Spike accounted for
\textasciitilde{}28\% of total RNA mass in the host-cell, which is
likely approaching the maximum achievable concentration. Although not a
direct comparison, during recombinant protein expression in CHO cells,
product molecules typically account for \textasciitilde{}30\% of
intracellular protein mass (in-house data). We concluded that
engineering efforts to further enhance intracellular product maintenance
are unlikely to be beneficial, and instead should focus on maximising
product accumulation rates. For SelfCirc-Spike, cell specific
productivity (product-mRNA produced per cell per hour) was relatively
constant throughout the first 4 hours of the production process, at
\textasciitilde{}5 fg cell\textsuperscript{-1} h\textsuperscript{-1},
equating to \textasciitilde{}10\% of total cellular RNA biosynthetic
activity during this time period. DNA vector engineering, for example T7
promoter re-design, may increase synthetic mRNA generation rates,
facilitating cells to reach the maximum intracellular product-mRNA
concentration level more quickly, permitting shorter production
processes with associated benefits in cost and manufacturing flexibility
(i.e., ability to rapidly switch between manufacture of different
products).

Capillary Gel Electrophoresis analysis clearly shows that product mRNA
accumulates intracellularly over time (Fig. 4 F-G). These data exemplify
that engineered product molecules are successfully protected from
nuclease-mediated decay, facilitating intracellular maintenance over
multi-hour time periods, as compared to the typical mRNA half-life
in\emph{E. coli} of \textasciitilde{}5 min (Bernstein et al., 2002;
Mohanty and Kushner, 2022). Moreover, the presence of a single sharp
peak at each sampling point indicates that the cell factory is producing
full-length Spike Protein-mRNA that is subject to minimal degradation
events. Accordingly, i) further system engineering to disrupt
the\emph{E. coli} degradasome-synthetic mRNA interactome is not
required, and ii) \emph{E. coli} is capable of synthesising homogenous
populations of large mRNA molecules, thereby simplifying downstream
processing steps. As expected, higher titres were obtained for
SelfCirc-Spike than TermtRNA-Spike, where maximum achieved yields were
15 mg/L and 10 mg/L respectively (Fig. 4E). As discussed, we anticipate
that significant increases in product yields will be obtained via
further DNA/cell/media engineering to increase maximum cell density,
integral of cell concentration, and cell specific productivity.
Synthetic mRNA yields \textgreater{} 100 mg/L should be relatively
straightforward to obtain, however achieving g/L titres, as is standard
for recombinant protein production in \emph{E. coli} , will likely
require significant process engineering.

\emph{3.4. Synthetic mRNA produced in E. coli can be purified at
large-scale and is functional in human cells}

To exemplify the utility of our \emph{E. coli} -based system for
large-scale mRNA synthesis we manufactured GFP-mRNA in 1 L production
processes. The first purification step required is total RNA extraction
from host cell factories. While this can be achieved with commercial
kits when production scales are \textless{} 1 ml, larger volumes require
a scalable cost-efficient procedure. To achieve this, we adapted the
RNASwift method previously described by Nwokeoji \emph{et. al} for
extraction of dsRNA products from \emph{E. coli} that utilises NaCL and
SDS to lyse cells and precipitate macromolecular contaminants (Nwokeoji
et al., 2016). To maximise both total RNA yield and RNA quality, we i)
introduced a lysozyme digestion step upstream of RNASwift, ii) lowered
the lysis incubation temperature from 90\selectlanguage{ngerman}ºC to 65ºC, and iii) added an
ethanol precipitation step downstream of RNASwift. Using this modified
RNASwift unit operation we were able to routinely obtain large yields
(10 mg per 0.5 g wet cell mass) of high-quality total RNA (RNA Integrity
Numbers \textgreater{} 9.5, as determined by capillary gel
electrophoresis).

While small amounts of product-mRNA can be purified from total RNA using
oligo-dT magnetic beads (see Fig 3C), larger quantities require
chromatographic operations. To show that mRNA manufactured in \emph{E.
coli} can be purified using a liquid chromatography separation step, we
utilised a 1 ml monolithic oligo-dT(18) column in combination with an
AKTA PCC system. Figure 5B shows a chromatogram representative of this
purification process, indicating conductivity as a measure of salt
concentration, and the UV trace of material eluted from the column.
Capillary gel electrophoresis analysis of pre- and post-purification
samples showed that both SelfCirc-GFP and TermtRNA-GFP molecules could
be efficiently purified by an affinity-capture chromatographic unit
operation (Fig. 5C-D). However, TermtRNA-GFP was isolated at much high
purity, 71\% as compared to 38\% for SelfCirc-GFP, where SelfCirc-GFP
samples showed a considerable wide peak of impurities representing
\textasciitilde{}30\% of total RNA. This may be due to SelfCirc-GFP
molecules having considerably smaller polyA tails than TermtRNA-GFP
species, 50 nt and 120 nt respectively, preventing use of elution
conditions that deliver both high yield and high purity. Further
mRNA/DNA engineering to increase the encoded polyA tail length should
permit product isolation with reduced process/product related
impurities. Either way, for both molecule-formats, it is clear that for
most applications a second chromatographic unit operation would be
needed to achieve requisite purity profiles, such as a size-exclusion
chromatography step. The use of two chromatographic unit operations is
standard for purification of other high-value macromolecules, including
recombinant proteins and IVT-derived mRNA (Fan et al., 2023; Rosa et
al., 2021; Sripada et al., 2022). A simplified process flow diagram for
large-scale and small-scale \emph{in vivo} mRNA production processes is
shown in Figure 5A.

Finally, to validate that mRNA products manufactured in \emph{E.
coli}were functional in mammalian cells, we transfected purified
SelfCirc-GFP and TermtRNA-GFP into Human embryonic kidney cells (HEK).
While SelfCirc-GFP contains an internal ribosome binding site (IRES),
obviating the need for post-purification processing, TermtRNA-GFP
required the enzymatic addition of a Cap-0 structure to enable
translation initiation. As shown in Figure 5E-F, both synthetic mRNA
molecular formats were translatable in HEK cells, facilitating similar
levels of GFP protein expression. Translational efficiency of
SelfCirc-GFP molecules would likely be further enhanced via
determination and selection of optimal IRES elements (Wesselhoeft et
al., 2018). Indeed, this may provide a route to encode cell-type
specificity into mRNA gene therapeutics (Plank et al., 2013).



\emph{4. Concluding remarks}

The \emph{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 \emph{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 \emph{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 \emph{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.

\emph{Data sharing statement}

The data that support the findings of this study are available from the
corresponding author upon reasonable request.

\emph{Conflict of interest statement}

The authors declare no conflict of interest

\emph{Author Contributions}

\textbf{Edward Curry} -- Conceptualisation, methodology, analysis,
investigation, manuscript preparation. \textbf{George Muir} --
Investigation, analysis. \textbf{Jixin Qu} -- Investigation,
analysis.\textbf{Zoltan Kis} - Investigation, analysis. \textbf{Martyn
Hulley} -- Conceptualisation, analysis. \textbf{Adam Brown} --
Conceptualisation, methodology, analysis, manuscript preparation,
funding acquisition.

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\textbf{Figure Legends}

\textbf{Figure 1} - mRNA structures were engineered to increase product
stability in \emph{E. coli} host-cells by \textbf{A)} including
`scaffold' tRNA-Lysine motifs at both the 5' and 3' termini,
\textbf{B)}incorporating ribozyme sequences to promote
self-circularisation, and\textbf{C)} inserting component parts of the
tRNA-Lysine motif at the 5' and 3' termini to facilitate formation of
pseudo-circular molecules. Designed elements were synthesized,
individually inserted into a GFP-expression plasmid and evaluated in 2.5
h production processes\textbf{(D)} . Data are expressed as a fold-change
of the production exhibited by the unengineered control system. Values
represent the mean\selectlanguage{english} +\selectlanguage{english} \emph{SD} ~of three independent experiments
(\emph{n} \selectlanguage{english} =\selectlanguage{english} 3, each performed in triplicate). UTR: Untranslated region;
IRES: Internal ribosome entry site; ORF: Open reading frame; GFP: Green
Fluorescent Protein.

\textbf{Figure 2} - mRNA manufacturing platform components were
sequentially optimised by evaluating the function of
\textbf{A)}engineered host cell chassis, \textbf{B)} synthetic DNA
expression vectors, and \textbf{C)} designed cell culture media
formulations. The relative performance of engineered systems was
evaluated in 2.5 h production processes. The additive impact of each
engineering step on overall product yield is shown in \textbf{D} . Data
are expressed as a fold-change of the production achieved using standard
control components. Values represent the mean\selectlanguage{english} +\selectlanguage{english} \emph{SD} ~of three
independent experiments (\emph{n} \selectlanguage{english} =\selectlanguage{english} 3, each performed in triplicate).
In all panels, the expressed product is SelfCirc-GFP (see Fig. 1); the
effect of whole system engineering on manufacture of TermtRNA-GFP is
shown in Supplementary figure 2.

\textbf{Figure 3} - \textbf{A)} Capillary electropherograms of total RNA
isolated from GFP-mRNA biomanufacturing systems comprising either i)
standard control components, or ii) an optimal combination of engineered
mRNA construct, DNA expression plasmid, cell host and media formulation
(Engineered system, see Fig. 2). \textbf{B)} Gel electrophoresis
analysis of total RNA isolated from Engineered systems producing Green
fluorescent protein (GFP), SARS-COV-2 Spike and Cypridina Luciferase
mRNA molecules. Full-length product mRNA molecules are highlighted by
red arrows; molecule sizes are enlarged due to the presence of IRES and
Intron elements in SelfCirc-mRNA (see Fig. 1). C) Comparison of relative
GFP-mRNA yields obtained from Standard control and Engineered systems,
quantified before (intracellular) and after purification using oligo-dT
magnetic beads. Intracellular yields are expressed as a fold-change of
the product yield obtained using the Standard control system. Values
represent the mean\selectlanguage{english} + \emph{SD} ~of three independent experiments
(\emph{n} \selectlanguage{english} =\selectlanguage{english} 3, each performed in triplicate).

\textbf{Figure 4} - SelfCirc-Spike and TermtRNA-Spike were produced in
biomanufacturing systems comprising an optimal combination of engineered
host cell, DNA plasmid and cell culture media (see Fig 2). Growth of
non-producer (uninduced) and producer cells was measured at 1 h
intervals during 6 h production processes \textbf{(A)} , to calculate
integral cell concentration (ICC) and cell specific growth rate (u)
values \textbf{(B)} . Total RNA \textbf{(C)} and product
mRNA\textbf{(E)} yields were also measured at 1 h intervals, and the
relative proportion of host cell RNA comprising Spike-mRNA molecules was
quantified \textbf{(D)} \emph{.} Total RNA samples from each measured
timepoint were analysed by capillary gel electrophoresis; dashed red
boxes on capillary electropherograms highlight SelfCirc-Spike (F) and
TermtRNA-Spike (G) product accumulating over time. In A-C, values
represent the mean +-\selectlanguage{english} SD of three independent experiments; D-G show data
from a single representative capillary gel electrophoresis timecourse
analysis.

\textbf{Figure 5} . \textbf{A)} Simplified process flow diagram for
large-scale and small-scale \emph{in vivo} mRNA production.
\textbf{B)}Typical chromatogram from oligo-d(T) affinity chromatography
purification of product mRNA manufactured in \emph{E. coli}
.\textbf{C-D)} Capillary electropherograms showing purification of
TermtRNA-GFP (C) and SelfCirc-GFP (D) products using oligo-d(T) affinity
chromatography. \textbf{E-F)} mRNA products manufactured in \emph{E.
coli} were purified and transfected into Human Embryonic Kidney cells.
24 hr posttransfection, GFP protein expression was measured by
fluorescent cell imaging (E) and fluorescent plate reader analysis (F).
Values in F represent the mean\selectlanguage{english} + \emph{SD} ~of three independent
experiments (\emph{n} \selectlanguage{english} =\selectlanguage{english} 3, each performed in triplicate).\selectlanguage{english}
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