Figure Legends
Figure 1 - mRNA structures were engineered to increase product
stability in E. coli host-cells by A) including
‘scaffold’ tRNA-Lysine motifs at both the 5’ and 3’ termini, B)incorporating ribozyme sequences to promote self-circularisation, andC) 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(D) . Data are expressed as a fold-change of the production
exhibited by the unengineered control system. Values represent the
mean + SD of three independent experiments (n = 3, each
performed in triplicate). UTR: Untranslated region; IRES: Internal
ribosome entry site; ORF: Open reading frame; GFP: Green Fluorescent
Protein.
Figure 2 - mRNA manufacturing platform components were
sequentially optimised by evaluating the function of A)engineered host cell chassis, B) synthetic DNA expression
vectors, and 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 D . Data are expressed as a
fold-change of the production achieved using standard control
components. Values represent the mean + SD of three independent
experiments (n = 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.
Figure 3 - 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). 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 + SD of three independent experiments
(n = 3, each performed in triplicate).
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 (A) , to calculate
integral cell concentration (ICC) and cell specific growth rate (u)
values (B) . Total RNA (C) and product mRNA(E) yields were also measured at 1 h intervals, and the
relative proportion of host cell RNA comprising Spike-mRNA molecules was
quantified (D) . 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 ± SD of three independent experiments; D-G show data
from a single representative capillary gel electrophoresis timecourse
analysis.
Figure 5 . A) Simplified process flow diagram for
large-scale and small-scale in vivo mRNA production. B)Typical chromatogram from oligo-d(T) affinity chromatography
purification of product mRNA manufactured in E. coli .C-D) Capillary electropherograms showing purification of
TermtRNA-GFP (C) and SelfCirc-GFP (D) products using oligo-d(T) affinity
chromatography. E-F) mRNA products manufactured in 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 + SD of three independent
experiments (n = 3, each performed in triplicate).