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 106 cells per ml in 24-shallow well plates, and incubated for 24 hours. 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.
3. Results and Discussion3.1) mRNA engineering to increase product stability in E. coli
The specific activity of RNAse E on a discrete mRNA species directly determines the half-life of that molecule in an 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 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 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 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 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 >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 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 E. coli , while also being associated with simplified downstream processing requirements, we concluded that this emerging product class was particularly well-suited for an in vivo biomanufacturing system. Accordingly, we focussed further optimisation of our platform on enhancing production of SelfCirc-mRNA.