Evaluation of synthetic mRNA with selected UTR sequences and alternative poly(A) tail, in vitro and in vivo

Abstract

Messenger RNA (mRNA) has emerged as an attractive new technology of drugs. The efficacy of mRNA technology depends on both the efficiency of mRNA delivery and translation. Untranslated regions (UTRs) and the poly(A) tail play a crucial role in regulating mRNA intracellular kinetics. Intending to improve the therapeutic potential of synthetic mRNA, we evaluated various UTRs and tail designs, using Pfizer-BioNTech coronavirus disease 2019 (COVID-19) vaccine sequences as a reference. First, we screened six 5' UTRs (cap-dependent/-independent), evaluated nine 5' UTR-3' UTR combinations, and a novel heterologous A/G tail in cell models, and in vivo using luciferase as a reporter gene. Then, to decipher the translation mechanism of selected UTRs, we correlated mRNA expression with ribosome load, mRNA half-life, mRNA immunogenicity, and UTR structures. Our results showed that the heterologous tail we introduced is as potent as the Pfizer-BioNTech tail and confirmed the high potency of the human α-globin 5' UTR. They also revealed the potential of the VP6 and SOD 3' UTRs. We validated our results using mRNA encoding the SARS-CoV-2 spike protein formulated as lipid nanoparticles (LNPs) for mouse immunization. Overall, the selected 3' UTRs and heterologous A/G tail have great potential as new elements for therapeutic mRNA design.

Keywords: 3′ UTR; 5′UTR; MT: Oligonucleotides: Therapies and Applications; mRNA; mRNA stability; poly(A) tail; translation efficiency; vaccine.

Graphical abstract

Figure 1

Stability of poly(A) tail sequences on DNA templates in bacteria and circular dichroism profiles of mRNA with the different poly(A) tails (A) Tail stability of A30-L-70, AG, and 120A plasmids in E. coli DH5-α. The percentage of tail stability represents the proportion of clones maintaining tail integrity relative to the total number of clones. mRNA with different tail sequences were analyzed by synchrotron radiation circular dichroism (SRCD) by performing a thermal scan from 15°C to 90 or 95°C. (B) Melting temperatures (Tm) of mRNA tails determined by SRCD analysis. (C) SRCD scans of A30-L-70 tail sequence, (D) SRCD scans of AG tail sequence, (E) SRCD scans of A120 tail sequence. DE, Delta Epsilon.

Figure 2

The potency of a novel heterologous poly A/G tail (A) Time course analysis of nano luciferase reporter gene (Nluc) expression in HeLa cells transfected with A30-L-70 and AG ARCA-mRNAs. The measurements were acquired at 4, 8, 24, 48, and 96 h post-transfection, and results are expressed as mean ± SD. Control is untransfected cells. (B) Based on these data, total protein expression over time (the area under the curve) was calculated and normalized to A30-L-70 mRNA. Statistical significance was assessed by nested one-way ANOVA. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001 (n = 3 independent experiments, each three technical replicates). (C) In vivo translation of firefly-luciferase-encoding (Fluc) mRNAs containing AG or A30-L-70 tails in BALB/c mice after i.m. injection (one experiment with three mice per group). (D) Quantification was done for all available mice per time point (6, 24, 48, and 72 h); (E) total protein expressed over time (the area under the curve) was calculated and is normalized to that of the A30-L-70 containing mRNA. One-way ANOVA, Tukey’s post-test; ∗p < 0.05; ∗∗∗p < 0.001.

Figure 3

In cellulo comparison of cap-dependent and cap-independent 5′UTRs (A) Schematic representation of 5′ UTR motifs cloned upstream of the nano luciferase reporter gene (Nluc). (B–D) After transfection of the respective mRNAs into cell lines, luciferase activity measured in relative light units (RLU) 24 h after transfection. Data are presented as the mean ± SD in HeLa (B), C2C12 (C), or DC2.4 cells (D). Statistical significance was assessed by nested one-way ANOVA. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. (n = 3 independent experiments, each three technical replicates).

Figure 4

Evaluation of 5′ and 3′UTR combinations in cell models (A) Schematic representation of nine constructs generated by cloning different 5′ UTR motifs upstream and 3′ UTR motifs downstream of the nano luciferase reporter gene (Nluc). (B) Time course analysis of Nluc expression in HeLa cells transfected with the nine mRNA constructs. Bioluminescence was acquired at 4, 8, 24, 48, and 96 h post-transfection, and results are expressed as mean ± SD. (C–F) Total protein expression over time (area under the curve) calculated from time course data and normalized to the hAg 5′UTR-AES 3′UTR containing mRNA in HeLa cells (C), or murine myoblasts (C2C12) (D), or murine dendritic cells (DC2.4) (E), or human myoblasts (AB1079) (F). Statistical significance was assessed by nested one-way ANOVA. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. (three independent experiments with three technical replicates). (G) The nine mRNAs were transfected to monocyte-derived dendritic cells (MoDCs). Relative light units (RLU) were measured 24 h post-transfection. Data are presented as mean ± SD. Statistical significance was assessed by nested one-way ANOVA. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. (n = 3 independent donors, each three technical replicates).

Figure 5

mRNA stability and ribosome load capacity (A) Average stability of the mRNA was assessed by RT-qPCR harvested at the indicated time points. The transcript levels were determined via ΔΔCT calculation relative to hAg-AES mRNA and are shown relative to the 2 h. (B) Nluc mRNA levels at 2, 24, and 96 h relative to 5′hAg-3′AES at 2 h Data are presented as mean ± SD. Statistical significance for 96 h was assessed by nested one-way ANOVA. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. (C) Polysome profile from HeLa cells 24 h after transfection, analyzed on a 10%–50% sucrose gradient. (D) Nluc mRNA levels assessed by RT-qPCR from each polysome fraction and presented in three groups: free mRNA, sub-polysomal-associated mRNA, and polysome-associated mRNA. Results are expressed as mean ± SD of two independent experiments with three technical replicates. Statistical significance for polysome fraction was assessed by one-way ANOVA, Dunnett’s post-test; ∗p < 0.05, ∗∗∗p < 0.001.

Figure 6

Evaluation of 5′ and 3′UTR combinations in mice Bioluminescence imaging of firefly luciferase (FLuc) expression in BALB/c mice following intramuscular (i.m.) injection of mRNA constructs. Representative images of three mice per group are shown at 6, 24, and 72 h post-injection. Quantification was done for all available mice per time point (6, 24, 48, and 72 h). Total protein expressed over time (the area under the curve) was calculated and is normalized to that of the hAg 5′UTR- AES 3′UTR containing mRNA. One-way ANOVA, Tukey’s post-test; ∗p < 0.05, ∗∗∗p < 0.001 (one experiment with three to seven mice as indicated).

Figure 7

Spike-encoded mRNA and investigating vaccine immunogenicity in mice (A) Schedule of vaccination and serum collection. (B and C) After transfection of the modified (m1Ψ)-spike mRNA into cells, the spike protein was measured using ELISA 24 h post-transfection. Data are presented as the mean ± SD for HeLa cells (B) and AB1079 cells (C). Statistical significance was assessed by nested one-way ANOVA. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. (three independent experiments with three technical replicates). BALB/c mice were immunized twice with 5 μg of spike-encoded modified (m1Ψ) mRNA-LNP at weeks 0 and 2. Mice in the mock group received nanoluciferase mRNA-LNP. Spike-protein-specific IgG titers were measured at weeks 1, 2, and 3 (E–G), while neutralizing antibodies were assessed at weeks 2 and 3 (D) using ELISA (n = 5). Statistical significance was assessed by Kruskal-Wallis test. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001 (one experiment with five mice per group). AES, AES-mtRNR1.