RNA and LNP Production

Figure 4. UTR optimization in IVT mRNAs for enhanced translation. Different 5' and 3' UTR combinations were tested regulating Gaussia luciferase expression in vitro. HEK293T cells were seeded on 12-well plates at a density of 2.3x10⁵ cells per well. Cells were transfected with 1 ug of mRNA per well. At 6 h, 24 h, 48 h, 72 h, and 96 h post-transfection, Gaussia luciferase activities were measured from cell culture medium.

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Figure 5. Incorporation of a miR-122 targeting site into the 3’ UTR reduces liver-specific gene expression. Mice were intravenously injected with 10 ug of LNP-mRNA coding for HiExpress^TM Firefly Luciferase, with or without a miR-122 targeting site in the 3’ UTR. Two different configurations with different positions of the miR-122 targeting site were tested (design A + B). 6 hours post-injection mice were sacrificed, organs were dissected, and luminescence was measured. miR-122 is highly and specifically expressed in the liver and the inclusion of the miR-122 targeting site led to reduced luciferase expression in liver tissue.

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Figure 6. Codon optimization in IVT mRNAs enhances gene expression both in vitro and in vivo. (A) Expression of HiExpress™ Firefly Luciferase and other luciferase in HEK293T cells from IVT mRNAs. Cells grown on a 12-well plate were transfected with 0.5 ug of mRNA per well and luciferase activity was measured at 6 h, 24 h, 48 h, and 72 h post-transfection. (B) Luciferase activity measured in adult C57BL/6 mice injected intramuscularly with 30 ug of LNP-mRNA at 6 h, 24 h, and 48 h post-injection. FLuc WT indicates wild-type firefly luciferase. FLuc WT (co) indicates codon-optimized wild-type firefly luciferase. FLuc2 indicates Luc2 firefly luciferase.

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Figure 7. PolyA tail size analysis. PolyA tails were cleaved from IVT mRNA using ribonuclease T1 and isolated by oligo dT affinity chromatography. (A) Isolated polyA tails analyzed by Urea-PAGE gel electrophoresis. (B) Isolated polyA tails analyzed by LC-MS. Deconvoluted spectrum was generated from 120 pmol of polyA tails with an expected size of 120 nt. (C) Size distribution of polyA tails with an expected size of 120 nt. Error bars represent standard deviation from triplicates. Weighted average length is 123 nt.

Figure 8. The influence of polyA length and structure on translational efficiency. HEK293T cells were transfected with IVT EGFP mRNA with the same Cap1 and UTRs but different polyA tail lengths as listed above.

Figure 9. Modified nucleotides increase gene expression and decrease dsRNA impurities. (A) Expression of firefly luciferase in HEK293T cells. mRNA was generated with or without modified nucleotides, N1-Methylpseudouridine (m1Ψ) and 5-Methylcytosine (m5C). Cells were grown on 12-well plates and transfected with 1 ug of mRNA per well. Luciferase activities in HEK293T cells at 6 h, 24 h, and 48 h post-transfection were measured. Error bars indicate standard deviations. (B) Equal amounts (750 ng per dot) of magnetic bead-purified EGFP IVT mRNA with or without nucleotide modification (m1Ψ) was assessed by dot blot assay for dsRNA impurities.

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Figure 10. Denaturing agarose gel result indicating high integrity IVT RNA >10,000 nt. 

Figure 11. IVT mRNA capping efficiency analyzed by LC-MS. Highly efficient capping can be achieved either using (A) co-transcriptional or (B) enzymatic approaches.

Double-stranded RNA (dsRNA) is a by-product of IVT and a major trigger of undesired immunogenicity. The dot blot result demonstrates that extra purification steps (e.g. IP-PR) may be necessary to achieve an ultra-purification scale with very low levels of dsRNA.

Figure 12. dsRNA removal efficiency of different purification processes. Equal amounts (1500 ng per dot) of hSpCas9 IVT mRNA purified by different processes was assessed by dot blot assay to estimate dsRNA impurities. Abbreviations: HIC, Hydrophobic interaction chromatography; IP-RP, Ion-pair reversed-phase liquid chromatography.

Figure 13. Gel electrophoresis analysis of precursor and circular EGFP RNA (circRNA-EGFP) after incubation with varying concentrations of RNase A as indicated above. RNase A will digest all linear but not circular RNA.

Figure 14. Cryo-TEM micrographs of LNP-mRNA. Scale bar=100 nm. 

Figure 15. Particle size and Zeta potential distribution analysis. PDI (A) and Zeta potential (B) were determined by DLS which measures the intensity differences of fluctuated light due to motion of particles. Data demonstrates homogeneous LNP mixtures.

Figure 16. Long-term (153 days) cryopreservation of anti-CD31 antibody-conjugated LNP-mRNA. (A) Firefly luciferase (FLuc)-expressing LNP-encapsulated mRNA was preserved under two conditions: 4 °C with no additives, and -80 °C in 7.5% sucrose solution. Particle size (pink bars) and PDI (teal dots) of both groups were compared to the original parameters. (B) Imaging of Fluc mRNA expression 6 hours post-injection. Female ICR mice were intravenously injected with PBS, LNPs preserved at 4 °C without additives, or LNPs preserved at -80 °C in 7.5% sucrose solution. (C) Encapsulation efficiency of LNP-mRNA after cryopreservation. Freshly prepared FLuc LNP-mRNA was used as the control. (D) Comparison of RNA integrity after cryopreservation. Overall, our data show that storing antibody-conjugated LNP-mRNA at -80 °C in 7.5% sucrose solution effectively maintains particle homogeneity, encapsulation efficiency, and mRNA expression for long-term preservation.

Figure 18. Efficient mRNA delivery in vitro using LNPs. Cells were transfected with LNP encapsulated EGFP mRNA or EGFP mRNA mixed with commercial transfection reagent. (A) Flow cytometry analysis of EGFP expression in Jurkat and HEK293T cells and (B) Fluorescent imaging of HEK293T cells at 24 hours post-transfection. MFI: median fluorescence intensity.