Therapeutic IVT RNA Development

Figure 1. UTR optimization for improved mRNA expression. 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 2. Codon optimization increases mRNA expression in vitro and in vivo. (A) Expression of HiExpress™ Firefly Luciferase mRNA and other luciferase mRNA in HEK293T cells. 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 3. 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 4. 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 5. Denaturing agarose gel result indicating high integrity IVT RNA >10,000 nt. 

Figure 6. IVT mRNA capping efficiency analyzed by LC-MS. Highly efficient capping (>99%) can be achieved either using (A) co-transcriptional or (B) enzymatic approaches.

Figure 7. Modified nucleotides increase mRNA 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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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 8. 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.