mRNA 유전자 전달 솔루션 (mRNA Gene Delivery Solutions)

Figure 2. UTR optimization for improved mRNA expression. Different 5' and 3' UTR combinations were tested for regulating Guassia luciferase expression in vitro. 293T 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 3. 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 encapsulated 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 4. 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. 60 ng of polyA tails with expected size of 120 nt and 150 nt were loaded on denaturing Urea-PAGE gel, respectively. (B) Isolated polyA tails analyzed by LC-MS. Deconvoluted spectrum was generated from a 120 pmol of polyA tails with an expected size of 120 nt. (C) Size distribution of the polyA tails with an expected size of 120 nt. The bar graph demonstrates the polyA tails have small heterogeneity. The error bars represent standard deviation from technical triplicates. The weighted average length is 123 nt.

Figure 5. Kozak sequence optimization increased IVT EGFP mRNA expression in zebrafish. One-cell stage zebrafish embryos were measured for EGFP expression at 6 h post-microinjection of 250 pg of zebrafish EGFP IVT mRNA. Fluorescent images indicated optimized Kozak sequence (version 2) greatly improved EGFP expression in vivo.

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Figure 6. Denaturing agarose gel result indicated high integrity was achieved for >10,000 nt IVT mRNA. 

Figure 7. IVT mRNA capping efficiency analyzed by LC-MS. Highly efficient capping (>95%) can be achieved either using (A) co-transcriptional or (B) enzymatic approaches.

Figure 8. Modified nucleotides increased mRNA expression and decreased dsRNA impurities. (A) Expression of firefly luciferase in 293T cells. The mRNA was generated with or without modified nucleotides, N1-Methylpseudouridine (m1Ψ) and 5-Methylcytosine (m5C). Cells grown on the 12-well plates were transfected with 1 ug of mRNA per well. Luciferase activities in 293T cells at 6 h, 24 h, and 48 h post-transfection were measured. Error bars indicate standard deviations. (B) Equal amount (750 ng per dot) of magnetic bead-purified EGFP IVT mRNA with or without nucleotide modification (m1Ψ) was blotted and subsequently detected by dot blot assay for estimating the dsRNA impurity.

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As a major trigger of undesired immunogenicity, double-stranded RNA (dsRNA) is a by-product of IVT. Dot blot result demonstrates extra purification steps (e.g. IP-PR) may be necessary for achieving an ultra-purification scale with very low levels of dsRNA.

Figure 9. dsRNA removal efficiency of different purification processes. Equal amounts (1500 ng per dot) of hSpCas9 IVT mRNA purified by different processes were blotted and subsequently detected by dot blot assay for estimating the dsRNA impurity. Abbreviations: HIC, Hydrophobic interaction chromatography; IP-RP, Ion-pair reversed-phase liquid chromatography. 

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Cryo-TEM results show good structural integrity and size homogeneity of our LNP-mRNA.

Figure 10. Cryo-TEM of LNP-mRNA. Scale bar=100 nm. 

Dynamic light scattering (DLS) analysis shows our LNP-mRNA products can reach a very low PDI (as low as PDI<0.1). 

Figure 11. Particle size and Zeta potential distribution analysis. PDI (A) and Zeta potential (B) was determined by DLS which measures the intensity differences of fluctuated light due to motion of particles. The Zeta potential of the sample is between -1.872 mV and +1.872 mV.

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