IVT mRNA

Figure 2. Expression of luciferase (Luc) mRNA and mRNA induced immune response in mice. (A) Luciferase activity visualized by live imaging at 6 h, 24 h, and 48 h post-injection. (B) Two pro-inflammatory cytokines, IL-6 and TNF-⍺, were quantified in the serum at 48 h post-injection. Error bars represent standard errors. Mice strain: C57BL/6J; mice age: 8 weeks; injection method: intramuscular injection. (C) IFN-γ ELISpot assay of splenocytes derived from Balb/C mice 14 days post intramuscular injection of 30 ug LNP-encapsulated mRNA coding for viral antigen A, viral antigen B, or control PBS.

Figure 3. Validation of hSpCas9 mRNA in vitro. (A) IVT Cas9 mRNA was transfected into 293T-EGFP cells with two types of EGFP-targeting gRNA. EGFP expression in non-treated (NC) and transfected cells was observed by microscopy (B) and quantified using flow cytometry (C + D). (E + F) The editing to EGFP genes on the genome was further confirmed by T7E1 assay and Sanger sequencing.

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Figure 4. Validation of IVT Chimeric Antigen Receptor (CAR) mRNA expression in 293T cells. (A) CD19 CAR mRNA with robust 5’/3’ UTRs and poly(A) was generated with or without N1-Methylpseudouridine (m1Ψ) and used to transfect 293T cells. (B) 24h post transfection, cells were incubated with PE-labeled human CD19 to quantify CAR expression using FACS. (C) The expression was further validated using an anti-CD3zeta antibody and western blot.

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Figure 5. Production and validation of fluorescently capped EGFP IVT mRNA. (A) Gel electrophoresis analysis of EGFP IVT mRNA samples without a fluorescent cap, with a Cy3-conjugated cap, and with a Cy5-conjugated cap. (B) Fluorescence microscopy images of EGFP expression in HEK293T cells 24 hours post-transfection with equal amounts of EGFP IVT mRNA, comparing samples with or without a fluorophore-conjugated cap. (C) Flow cytometry analysis of EGFP mean fluorescence intensity and (D) % of EGFP-positive cells at specified timepoints following transfection with either unconjugated or fluorophore-conjugated cap EGFP IVT mRNA. (E) Flow cytometry results indicating Cy3 mean fluorescence intensity (bars) and % of Cy3-positive cells (circles) at designated timepoints post-transfection with Cy3-conjugated cap EGFP IVT mRNA. (F) Flow cytometry analysis indicating Cy5 mean fluorescence intensity (bars) and % of Cy5-positive cells (circles) at designated timepoints post-transfection with Cy5-conjugated cap EGFP IVT mRNA.