LNP Encapsulation

Figure 3. Efficient mRNA delivery and expression using LNP in vitro. 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.

Figure 4. 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 5. Transfection of primary T cells with EGFP mRNA. (A) Histogram count of cells expressing EGFP as measured by flow cytometry 24 hours after transfection. Primary T cells were subjected to three different treatments: no treatment control (NC), mRNA encapsulated with a commercial transfection reagent, and lipid nanoparticle encapsulated mRNA (LNP). (B) Corresponding quantification of median fluorescence intensity (MFI) (black) and the % of EGFP-positive cells (grey) as measured by flow cytometry.

Figure 6. Anti-CD31 conjugated firefly luciferase (FLuc) LNP-mRNA showed improved luciferase expression in lung. Mice strain: C57BL/6J; mice age: 6-8 weeks; mice gender: female; administration route: tail vein. Negative controls: IgG2a-conjugated FLuc LNP-mRNA.

Figure 7. LNP formulations achieve efficient plasmid transfection in vitro. (A) A plasmid overexpressing EGFP (pDNA-CMV>EGFP) was encapsulated with SM102 or ALC-0315 LNP and transfected to HEK293T cells. The EGFP expression was captured 24 h post-transfection. (B) Optimized SM102-based LNP formulation achieved ~6.6 times better transfection to HEK293T cells than a commercial PEI transfection reagent. A firefly luciferase expressing plasmid (pDNA-CMV>Fluc) was used as the reporter.

Figure 8. A Luciferase expressing plasmid (pDNA-CAG>Luc+) was encapsulated with VectorBuilder’s novel LNP formulation and was intravenously injected at 0.6 mg/kg body weight. Luciferase expression was detected 96 h post-injection.