LNP Encapsulation

Figure 4. Human CAR-T cells generated using VectorBuilder’s LNP-mRNA exhibited cytotoxicity against CD19⁺ cells. (A) Primary human T cells were transfected with lipid nanoparticle (LNP) encapsulated anti-CD19 CAR mRNA. The killing function of the CAR-T cells was validated by co-incubation with CD19⁺ Raji cells and measuring the released lactate dehydrogenase (LDH). (B) Two IVT mRNAs coding anti-CD19 CAR with different co-stimulatory domains, 4-1BB (CD19-BBz, 1949 nt) or CD28 (CD19-28z, 1931 nt), were encapsulated in LNPs. (C) Activated human T cells were transfected with the corresponding LNP-mRNA and validated for CD19 CAR expression. (D) T cell-induced cytotoxicity was measured by LDH assay 18 hours after co-incubation of CAR-T cells with Raji cells at various effector-to-target (E:T) ratios.

Figure 5. Highly efficient human T cell transfection using EGFP LNP-mRNA. (A) Activated T cells were transfected with LNP-encapsulated EGFP mRNA at 6 ug mRNA per 1×10⁶ cells. (B) Denaturing agarose gel electrophoresis confirmed the correct length and integrity of the EGFP mRNA before encapsulation. (C) The particle size and Zeta potential of the LNPs were measured before T cell transfection. (D) EGFP expression was imaged and analyzed 24 hours post-transfection using fluorescence microscopy and flow cytometry.

Figure 6. 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 7. 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 8. 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 9. 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 10. 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.

Figure 11. Tissue specific mRNA delivery using antibody conjugated lipid nanoparticles (LNP). (A) Ai9 mice harboring a cre-dependent tdTomato expression cassette were i.v. injected with Cre mRNA encapsulated in LNP conjugated with or without anti-CD31 antibodies (0.4 mg/kg). (B) tdTomato expression in lung was visualized 72 h post injection.