ReadyEdit LNP Formulation

Espcas9 mRNA/sgRNA targeting TARC (GenScript) were formulated in LP01-LNP formulation at 1:1 mass ratio achieved 97% editing efficiency, comparing to 61% by lipofectamine. 2.4 ug of RNA/LNP formulation were incubated with HEK293T cells, cell were lysed at day 3 and day 5 for PCR and sanger sequencing. Transfection efficiency was compared by delivering eGFP mRNA (100% N1-methyl-pseU modified, GenScript) into Jurkat and THP-1 immune cells using SM102-LNP (GenScript), Lipofectamine™ MessengerMAX™ (ThermoFisher Scientific) and TransIT® (Mirus). 100ng of mRNA were transfected into cells in 24 well plates. Starvation medium (OptiMEM) was used for lipofectamine and TransIT per the manufacturer’s instruction, and complete medium was used for LNP. The expression of eGFP was measured by flow cytometry after 48 hours. GS-MC3 LNP formulation has strong mRNA expression GS-MC3 LNP as generic formulation for customers’ mRNA screening project In vivo delivery efficiency of four different LNP formulations was compared by delivering Fluc mRNA (100% N1-methyl-pseU modified, GenScript) into Balb-C mice via IM injection at a dose of 0.25mg/kg. The expression of Fluc mRNA was measured by whole-body bioluminescence imaging. Fluc mRNA delivered by MC3-LNP showed the highest expression level after 8 and 48 hours by imaging at both Dorsal and Ventral pose of mice. To evaluate the biodistribution profile of different formulations, the heart, liver, lung, spleen, kidney, and brain of the mice were collected and imaged after 48 hours, MC3 -LNP formulation showed the strongest liver accumulation compared to others after 48 hours. Both LNP formulations are safe for IV injection SM102 has stronger liver accumulation among the two tested formulations In vivo delivery efficiency of two different LNP formulations was compared by delivering Fluc mRNA (100% N1-methyl-pseU modified, GenScript) into Balb-C mice via IV injection at a dose of 0.3mg/kg. The expression of Fluc mRNA was measured by whole-body bioluminescence imaging. In imaging analysis, Fluc-mRNA delivered by SM102-LNP showed higher expression efficiency than MC3 after 8 and 48 hours. To evaluate the biodistribution profile of different formulations, heart, liver, lung, spleen, kidney, and brain of the mice were collected and imaged after 48 hours, SM102 -LNP showed stronger liver accumulation compared to MC3-LNP after 48 hours. MC3-LNP showed stronger spleen accumulation after 48 hrs. Neither formulation caused significant body weight loss during the experiment period for 3 days.

Fluc mRNA(N1-methyl-pseU) was encapsulated with SM102-LNP or SM102-LNP decorated with anti-Human CD3 Antibody (Clone: OKT3, mAb, Mouse, GenScript, A02199), 100ng of mRNA encapsulated in LNP were incubated with jurkat cells in RPMI140 +10%FBS at 37C for 24 hrs. The expression of Fluc was detected using Luciferase Assay (Promega, E4030). eGFP mRNA(N1-methyl-pseU) was encapsulated with SM102-LNP or SM102-LNP decorated with anti-Human CD3 Antibody (Clone OKT3, mAb, Mouse, GenScript), 10-150ng of mRNA encapsulated in LNP were incubated with primary T cells for 24 hrs. The expression of eGFP was detected using flow cytometry. W1, W2 and W3 indicate different wash conditions. Fluc mRNA(N1-methyl-pseU) was encapsulated with SM102-LNP or SM102-LNP decorated with anti-mouse CD3 Antibody, 0.3mg/kg of mRNA encapsulated in LNP were injection through tail vein into mice, after 24 hrs, mice were sacrificed, and the expression of Fluc mRNA was measured with whole body imaging with different organs. *Not sure which T-cell targeted LNP fits your application? Check our LNP selector tool for guidance >> Resting primary T cells were seeded at 1 × 10⁶ cells per well in a 24-well plate and transfected with 500 ng of generic LNP, CD3Ab-LNP, or CD5Ab-LNP. The anti-human CD3 antibody (OKT3, mAb, mouse, GenScript) and anti-human CD5 antibody (mAb, Rabbit, GenScript) were used for LNP surface decoration. T-cell expression was assessed by flow cytometry (FACS) at 48 h post-transfection. Because CD3Ab (OKT3) is known to activate primary T cells, CD3Ab-LNP exhibited a higher expression level compared with CD5Ab-LNP. Notably, despite the intrinsic difficulty of transfecting resting T cells, CD5Ab-LNP still achieved over 80% transfection-positive cells. Resting primary T cells were seeded at 1 × 10⁶ cells per well in a 24-well plate and transfected with 500 ng of generic LNP, CD3Ab-LNP, CD4Ab-LNP, or CD8Ab-LNP. The anti-human CD3 antibody (OKT3, mAb, mouse, GenScript), anti-human CD4 antibody (F001, mAb, mouse, GenScript), and anti-human CD8 antibody (F002, mAb, mouse, GenScript) were used for LNP surface decoration. T-cell expression was assessed by flow cytometry (FACS) at 48 h post-transfection. CD4Ab-LNP and CD8Ab-LNP exhibited selective delivery to CD4⁺ and CD8⁺ T cells, respectively, with minimal off-target expression. EGFP mRNA(N1-methyl-pseU) was encapsulated with SM102-LNP or SM102-LNP decorated with anti-Human CD34 Antibody (Purified anti-human CD34 antibody(Anti-Human CD34 Antibody, mAb, Mouse, GenScript, CP0001) 100ng of mRNA encapsulated in LNP were incubated with Kasumi-1 cells for 24 hrs. The expression of eGFP was detected using flow cytometry. EGFP mRNA(N1-methyl-pseU) was encapsulated with ALC0315-LNP or ALC0315-LNP decorated with anti-Human CD34 Antibody (Purified anti-human CD34 antibody(Anti-Human CD34 Antibody, mAb, Mouse, GenScript, CP0001) 100ng of mRNA encapsulated in LNP were incubated with primary human CD34+ cells for 24 hrs. The expression of eGFP was detected using flow cytometry. Fluc mRNA(N1-methyl-pseU) was encapsulated with LNP , 0.3mg/kg of mRNA encapsulated in LNP were injection through tail vein into mice, after 24 hrs, mice were sacrificed, and the expression of Fluc mRNA was measured with whole body imaging with different organs. EGFP mRNA(N1-methyl-pseU) was encapsulated with SM102-LNP or SM102-LNP decorated with TriGalNac, 0.3mg/kg of mRNA encapsulated in LNP were injection through tail vein into mice, after 24 hrs, mice were sacrificed, and liver organs were lysed. The eGFP expression in lysate was detected using western blot method. Fluc mRNA (m1Ψ) was encapsulated with LNP, 0.3mg/kg of mRNA encapsulated in LNP were injection through tail vein into mice. Conjugation of anti-CD31 antibodies to LNPs can enhance mRNA expression levels in the lungs of mice. FACS analysis of lung cells revealed that CD31-Ab/LNPs exhibit clear targeting specificity toward CD31⁺ cells. Fluc mRNA (m1Ψ) was encapsulated with LNP, 0.3mg/kg of mRNA encapsulated in LNP were injection through tail vein into mice. Mannose/LNP improved Fluc mRNA expression in spleen and lymph node system. Mannose/LNPs delivering sgRNA targeting Zeb2 achieved efficient gene editing in Cas9⁺ BMDM cells and significantly suppressed tumors in a Cas9-GFP mouse model. Fluc mRNA (m1Ψ) was encapsulated with LNP, 0.3mg/kg of mRNA encapsulated in LNP were injection through tail vein into mice. KKEEE/SM102 LNP enhanced Fluc mRNA expression in kidney organ ~4.6 times comparing undecorated LNP.