transfer plasmid vectors encoding the second-generation car constructs  (GenScript corporation)

Journal: International Journal of Nanomedicine

Article Title: Delivery of Plasmid DNA by Ionizable Lipid Nanoparticles to Induce CAR Expression in T Cells

doi: 10.2147/IJN.S424723

Figure Lengend Snippet: Top-performing LNPs generate CAR-Jurkat cells with high efficiency. Jurkat cells were transfected with top-performing LNPs using 420 ng/60,000 cells, collected after 3 days and co-cultured at 1:1 or 1:0 ratios with Raji cells. ( A ) Schematic of Jurkat cell transfection with LNP-CAR and effector cell activation after co-culture with Raji cells. ( B ) Representative histogram of Jurkat and Raji cells distinguished based on the expression of CD19. ( C ) Expression of activation markers PD-1 and CD69 in Jurkat cells expressing CAR-CD19BBz. ( D and E ) Activation of Jurkat cells expressing CAR-CD19BBz measured by mean fluorescence intensity (MFI) of CD69 ( D ) and PD-1 ( E ) after 24 hours co-cultured with Raji cells. * p<0.05; ** p<0.01; ***p<0.001; **** p<0.0001 Not significant (ns) p>0.05 versus LNP-9-GN1; “a” = p<0.001 compared to 1:1; “b” = p<0.0001 compared to 1:1; evaluated by one-way ANOVA with n=3. ( F and G ) Activation of Jurkat cells transfected with the optimized formulation LNP-9 24 hours after co-culture with Raji cells at different effector-to-target ratios. Activation was assessed by the expression of CD69 ( F ) and PD-1 ( G ). *p<0.05; **p<0.01; ***p<0.001; Not significant (ns) p>0.05 evaluated by two-way ANOVA with n=3. Graphs represent mean ± SD.

Article Snippet: We encapsulated a pDNA encoding for a second-generation CAR targeting CD19 (CAR-CD19BBz, Addgene no. 135992) in our lead formulation LNP-5 (LNP-5-CAR).

Techniques: Transfection, Cell Culture, Activation Assay, Co-Culture Assay, Expressing, Fluorescence, Formulation

Journal: International Journal of Nanomedicine

Article Title: Delivery of Plasmid DNA by Ionizable Lipid Nanoparticles to Induce CAR Expression in T Cells

doi: 10.2147/IJN.S424723

Figure Lengend Snippet: LNP-9-CAR promotes functional delivery of pDNA to primary human T cells. ( A ) Schematic of primary human T cell transfection with LNP-9-CAR ( B ) Representative plots of CAR-CD19BBz expression 3 days after transfection of primary human T cells with LNP-9-CAR. ( C ) Cell viability of CD3 + cells 3 days after transfection with LNP-9-CAR. Not significant (ns) p>0.05 evaluated by unpaired t -test compared to NTC. ( D ) Frequency of Live/CD3 + /CAR-CD19BBz + T cells analyzed 3 days after transfection with LNP-9-CAR. ***p<0.001 evaluated by unpaired t -test compared to NTC. ( E ) Representative plots of T cell subsets expressing CAR-CD19BBz 3 days after transfection with LNP-9-CAR identified by the expression of CD4 and CD8. ( F ) Frequency of Live/CD3 + /CAR-CD19BBz + CD4 + and CD8 + T cells analyzed 3 days after transfection with LNP-9-CAR. **p<0.01 evaluated by unpaired t -test compared to NTC. ( G ) Schematic of T cell transfection with LNP-9-CAR and tumor cell specific killing in co-culture with Raji cells. ( H ) Representative plots of Annexin V/7AAD labeled cells gated on the CD3 − /CD19 + population. Dead cells are labeled as double positive for both markers. ( I ) Results of specific killing of CD19 + 24 hours after coplating with CAR T cells or non-transfected cells at different ratios. *p<0.05; ****p<0.0001; Not significant (ns) p>0.05 evaluated by two-way ANOVA with n=4 donors. NTC = non-transfected control. Graphs represent mean ± SD.

Article Snippet: We encapsulated a pDNA encoding for a second-generation CAR targeting CD19 (CAR-CD19BBz, Addgene no. 135992) in our lead formulation LNP-5 (LNP-5-CAR).

Techniques: Functional Assay, Transfection, Expressing, Co-Culture Assay, Labeling, Control