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

Abstract

Introduction: Chimeric antigen receptor (CAR) cell therapy represents a hallmark in cancer immunotherapy, with significant clinical results in the treatment of hematological tumors. However, current approved methods to engineer T cells to express CAR use viral vectors, which are integrative and have been associated with severe adverse effects due to constitutive expression of CAR. In this context, non-viral vectors such as ionizable lipid nanoparticles (LNPs) arise as an alternative to engineer CAR T cells with transient expression of CAR.

Methods: Here, we formulated a mini-library of LNPs to deliver pDNA to T cells by varying the molar ratios of excipient lipids in each formulation. LNPs were characterized and screened in vitro using a T cell line (Jurkat). The optimized formulation was used ex vivo to engineer T cells derived from human peripheral blood mononuclear cells (PBMCs) for the expression of an anti-CD19 CAR (CAR-CD19BBz). The effectiveness of these CAR T cells was assessed in vitro against Raji (CD19⁺) cells.

Results: LNPs formulated with different molar ratios of excipient lipids efficiently delivered pDNA to Jurkat cells with low cytotoxicity compared to conventional transfection methods, such as electroporation and lipofectamine. We show that CAR-CD19BBz expression in T cells was transient after transfection with LNPs. Jurkat cells transfected with our top-performing LNPs underwent activation when exposed to CD19⁺ target cells. Using our top-performing LNP-9-CAR, we were able to engineer human primary T cells to express CAR-CD19BBz, which elicited significant specific killing of CD19⁺ target cells in vitro.

Conclusion: Collectively, our results show that LNP-mediated delivery of pDNA is a suitable method to engineer human T cells to express CAR, which holds promise for improving the production methods and broader application of this therapy in the future.

Keywords: CAR T cells; cancer immunotherapy; cell engineering; lipid nanoparticles; pDNA delivery.

Figure 1

Schematic workflow for the optimization of DNA-loaded LNPs for the expression of CAR-CD19BBz. (A) Schematic representation of the components used for LNP synthesis by microfluidic. (B) Representation of the mini-library of LNPs (left) and DLS measurement of the optimized LNP-9-CAR. (C) Schematic representation of LNP screening using Jurkat cells. (D) Representation of the transfection of T cells using the optimized LNP for the expression of CAR-CD19BBz and co-culture experiments with Raji cells to assess effector cell activation and tumor cell specific killing.

Figure 2

DNA loaded LNPs induce transient expression of CAR-CD19BBz in Jurkat cells. (A) Schematic of LNP-CAR mediated transfection of Jurkat cells. (B) CAR-CD19BBz expression in live (PI⁻) Jurkat cells 3 days after transfection with LNP-5-CAR with different amounts of pDNA identifying the best concentration to be used. * p<0.05; **** p<0.0001 versus 420 ng/ 60,000 cells evaluated by one-way ANOVA with n=3. (C) Cell viability of Jurkat cells assessed by the frequency of PI⁻ cells after transfection with LNP-5-CAR. (D) CAR-CD19BBz expression over time in Jurkat cells treated with 420 ng/60,000 cells of LNP-5-CAR confirming the transient expression of CAR in these cells. ** p<0.01; ***p<0.001; ****p<0.0001 versus NTC on the same timepoint evaluated by one-way ANOVA with n=3. (E) Cell viability of Jurkat cells over time. * p<0.05; ****p<0.0001; Not significant (ns) p>0.05 versus NTC on the same timepoint evaluated by one-way ANOVA with n=3.

Figure 3

Higher DOPE molar ratios facilitate efficient transfection with CAR-CD19BBz in Jurkat cells. (A) Schematic of the components used to prepare LNPs by microfluidic mixing. (B) Schematic of LNP-CAR screening using Jurkat cells. (C) Hydrodynamic diameter (Size) and polydispersity index (PDI) of different LNPs encapsulating CAR-CD19BBz pDNA. (D) Zeta potential of different LNPs. (E) Representative cryo-TEM of LNPs encapsulating pDNA. (F) CAR-CD19BBz expression in live (PI⁻) Jurkat cells 3 days after transfection with different LNPs identifying top-performing LNPs. **** p<0.0001 versus NTC evaluated by one-way ANOVA with n=3. (G) Cell viability of Jurkat cells assessed by the frequency of PI⁻ cells after transfection with top-performing LNPs **** p<0.0001 versus NTC; evaluated by one-way ANOVA with n=3. (H) Representative histograms of CAR-CB19BBz expression after transfection. NTC = non-transfected control; EP = Electroporation; Lipo = Lipofectamine. Graphs represent mean ± SD. (I) CAR-CD19BBz expression measured by mean fluorescence intensity (MFI) after transfection with top-performing LNPs and controls. **p<0.01; ****p<0.0001; Not significant (ns) p>0.05 versus NTC evaluated by one-way ANOVA with n=3. “a” = p<0.001 compared to EP; “b” = p<0.001 compared to Lipo evaluated by one-way ANOVA with n=3. NTC = non-transfected control; Graphs represent mean ± SD.

Figure 4

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.

Figure 5

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.