학술논문
Delivery of Plasmid DNA by lonizable Lipid Nanoparticles to Induce CAR Expression in T Cells
ORIGINAL RESEARCH
ORIGINAL RESEARCH
Document Type
Academic Journal
Author
Prazeres, Pedro Henrique Dias Moura; Ferreira, Heloisa; Costa, Pedro Augusto Carvalho; da Silva, Walison; Alves, Marco Tullio; Padilla, Marshall; Thatte, Ajay; Santos, Anderson Kenedy; Lobo, Anderson Oliveira; Sabino, Adriano; Del Puerto, Helen Lima; Mitchell, Michael J.; Guimaraes, Pedro Pires Goulart
Source
International Journal of Nanomedicine. October 31, 2023, Vol. 18, p5891, 14 p.
Subject
Language
English
ISSN
1178-2013
Abstract
Introduction Immunotherapy, using T cells expressing the chimeric antigen receptor (CAR) targeting CD19, has been approved since 2017 by the FDA for the treatment of B-cell acute lymphoblastic leukemia (B-ALL), [...]
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.sup.+]) 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.sup.+] 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.sup.+] 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: lipid nanoparticles, pDNA delivery, CAR T cells, cell engineering, cancer immunotherapy
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.sup.+]) 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.sup.+] 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.sup.+] 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: lipid nanoparticles, pDNA delivery, CAR T cells, cell engineering, cancer immunotherapy