International Journal of Nanomedicine (Oct 2023)

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

  • Prazeres PHDM,
  • Ferreira H,
  • Costa PAC,
  • da Silva W,
  • Alves MT,
  • Padilla M,
  • Thatte A,
  • Santos AK,
  • Lobo AO,
  • Sabino A,
  • Del Puerto HL,
  • Mitchell MJ,
  • Guimaraes PPG

Journal volume & issue
Vol. Volume 18
pp. 5891 – 5904

Abstract

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Pedro Henrique Dias Moura Prazeres,1,2 Heloísa Ferreira,2 Pedro Augusto Carvalho Costa,2 Walison da Silva,2 Marco Túllio Alves,2 Marshall Padilla,3 Ajay Thatte,3 Anderson Kenedy Santos,4,5 Anderson Oliveira Lobo,6 Adriano Sabino,7 Helen Lima Del Puerto,1 Michael J Mitchell,3 Pedro Pires Goulart Guimaraes1,2 1Department of Pathology, Federal University of Minas Gerais, Belo Horizonte, MG, Brazil; 2Department of Physiology and Biophysics, Federal University of Minas Gerais, Belo Horizonte, MG, Brazil; 3Department of Bioengineering, University of Pennsylvania, Philadelphia, PA, USA; 4Department of Pediatrics/Gastroenterology and Hepatology, Yale School of Medicine, New Haven, CT, USA; 5Department of Genetics, Yale School of Medicine, New Haven, CT, USA; 6Department of Materials Engineering, Federal University of Piauí, Teresina, PI, Brazil; 7Department of Clinical and Toxicological Analysis, Federal University of Minas Gerais, Belo Horizonte, MG, BrazilCorrespondence: Pedro Pires Goulart Guimaraes, Department of Physiology and Biophysics, Federal University of Minas Gerais, Belo Horizonte, MG, Brazil, Email [email protected]: 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: lipid nanoparticles, pDNA delivery, CAR T cells, cell engineering, cancer immunotherapy

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