Citation

  • Authors: Si K. et al.
  • Year: 2023
  • Journal: Cytotherapy S1465-3249 00004-X
  • Applications: in vitro / DNA / jetPRIME
  • Cell type: HEK-293T
    Description: Human embryonic kidney Fibroblast
    Known as: HEK293T, 293T

Method

Lentiviral vector manufacture and T-cell transduction The lentiviral vector was generated by transfection of HEK293T cells using the jetPRIME protocol. Briefly, HEK293T cells were cultured in 10-cm dishes at a cell density of 70–80%, and lentiviral backbone plasmids and psPAX2 and pMD2.G packaging plasmids were transfected in a 4:3:1 ratio, with total plasmids controlled at 20 μg. Viral supernatants were harvested 24 h and 48 h post-transfection, filtered through a 0.45-µm filter and ultracentrifuged at 12 000 × g overnight at 4°C. The pellets were resuspended in frozen PBS and stored at –80°C. T cells were co-incubated with lentiviral vectors after 1 day of activation and infected with multiplicity of infection values between 10 and 50.

Abstract

Background aims: Most current chimeric antigen receptor (CAR) T cells are generated by viral transduction, which induces persistent expression of CARs and may cause serious undesirable effects. Messenger RNA (mRNA)-based approaches in manufacturing CAR T cells are being developed to overcome these challenges. However, the most common method of delivering mRNA to T cells is electroporation, which can be toxic to cells. Methods: The authors designed and engineered an exosome delivery platform using the bacteriophage MS2 system in combination with the highly expressed protein lysosome-associated membrane protein 2 isoform B on exosomes. Results: The authors' delivery platform achieved specific loading and delivery of mRNA into target cells and achieved expression of specific proteins, and anti-CD3/CD28 single-chain variable fragments (scFvs) expressed outside the exosomal membrane effectively activated primary T cells in a similar way to commercial magnetic beads. Conclusions: The delivery of CAR mRNA and anti-CD3/CD28 scFvs via designed exosomes can be used for ex vivo production of CAR T cells with cancer cell killing capacity. The authors' results indicate the potential applications of the engineered exosome delivery platform for direct conversion of primary T cells to CAR T cells while providing a novel strategy for producing CAR T cells in vivo.
Keywords: anti-CD3/CD28 scFvs; exosomes; mRNA CAR T; mRNA delivery.

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