Engineered exosome-mediated messenger RNA and single-chain variable fragment delivery for human chimeric antigen receptor T-cell engineering

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 .

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