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. 2022 May 4;100(6):424–439. doi: 10.1111/imcb.12551

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© 2022 The Authors. Immunology & Cell Biology published by John Wiley & Sons Australia, Ltd on behalf of Australian and New Zealand Society for Immunology, Inc.

This is an open access article under the terms of the http://creativecommons.org/licenses/by-nc/4.0/ License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.

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Strategies to express miRNA in CAR T cells. One single promoter drives both miRNA and CAR genes in a, b, c & d strategies. (a) A promoter derives miRNA and green fluorescent protein (GFP)‐P2A‐CAR coding sequence and internal ribosome entry site (IRES) sequence enhance the translation, (b) Intronic miRNAs use alternative splicing for maturation result in 5'UTR of mRNA vital for preserved translation, (c) placing miRNA downstream of CAR sequence, (d) bidirectional promoters to express miRNA and GFP‐P2A‐CAR with a single promoter. Alternatively, miRNAs and GFP‐P2A‐CAR can be expressed separately using two promoters in (e) uni‐directional or (f) reverse‐orientation fashion. Controlled miRNA expression using (g) Tet‐On system with G72V‐rtTA or (h) an auto‐inducible promoter. Clusters of natural miRNAs to express (i) six miRNAs, (j) three miRNAs, or (k) two miRNAs. (l) Structure of artificial polycistronic miR31 by joining several repeats of pri‐miR31 sequences.