Georgiadis et al. developed a lentivirus construct containing a CD19 CAR-encoding region and a TRAC-targeted sgRNA to achieve simultaneous transgene expression with TCR knockout. Following transduction and electroporation of Cas9 mRNA, a highly homogenous and effective CD19 CAR+TCR- T cell population was produced. When adoptively transferred into a mouse leukemia model, CAR+TCR- T cells rapidly cleared tumors, expanded more readily, and expressed less PD-1 compared to CAR+TCR+ T cells. This strategy could be incorporated into an automated manufacturing platform.
Gene editing can be used to overcome allo-recognition, which otherwise limits allogeneic T cell therapies. Initial proof-of-concept applications have included generation of such "universal" T cells expressing chimeric antigen receptors (CARs) against CD19 target antigens combined with transient expression of DNA-targeting nucleases to disrupt the T cell receptor alpha constant chain (TRAC). Although relatively efficient, transgene expression and editing effects were unlinked, yields variable, and resulting T cell populations heterogeneous, complicating dosing strategies. We describe a self-inactivating lentiviral "terminal" vector platform coupling CAR expression with CRISPR/Cas9 effects through incorporation of an sgRNA element into the DeltaU3 3' long terminal repeat (LTR). Following reverse transcription and duplication of the hybrid DeltaU3-sgRNA, delivery of Cas9 mRNA resulted in targeted TRAC locus cleavage and allowed the enrichment of highly homogeneous (>96%) CAR(+) (>99%) TCR(-) populations by automated magnetic separation. Molecular analyses, including NGS, WGS, and Digenome-seq, verified on-target specificity with no evidence of predicted off-target events. Robust anti-leukemic effects were demonstrated in humanized immunodeficient mice and were sustained longer than by conventional CAR(+)TCR(+) T cells. Terminal-TRAC (TT) CAR T cells offer the possibility of a pre-manufactured, non-HLA-matched CAR cell therapy and will be evaluated in phase 1 trials against B cell malignancies shortly.
Author Info: (1) Molecular and Cellular Immunology Unit, UCL Great Ormond Street Institute of Child Health, London WC1N 1EH, UK. (2) Molecular and Cellular Immunology Unit, UCL Great Ormond Str
Author Info: (1) Molecular and Cellular Immunology Unit, UCL Great Ormond Street Institute of Child Health, London WC1N 1EH, UK. (2) Molecular and Cellular Immunology Unit, UCL Great Ormond Street Institute of Child Health, London WC1N 1EH, UK. (3) Molecular and Cellular Immunology Unit, UCL Great Ormond Street Institute of Child Health, London WC1N 1EH, UK. (4) Molecular and Cellular Immunology Unit, UCL Great Ormond Street Institute of Child Health, London WC1N 1EH, UK. (5) Molecular and Cellular Immunology Unit, UCL Great Ormond Street Institute of Child Health, London WC1N 1EH, UK. (6) NIHR Great Ormond Street Hospital Biomedical Research Centre, 30 Guilford Street, London WC1N 1EH, UK. (7) Desktop Genetics Ltd., 28 Hanbury Street, London E1 6QR, UK. (8) Desktop Genetics Ltd., 28 Hanbury Street, London E1 6QR, UK. (9) Desktop Genetics Ltd., 28 Hanbury Street, London E1 6QR, UK. (10) Department of Chemistry, Seoul National University, Seoul, South Korea. (11) Department of Chemistry, Seoul National University, Seoul, South Korea. (12) Molecular and Cellular Immunology Unit, UCL Great Ormond Street Institute of Child Health, London WC1N 1EH, UK; NIHR Great Ormond Street Hospital Biomedical Research Centre, 30 Guilford Street, London WC1N 1EH, UK. Electronic address: w.qasim@ucl.ac.uk.
