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Long terminal repeat CRISPR-CAR-coupled "Universal" T cells mediate potent anti-leukemic effects

Cited 43 time in Web of Science Cited 47 time in Scopus
Authors
Georgiadis, Christos; Preece, Roland; Nickolay, Lauren; Etuk, Aniekan; Petrova, Anastasia; Ladon, Dariusz; Danyi, Alexandra; Humphryes-Kirilov, Neil; Ajetunmobi, Ayokunmi; Kim, Daesik; Kim, Jin-Soo; Qasim, Waseem
Issue Date
2018-05
Citation
Molecular Therapy, Vol.26 No.5, pp.1215-1227
Abstract
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 Delta U3 3' long terminal repeat (LTR). Following reverse transcription and duplication of the hybrid Delta U3-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.
ISSN
1525-0016
URI
https://hdl.handle.net/10371/165704
DOI
https://doi.org/10.1016/j.ymthe.2018.02.025
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