Molecular therapy : the journal of the American Society of Gene TherapyApr 2, 2018

Engineered Universal T Cells Using CRISPR Show Strong Anti-Leukemia Effects

CRISPR Gene Editing Weekly Brief ↗PubMed ↗DOI ↗

Updated Mar 21, 2026

Abstract

More than 96% of the CAR T cell populations were highly homogeneous following targeted gene editing.

Gene editing can potentially address the challenges of allo-recognition in allogeneic T cell therapies.
A self-inactivating lentiviral vector was developed to link CAR expression with CRISPR/Cas9 gene editing.
The method achieved targeted cleavage of the T cell receptor alpha constant chain (TRAC), leading to consistent T cell populations.
Molecular analyses confirmed on-target gene editing with no identified off-target effects.
In humanized immunodeficient mice, the engineered T cells demonstrated robust and sustained anti-leukemic effects.

Simplified

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 ΔU3 3' long terminal repeat (LTR). Following reverse transcription and duplication of the hybrid ΔU3-sgRNA, delivery of Cas9 mRNA resulted in targeted TRAC locus cleavage and allowed the enrichment of highly homogeneous (>96%) CAR(>99%) TCRpopulations 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 CARTCRT 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. + -+ +

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Engineered Universal T Cells Using CRISPR Show Strong Anti-Leukemia Effects

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