Genome medicine · 2026
CRISPR integration of an FLT3 CAR into the TRAC locus generates CAR T cells for AML.
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Noer Maya G, Christensen Kimmie B, Mikkelsen Nanna S, Skjelbostad Sigrid Fu, Rothemejer Frederik H, Wittenborn Thomas, Fammé Simon, Mikkelsen Kasper, Barington Torben, Thomsen Martin K, Barnkob Mike B, Møller Bjarne K, Bak Rasmus O
Аннотация
BACKGROUND: Acute myeloid leukemia (AML) remains a therapeutically challenging hematological malignancy with high relapse rates, especially in patients harboring mutations in the FMS-like tyrosine kinase 3 (FLT3) gene. FLT3, a receptor tyrosine kinase involved in hematopoietic cell proliferation and survival, is frequently mutated or overexpressed in AML, contributing to leukemic proliferation and poor prognosis. Although FLT3 inhibitors have demonstrated clinical benefit, resistance often develops, underscoring the need for alternative treatment strategies.
METHODS: In this study, we developed and evaluated allogeneic anti-FLT3 CAR T cells engineered by CRISPR/Cas9-mediated targeted integration into the T cell receptor α constant (TRAC) locus (TRAC-CAR T). This approach enables CAR expression under the endogenous TRAC promoter while simultaneously disrupting native TCR expression, thereby allowing the use of allogeneic T cells with reduced risk of graft-versus-host disease.
RESULTS: TRAC-CAR T cells demonstrated efficient disruption of TCRαβ expression, robust surface CAR expression, and superior integration efficiency compared to safe harbor AAVS1-targeted and lentivirally transduced CAR T cells (LV-CAR T). Functionally, TRAC-CAR T cells exhibited potent and selective cytotoxicity against FLT3-positive AML cell lines in vitro, achieving comparable killing relative to LV-CAR T cells. In a xenograft AML mouse model, TRAC-CAR T cells effectively reduced leukemia burden and prolonged mouse survival, with efficacy matching that of LV-CAR T cells. Notably, TRAC-CAR T cells exhibited reduced exhaustion marker expression.
CONCLUSIONS: Together, these results highlight TRAC-integrated anti-FLT3 CAR T cells as a promising off-the-shelf immunotherapy candidate, combining precise gene editing, efficient manufacturing, and potent anti-leukemic activity.
METHODS: In this study, we developed and evaluated allogeneic anti-FLT3 CAR T cells engineered by CRISPR/Cas9-mediated targeted integration into the T cell receptor α constant (TRAC) locus (TRAC-CAR T). This approach enables CAR expression under the endogenous TRAC promoter while simultaneously disrupting native TCR expression, thereby allowing the use of allogeneic T cells with reduced risk of graft-versus-host disease.
RESULTS: TRAC-CAR T cells demonstrated efficient disruption of TCRαβ expression, robust surface CAR expression, and superior integration efficiency compared to safe harbor AAVS1-targeted and lentivirally transduced CAR T cells (LV-CAR T). Functionally, TRAC-CAR T cells exhibited potent and selective cytotoxicity against FLT3-positive AML cell lines in vitro, achieving comparable killing relative to LV-CAR T cells. In a xenograft AML mouse model, TRAC-CAR T cells effectively reduced leukemia burden and prolonged mouse survival, with efficacy matching that of LV-CAR T cells. Notably, TRAC-CAR T cells exhibited reduced exhaustion marker expression.
CONCLUSIONS: Together, these results highlight TRAC-integrated anti-FLT3 CAR T cells as a promising off-the-shelf immunotherapy candidate, combining precise gene editing, efficient manufacturing, and potent anti-leukemic activity.