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A versatile one-shot high-multiplex base editing strategy enables the generation of 20-plex knockout CAR T cells with enhanced anti-tumor efficacy

ABSTRACT: Advances in genomic editing have expanded the therapeutic potential of cell therapies such as chimeric antigen receptor (CAR) T cells, and integrating multifunctional enhancements and allogeneic properties into next-generation therapeutic products requires high-multiplex gene editing strategies that minimize editing-associated toxicity. Here, we pioneered a viral vector-free, one-shot adenine base editing (ABE) approach to enable the simultaneous high-multiplex knockout of at least 20 distinct genes in primary human T cells. The incorporation of this strategy into CAR T cells produced cells with a hyperfunctional phenotype based on a rationally selected 20-plex knockout gene-set. Refinement of this gene-set attenuated initial in vivo toxicity, revealing superior in vivo tumor control of 20-plex edited CAR T cells compared to mock-edited controls in a mouse model of pancreatic ductal adenocarcinoma. We further extended this approach to primary human hematopoietic stem cells (HSCs) and natural killer (NK) cells, demonstrating the method’s broad utility for ex vivo gene editing at unprecedented multiplexity levels across diverse cell types of translational relevance.

ORGANISM(S): Homo sapiens

PROVIDER: GSE308760 | GEO | 2025/09/24

REPOSITORIES: GEO

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Project description:Despite the potential of CAR-T therapies for hematological malignancies, their efficacy in patients with relapse and refractory Acute Myeloid Leukemia has been limited. The aim of our study has been to develop and manufacture a CAR-T cell product that addresses some of the current limitations. We initially compared the phenotype of T cells from AML patients and healthy young and elderly controls. This analysis showed that T cells from AML patients displayed a predominantly effector phenotype, with increased expression of activation (CD69 and HLA-DR) and exhaustion markers (PD1 and LAG3), in contrast to the enriched memory phenotype observed in healthy donors. This differentiated and more exhausted phenotype was also observed, and corroborated by transcriptomic analyses, in CAR-T cells from AML patients engineered with an optimized CAR construct targeting CD33, resulting in a decreased in vivo antitumoral efficacy evaluated in xenograft AML models. To overcome some of these limitations we have combined CRISPR-based genome editing technologies with virus-free gene-transfer strategies using Sleeping Beauty transposons, to generate CAR-T cells depleted of HLA-I and TCR complexes for allogeneic approaches. Our optimized protocol allows one-step generation of edited CAR-T cells that show a similar phenotypic profile than non-edited CAR-T cells, with equivalent in vitro and in vivo antitumoral efficacy. Moreover, genomic analysis of edited CAR-T cells revealed a safe integration profile of the vector, with no preferences for specific genomic regions, with a highly specific editing of the HLA-I and TCR, without significant off-target sites. Finally, production of edited CAR-T cells at a larger scale allowed the generation and selection of enough HLA-IKO/TCRKO CAR-T cells that would be compatible with clinical applications. In summary, our results demonstrate that CAR-T cells from AML patients, although functional, present phenotypic and functional features that could compromise their antitumoral efficacy, compared to CAR-T cells from healthy donors. The combination of CRISPR technologies with transposon-based delivery strategies allow the generation of HLA-IKO/TCRKO CAR-T cells, compatible with allogeneic approaches, that would represent a promising option for AML treatment.

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A versatile one-shot high-multiplex base editing strategy enables the generation of 20-plex knockout CAR T cells with enhanced anti-tumor efficacy

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