Study Identifies New Targets for More Potent CAR-T Therapy

DUAL gene knockout approach has enhanced the antitumour activity of chimeric antigen receptor (CAR)-T cells across multiple tumour models, offering a potential strategy to improve cellular cancer therapies. 

Although CAR-T cell therapy has transformed the treatment of haematological malignancies, its effectiveness remains limited by several negative regulatory mechanisms within T cells. Identifying combinations of genetic modifications that overcome these restrictions could help produce more effective and durable responses. 

In a study, researchers conducted a high-throughput in vivo CRISPR screen to identify synergistic gene pairs that could improve CAR-T cell function. The approach identified multiple promising combinations, with the NR4A1-SOCS3 double knockout (DKO) emerging as the leading candidate. 

Screening for Synergistic Gene Targets 

The researchers used an in vivo CAR-T double-knockout CRISPR screen to identify gene pairs that were both effective and synergistic in enhancing antitumour activity. 

Among the combinations identified, NR4A1-SOCS3 DKO demonstrated several favourable immunological features. The findings suggest that simultaneously targeting these two intracellular checkpoints could address multiple regulatory mechanisms that otherwise limit T-cell activity. 

The approach also demonstrated the value of high-throughput in vivo screening for identifying combinations of genetic targets, rather than relying solely on individual gene modifications. 

Improved Metabolic Fitness and Mitochondrial Activity 

Whole-transcriptome profiling and in vivo single-cell RNA sequencing revealed that NR4A1-SOCS3 DKO specifically increased metabolic pathway activity in CAR-T cells. 

The modified cells demonstrated significantly greater metabolic fitness and mitochondrial activity, indicating improvements in cellular functions associated with their antitumour performance. 

These changes were accompanied by exceptionally potent antitumour efficacy. Both NR4A1-SOCS3 DKO CAR-T cells and conventional T cells demonstrated strong activity across different tumour models. 

Sustained Tumour Control Without Compromising Safety 

NR4A1-SOCS3 DKO cells achieved long-term tumour control across the tested models without compromising safety features. The combination also outperformed several benchmark single-gene and dual-gene modifications in both T cells and CAR-T cells. 

The findings establish NR4A1-SOCS3 as a promising pair of intracellular checkpoints for engineering higher-performance CAR-T therapies. 

The researchers concluded that high-throughput in vivo double-knockout screening could accelerate the discovery of combination targets for cellular immunotherapy. Further investigation will be needed to determine how these findings translate into clinical applications, but the results provide a potential framework for developing more effective CAR-T treatments against cancer. 

Reference 

Zhou L et al. In vivo CAR-T double knockout screen maps synergistic antitumour gene pairs. Nature. 2026. [Epub ahead of print].