Recently, chimeric antigen receptor (CAR)-T cell therapy has shown great promise in treating haematological malignancies1–7. However, CAR-T cell therapy currently has several limitations8–12. Here we successfully developed a two-in-one approach to generate non-viral, gene-specific targeted CAR-T cells through CRISPR–Cas9. Using the optimized protocol, we demonstrated feasibility in a preclinical study by inserting an anti-CD19 CAR cassette into the AAVS1 safe-harbour locus. Furthermore, an innovative type of anti-CD19 CAR-T cell with PD1 integration was developed and showed superior ability to eradicate tumour cells in xenograft models. In adoptive therapy for relapsed/refractory aggressive B cell non-Hodgkin lymphoma (ClinicalTrials.gov, NCT04213469), we observed a high rate (87.5%) of complete remission and durable responses without serious adverse events in eight patients. Notably, these enhanced CAR-T cells were effective even at a low infusion dose and with a low percentage of CAR+ cells. Single-cell analysis showed that the electroporation method resulted in a high percentage of memory T cells in infusion products, and PD1 interference enhanced anti-tumour immune functions, further validating the advantages of non-viral, PD1-integrated CAR-T cells. Collectively, our results demonstrate the high safety and efficacy of non-viral, gene-specific integrated CAR-T cells, thus providing an innovative technology for CAR-T cell therapy.

The development of luminescent materials for anticounterfeiting and encryption is of great importance. Herein, we develop a multistimuli-responsive luminescent material, NaCaGeO:Pb/Er, and use it to print luminescent images. The photoluminescence and upconversion luminescence of these images show different patterns and colors under different stimuli. The photostimulated luminescence (PSL) of the printed images causes dynamic changes in appearance and is accordingly applied for dynamic multimodal anticounterfeiting on banknotes. The PSL of these luminescent images is also applied in a virtual war scenario to demonstrate that the dynamic PSL-encrypted information in the fabricated image is sufficiently safe even in extreme cases and that spies will be detected. These results can inspire us with more creative security designs based on this luminescent material.

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In recent years, chimeric antigen receptor (CAR) T cell therapy has shown great promise in treating hematological malignancies. However, using virus in manufacture of CAR T cells brings about several problems. The application of CRISPR/Cas9 genome editing technology emerges in constructing novel CAR T cells by disrupting endogenous genes. Here we successfully develop a two-in-one approach to generate non-viral genome specific targeted CAR T cells through CRISPR/Cas9. By targeting a CAR in AAVS1 safe harbor locus, we demonstrated that these CAR T cells behave comparable to those conventionally produced by lentivirus. Furthermore, PD1-knockin anti-CD19 CAR T cells show a superior ability to eradicate tumor cells with high PD-L1 expression. In the adoptive therapy for relapsed/refractory (r/r) aggressive B-cell non-Hodgkin lymphoma (B-NHL), we observed durable responses without serious adverse events and complete remission (CR) in patients treated with these PD1 knockout CAR T cells. Collectively, our results prove the safety and feasibility of non-viral genome specific integrated CAR T cells, thus providing a new potential strategy for cancer treatment using these novel CAR T cells.

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