Dali Li

CRISPR/Cas9 genome editing has gained rapidly increasing attentions in recent years, however, the translation of this biotechnology into therapy has been hindered by efficient delivery of CRISPR/Cas9 materials into target cells. Direct delivery of CRISPR/Cas9 system as a ribonucleoprotein (RNP) complex consisting of Cas9 protein and single guide RNA (sgRNA) has emerged as a powerful and widespread method for genome editing due to its advantages of transient genome editing and reduced off-target effects. In this review, we summarized the current Cas9 RNP delivery systems including physical approaches and synthetic carriers. The mechanisms and beneficial roles of these strategies in intracellular Cas9 RNP delivery were reviewed. Examples in the development of stimuli-responsive and targeted carriers for RNP delivery are highlighted. Finally, the challenges of current Cas9 RNP delivery systems and perspectives in rational design of next generation materials for this promising field will be discussed.

Glioblastoma (GBM) is one of the most deadly brain tumors. The convenient access to The Cancer Genome Atlas (TCGA) database allows for large-scale global gene expression profiling and database mining for potential correlation between genes and overall survival of a variety of malignancies including GBM. Previous reports have shown that tumor microenvironment cells and the extent of infiltrating immune and stromal cells in tumors contribute significantly to prognosis. Immune scores and stromal scores calculated based on the ESTIMATE algorithm could facilitate the quantification of the immune and stromal components in a tumor. To better understand the effects of genes involved in immune and stromal cells on prognosis, we categorized GBM cases in the TCGA database according to their immune/stromal scores into high and low score groups, and identified differentially expressed genes whose expression was significantly associated with prognosis in GBM patients. Functional enrichment analysis and protein-protein interaction networks further showed that these genes mainly participated in immune response, extracellular matrix, and cell adhesion. Finally, we validated these genes in an independent GBM cohort from the Chinese Glioma Genome Atlas (CGGA). Thus, we obtained a list of tumor microenvironment-related genes that predict poor outcomes in GBM patients.

Abstract Recently, chimeric antigen receptor (CAR)-T cell therapy has shown great promise in treating haematological malignancies 1–7 . However, CAR-T cell therapy currently has several limitations 8–12 . 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.