Fig. 11

The ex vivo CRISPR manipulation of human T cells for adoptive T cell therapy. Ongoing clinical trials are currently dedicated to assessing the safety and effectiveness of CRISPR-engineered T cells through ex vivo modification and subsequent transfer. The goal is to enhance the anti-cancer response of T cells taken from healthy donors or patients. These trials investigate the potential of both allogeneic (from different donors) and autologous (from the patient themselves) T cells in various approaches, including tumor-infiltrating lymphocytes (TILs) and chimeric antigen receptor (CAR) T cells. One of the methods involves using CRISPR-Cas9 to remove immunosuppressive factors, like the programmed cell death protein 1 (PD1) ligand, from human primary T cells. This approach is being tested for adoptive T cell therapy involving both TILs and CAR T cells. The delivery of CRISPR-Cas9 ribonucleoproteins (RNPs) allows precise editing of immunosuppressive factors such as PD1 by guiding Cas9 to specific locations. Researchers are also exploring the deletion of the endogenous T cell receptor (TCR) using CRISPR-Cas9 to prevent TCR priming or immune rejection in the case of allogeneic T cells. Another avenue being explored is the replacement of the endogenous TCR with a cancer antigen-specific TCR, either through a TCR transgene or a CAR element. This has been shown to enhance the killing of cancer cells by T cells. In clinical trials, CRISPR-Cas9 homology-directed repair (HDR)-mediated knock-in to the T cell receptor α-chain constant (TRAC) locus is used to deliver CAR elements, and its efficacy is being tested. Additionally, CRISPR is used to delete the endogenous T cell receptor-β constant (TRBC) locus and endogenous major histocompatibility complex class I (MHC-I) to prevent immune rejection after transplant, and to remove immunosuppressive factors, all aimed at improving T cell activity in CAR T cells. Next-generation sequencing (NGS) is employed to confirm the engineered T cells, ensuring accurate on-target editing with minimal off-target effects. The expanded and validated T cells are then transplanted into the cancer patient, and disease progression is closely monitored to assess the safety and efficacy of the engineered T cells. Reprinted from [11] with permission from Springer Nature