Exploiting the CRISPR-Cas9 gene-editing system for human cancers and immunotherapy

Abstract

The discovery of clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9 (CRISPR-Cas9) technology has brought advances in the genetic manipulation of eukaryotic cells, which has revolutionised cancer research and treatment options. It is increasingly being used in cancer immunotherapy, including adoptive T and natural killer (NK) cell transfer, secretion of antibodies, cytokine stimulation and overcoming immune checkpoints. CRISPR-Cas9 technology is used in autologous T cells and NK cells to express various innovative antigen designs and combinations of chimeric antigen receptors (CARs) targeted at specific antigens for haematological and solid tumors. Additionally, advanced engineering in immune cells to enhance their sensing circuits with sophisticated functionality is now possible. Intensive research on the CRISPR-Cas9 system has provided scientists with the ability to overcome the hostile tumor microenvironment and generate more products for future clinical use, especially off-the-shelf, universal cellular products, bringing exciting milestones for immunotherapy. This review discussed the application and challenges of CRISPR technology in cancer research and immunotherapy, its advances and prospects for promoting new cell-based therapeutic beyond immune oncology.

Keywords: CRISPR‐Cas9; T cells; cancer; genetic manipulation; immunotherapy; natural killer cells.

Figure 1

Application of the CRISPR‐Cas9 system in cancer research and therapeutics. (a) Various delivery methods of the CRISPR‐Cas9 material. They range from lentivirus, adeno‐associated virus (AAV), nanoparticles and Cas9‐mRNA. The CRISPR system can employ either the non‐homologous end joining (NHEJ) or the homology‐directed repair (HDR) for gene knockout and knock‐in, respectively. (b) The identification of essential genes or gene clusters peculiar to individual cancer cells. (c) Target validation mediated by degron tag knock‐in in a gene subjecting its expression to the presence of a small molecule. (d) Schematic workflow of DrugTargetSeqR application in identifying a drug's direct target gene curated from recurring gene mutation between parental cancer cells and non‐MDR clones, which can be validated by biochemical assays to ascertain whether mutations are sufficient to confer resistance. (e) CRISPR‐Cas9 mediated generation of humanised mouse strains carrying physiological levels of gene expression. Their endogenic gene expression levels make them essential components for human biology and pathology modelling, including the study of dosage‐sensitive genes such as aggregate sensitive proteins and RNA‐binding proteins (f) Cas9 mediated transgenic mouse models mediated by the delivery of viral sgRNA. Co‐expressing and/or inducible Cas9 enzymes can cause tissue‐specific gene knockout in different organs. (g) CRISPR‐Cas9 generation of mutation (point or compound) by chromosome translocation or deletion in different mouse tissues, generating a panel of isogenic cell lines with a variety of oncogenic lesions. (h) Generation of germline mouse models harbouring several genetic mutations mediated by CRISPR‐Cas9 engineered embryonic stem (EM) cells.

Figure 2

CRISPR‐Cas9 genome‐editing strategies in adoptive T‐cell immunotherapy for cancer. Applications of the CRISPR‐Cas9 in T‐cell cancer immunotherapy. (a) Isolated patient‐derived T cells are genetically engineered with CRISPR‐Cas9 to knockout endogenous genes, for example PD‐1, and knock‐in therapeutic TCR, and CARs, followed by ex vivo expansion and adoptive transfer. (b) CRISPR‐Cas9 inspired dual‐specific tumor recognition to overcome tumor heterogeneity or antigen loss. This can be achieved by transducing a single CAR molecule into two T‐cell populations (separate transduction), incorporating two CAR molecules into a single‐cell population either individually or by bicistronic (co‐transduction) and linking two separate CAR molecules to produce a single signalling chain (tandem transduction). (c) To surmount the off‐target effect and fine‐tune antigen sensing of tumor‐specific T cells, incorporating a synNotch receptor specific for a first antigen that can trigger the production of CAR upon interaction with a second antigen – this triggers its activation with a licence to kill the tumor. (d) Genetically reprogrammed T cells to overcome the hostile tumor microenvironment. The incorporation of genes capable of local cytokines or antibody release. Similarly, switched receptor strategies enhance sustained antitumor response and the deletion of inhibitory molecules or immune checkpoints to generate off‐the‐shelf T‐cell therapies.

Figure 3

Overview of CRISPR‐Cas9 genome‐editing strategies for NK cell immunotherapy. (a) NK cell sources (UCB, umbilical cord blood; hESCs, hematopoietic embryonic stem cells; iPSCs, induced pluripotent stem cells; NK‐92, NK‐92 cell line; PBNK, peripheral blood mononuclear cells) and its manipulation via the multiplex capability of the CRISPR system. (b) Engineered NK cells with augmented antitumor capabilities such as tumor specificity, cytotoxicity, expansion and tumor infiltration. (c) Engineered NK cells adoptively transferred to confer tumor regression and clearance.