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March 05, 2018

Applications of CRISPR/Cas9 Technology in Translational Research on Solid-Tumor Cancers

Efforts Are Underway to Move CRISPR/Cas9 from Bench to Bedside

Applications of CRISPR/Cas9 Technology in Translational Research on Solid-Tumor Cancers

Figure 1. Summary of known immune checkpoint regulators in the tumor microenvironment. The major negative checkpoint regulators in the Ig superfamily are shown in CD8+ T cells and interacting cells (e.g., tumor cells, DCs, etc.). CRISPR/Cas9-mediated knockout of these targets shows great promise in immunotherapy.

  • Genome editing has become a powerful tool for biological research and a potential avenue for treating many diseases.1 Compared to previous tools such as zinc finger nucleases and transcription activator-like effector nucleases (TALENs), the CRISPR*/Cas9 system is simpler to design, easier to use, less time-consuming, and less costly.2,3

    CRISPR/Cas9's versatility and convenience as a genome editing platform means it can be harnessed to study genome abnormality in the genesis and progression of cancers, and potentially even to correct harmful gene mutations for clinical applications. Another possible treatment strategy is to target interactions between cancer cells and the tumor microenvironment. Activated T cells play a key role in antitumor immunity, but are subject to various regulatory signals in the tumor microenvironment (Figure 1). One negative regulator of T-cell activity is programmed cell death 1 (PDCD1 or PD-1), a surface receptor on activated T cells. Certain cancer cells exploit this immune checkpoint by expressing the ligand CD274 (PD-L1 or B7-H1), which binds PD-1 and transmits an inhibitory signal to the T cell, thus downregulating immune responses against them and escaping destruction.4 Indeed, PD-L1 expression on mouse tumor cells increases apoptosis of activated antitumor T cells and thus suppresses local antitumor T cell–mediated responses.5,6 Therefore, this PD-L1/PD-1 interaction between cancer cells and immune cells may also be an attractive target for CRISPR/Cas9 in enhancing cancer immunotherapies.

    Here, the CRISPR/Cas9 system and its applications in basic and translational research on solid-tumor cancers are reviewed. The challenges of translating therapeutic CRISPR/Cas9 into clinical use are also discussed.

  • CRISPR/Cas9 Overview

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    Table 1. Applications of CRISPR/Cas9 in translational research on solid-tumor cancers

    CRISPR/Cas9 consists of two biological components: the RNA-guided DNA endonuclease Cas9 and a chimeric single-guide RNA (sgRNA). Cas9, usually derived from Streptococcus pyogenes, is a large protein with two nuclease domains, each of which cleaves one strand of the double helix. The sgRNA is a dual RNA structure formed by CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA) and contains a 20-nucleotide sequence complementary with the DNA target site.3,7

    Single-guide RNA locates the genomic target sequence via Watson–Crick base pairing and directs Cas9 to it. Cleavage of the target by Cas9 yields DNA double-stranded breaks (DSBs), which the host cell repairs either by non-homologous endjoining (NHEJ), which frequently generates frameshift mutations and a gene knockout, or by the more precise homology-directed repair (HDR), which uses an exogenous template that can introduce a gene knockin or point mutations.8 CRISPR/Cas9 is the most convenient and flexible genome editing system developed so far, needing just a customized sgRNA sequence to introduce edits virtually anywhere the user desires. The only constraint is that the selected target sequence must be immediately downstream of a protospacer adjacent motif (PAM), a two to six nucleotide sequence, for Cas9 to recognize.7–9 The applications of CRISPR/Cas9 in the context of solid-tumor cancers are the focus of much current research (see Table 1).

  • Applications in Solid-Tumor Cancer Research

    The process of tumorigenesis involves polygenic and multistage changes, including oncogene activation, suppressor gene inactivation, and chromosomal rearrangement.10–12 Additionally, the interaction between cancer cells and their tumor microenvironment plays an important role in tumor genesis and progression. Accordingly, CRISPR/Cas9 marks a breakthrough in cancer therapy exploration, as it can introduce loss-of-function mutations and gain-of-function mutations to better our understanding of cancer and help eliminate tumors (Table 1).

  • Lung Cancer

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    Figure 2. Cancer immunotherapy by PD-1 knockout in T cells via CRISPR/Cas9. T cells are collected from the patient and edited by CRISPR/Cas9, then reinfused to the patient. PD-1-knockout T cells escape inhibition by tumor cells expressing PD-L1 and exert an antitumor action.

    The ability of CRISPR/Cas9 to locate virtually any DNA sequence in the genome allows scientists to target and study any genes they wish. For example, KRASTP53STK11, and EML4-ALK, among others, could all be targets of CRISPR/Cas9 technologies to model lung cancer. Loss-of-function mutations in TP53 and STK11 and HDR-mediated KRAS(G12D) mutations generated via CRISPR/Cas9 have been shown to produce lung adenocarcinoma.13 CRISPR/Cas9 is also able to produce chromosome structural aberrations, as shown in a mouse model of lung cancer that expressed the EML4-ALK fusion gene and responded to ALK inhibitors.14 Furthermore, CRISPR/Cas9 has been used to knockout oncogenes and oncosuppressors such as NESJUNB, and MFN2 systematically to investigate their effect on cancer phenotype.15–17

    In addition, CRISPR/Cas9 can be used as a therapeutic approach to lung cancer and hasten its progression from bench to bedside. CRISPR/Cas9-mediated knockout of the PDCD1 (PD-1) gene in T cells significantly reduces their PD-1 expression and enhances cellular immune response in vitro.4 These findings are being tested in human subjects for the first time: A recently launched Phase I trial led by one of the authors (Y.L.) at Sichuan University's West China Hospital will investigate the safety and efficacy of PD-1-knockout gene-edited T cells in metastatic non–small cell lung cancer (NSCLC) patients.18 The aim is to collect lymphocytes from patients' peripheral blood and knockout PD-1, select and expand the cells ex vivo, and then infuse them back into patients for treatment (Figure 2). Safety events, curative effect, biomarkers, and immunological markers will be measured and evaluated after infusion.19

    Similar clinical trials conducted by researchers at Hangzhou Cancer Hospital and Peking University plan to evaluate PD-1 knockout T cells in esophageal, bladder, renal cell, and prostate cancers.20–23 As PD-1-blocking antibodies already exist and have been approved for immunotherapy in melanoma and NSCLC, PD-1-inactivated T cells generated via CRISPR/Cas9 will need to show greater efficiency, greater stability, and fewer side effects to justify their clinical use. 24 If so, then it is envisaged that CRISPR/Cas9-mediated knockout of other immune checkpoint genes, such as CTLA4 and HAVCR2, could exert an anticancer effect.25 Hence, this system may serve as a boon for lung cancer therapeutics exploration, as well as for treating other solid tumors that do not rely on the PD-1/PD-L1 pathway for immunosuppression.

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