The principle of CAR-T cell immunotherapy is to use genetic engineering methods to express chimeric antigen receptor (CAR) molecules on the surface of T cells. CAR-T cells can specifically recognize the antigen on the surface of tumor cells through the single-chain variable fragment (scFv) antibody expressed on their surface, thereby killing tumor cells.
After years of development, CAR-T cell immunotherapy has achieved remarkable results in the field of tumor treatment, but it still faces many challenges. During clinical use, CAR-T cells usually have serious side effects, such as cytokine release syndrome, off-target toxicity, and tumor lysis syndrome, which limit its clinical applications. Therefore, the development direction of next-generation CAR-T cells is how to reduce toxicity through structural optimization of CAR molecules and T cell modification, while enhancing the anti-tumor effect of CAR-T cells. In addition, the patient's own T cells usually have insufficient cell numbers or loss of function due to the disease or the treatment, resulting in the failure of CAR-T cell preparation.
Therefore, the development of universal next-generation CAR-T cells is important for the advancement of CAR-T cell immunotherapy. Although the use of allogeneic T cells usually causes severe graft versus host disease (GvHD), the emergence of CRISPR/Cas9 technology has provided the possibility to solve this problem. At present, universal CAR-T cells prepared by CRISPR/Cas9-mediated knockout of the endogenous αβ T cell receptors (TCR) to generate TCR-negative CAR T cells have achieved good therapeutic effects in preclinical trials. In this article, we will introduce the research status of CAR-T cell immunotherapy and describe the application of CRISPR/Cas9 gene editing in enhancing the safety and efficacy of CAR-T cell therapy.
The chimeric antigen receptor (CAR) molecule is a type of genetically engineered fusion protein; its structure is similar to TCR protein. The CAR molecule contains an extracellular domain that recognizes tumor antigens and one or several intracellular signaling pathway domains. The extracellular protein domains of CAR molecules are usually derived from antibody single-chain variable regions, and their intracellular signal pathway domains usually include CD28, 4-1BB or OX40 and other costimulatory signal domains and CD3ζ signal domains.
The signal pathway domain of the first-generation CAR molecule only contained CD3ζ signal, so it could not completely activate a T cell and did not achieve good application effects. On this basis, scholars have further improved the structure of CAR molecules, introducing one or two costimulatory molecules to form the second-generation CAR and the third-generation CAR molecules, respectively. In recent years, the fourth generation of CAR molecules have evolved: In addition to the elements of traditional CAR molecules, they can induce T cells to secrete cytokines upon CAR signaling. In addition, the study of universal CAR-T cells with TCRα (TRAC) or TCRβ (TRBC) knockout represents an important direction for the next generation of CAR-T cell research, which is called the fifth-generation CAR-T technology . The evolution of CAR-T cells in successive generations is shown in Figure 1 .
Figure 1. The development history of CAR-T cells 
CRISPR/Cas9 technology is widely used in the development of universal CAR-T cells, expanding the application range of CAR-T cells, and also in the modification of immune cells (including T cells) to enhance anti-tumor effects. Although the application of CRISPR/Cas9 technology still faces many issues with safety and efficacy, there is no doubt that this technology will play an important role in the development of the next generation of new CAR-T technology.
1. Using CRISPR/Cas9 technology to destroy inhibitory molecules that affect T cell function
Tumor cells can suppress the immune response or escape being killied by immune cells by expressing negative regulators. These negative regulators are called immune checkpoints. PD-1 (PDCD1) is an important immune checkpoint molecule, and the activation of its signaling pathway can significantly inhibit T cell proliferation and immune response.
At present, the safety of clinical application of CRISPR/Cas9-edited TRAC gene and PDCD1 gene knockout CAR-T cells and their therapeutic feasibility have been verified in a study. Researchers found that CRISPR/Cas9 has a higher editing efficiency for endogenous TRAC and PDCD1 genes, but a lower editing efficiency for TRBC genes. At the same time, the results of this study showed that CRISPR/Cas9-edited T cells can survive in the body for nearly 9 months, and only have low levels of toxic side effects in the clinical study .
In another study, Rupp et al. transferred Cas9 and sgRNA targeting exon 1 of the PD-1 gene into T cells by electroporation, and then transfected the cells with a lentivirus (LV) containing the CD19 CAR gene. The results in the mouse model constructed by PDL1+CD19+ tumor cells showed that CD19 CAR-T cells with PD-1 gene knockout can effectively eliminate tumor cells. This result indicates that the PD-1/PDL1 signaling pathway plays an important role in regulating T cell function .
2. Construction of anti-suicide CAR-T cells
Recently, a research report described a new type of anti-suicide "off-the-shelf" CAR-T cell (or UCART7) that targets CD7+ T cell malignancies. Researchers used CRISPR/Cas9 technology to knock out TCR and CD7 molecules of T cells. The knockout of TCR avoids the risk of GVHD caused by CAR-T used in allogeneic therapy, and the knockout of CD7 enables CAR-T cells to avoid mutual suicide.
In this study, CD7 CAR-T cells showed good anti-tumor activity in a variety of CD7+ leukemia cell lines and tumor animal models in vivo and in vitro, and they did not cause GVHD. Therefore, the development of the CD7 CAR-T technology could become extremely important for the treatment of relapsed and refractory acute T lymphocytic leukemia and non-Hodgkin T cell lymphoma .
3. Reduce the cytokine storm in the clinical application of CAR-T cells
Considering the important role of granulocyte-macrophage colony stimulating factor (GM-CSF) in inducing cytokine storm, some researchers focused on how to reduce the induction of cytokine storm by targeting GM-CSF. Notably, GM-CSF plays an important role in the differentiation and proliferation of hematopoietic stem cells.
In 2019, Sterner et al. used CRISPR/Cas9 to knock out the GM-CSF gene of CD19 CAR-T cells. The results of this study showed that the secretion of GM-CSF from CAR-T cells with GM-CSF gene knockout was indeed significantly reduced, but it did not affect the important functions of CAR-T cells. More importantly, the GM-CSF defective CD19 CAR-T cells have a significant anti-tumor effect in vivo compared to the wild-type CD19 CAR-T cells .
4. Preparation of universal CAR-T cells
Universal CAR-T (UCAR-T) cells can be prepared using T cells from healthy donors, and can also be mass-produced, which greatly broadens the application range of CAR-T cell immunotherapy. Therefore, universal CAR-T cell therapy products are an important direction for the development of next-generation CAR-T cells.
At present, the development of UCAR-T cells is mainly achieved by knocking out or silencing the TCR gene and β2M gene of T cells using CRISPR/Cas9 gene knockout technology. Eyquem et al. used CRISPR-Cas9 technology to knock out the TCRα constant region: The sgRNA target position was the 5'end of the first exon of TCRα, AAV was used to deliver the CD19 CAR gene sequence surrounded by the homology arm to the cell, which was inserted into the TCR site by homologous recombination, and the results showed that nearly 95% of T cells had TCR knocked out. Further results of NALM6 mouse in vivo tumor model experiments showed that only 1×105 CAR-T cells can be injected back to effectively control the growth of tumor cells, at the same time, only 2% of T cells express inhibitory receptors such as PD1, LAG3, and TIM3. The low expression of these inhibitory receptors may be the main reason for the good anti-tumor effect. The results of this study provide us with a beautiful picture of how to enhance the anti-tumor effect of CAR-T cells through gene editing technology.
With the growing application of CRISPR/Cas9 technology in the field of CAR-T cell immunotherapy, it will play an important role in aspects such as solving the rejection of allogeneic therapy, overcoming the tonic signal of CAR-T, improving T Cell function exhaustion, overcoming the suppression of tumor microenvironment, and reducing the toxic side effects of CAR-T therapy.
In addition, the development of a large-scale high-throughput genetic screening method based on CRISPR/Cas9 provides the possibility for efficient and specific screening of hundreds of genes in T cells. At the same time, there is a great research space for the application of CRISPR/Cas9 technology in the identification of potential genes that can reverse the depletion of CAR-T cells. However, as the CRISPR/Cas9 system is an emerging technology in cell therapy, it is currently mostly used in research laboratories in the academic and pharmaceutical industries. With the rapid development of CRISPR/Cas9 applications and the emergence of new gene editing technologies, we hope to witness CAR-T cells make greater breakthroughs in the field of tumor immunotherapy.
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