CRISPR-Cas9 gene editing in three major tumor immunotherapy approaches

Gene editing technology is an emerging molecular biotechnology that artificially changes specific gene loci in a nucleotide sequence, inserting, deleting, replacing or modifying specific target genes in the genome to alter their expressed traits. CRISPR-Cas9, as an emerging gene editing technology, is widely used in the field of cancer treatment through targeted knockout of tumor immune checkpoint molecules or through rapid and simple gene editing. It has significantly reduced the operational difficulty of tumor immunotherapy, while also greatly promoting the development of tumor immunotherapy research. Therefore, this article briefly discusses its application in the three major tumor immunotherapy approaches: CAR-T, immune checkpoints and antibody-targeted therapy.

Application in CAR-T therapy

CAR-T cell immunotherapy is the hottest research topic among tumor immunotherapies. CAR-T technology obtains modified T cells through gene editing, which can specifically recognize specific receptors on the surface of tumor cells and enhance their defense against tumor cells.

Improving the preparation efficiency of CAR-T cells. CAR-T therapy collects the patient's T cells for genetic modification, which become CAR-T cells and are then infused into the patient. The process is time-consuming, difficult and costly, and is also limited by the number of the patient's own T cells available for treatment. CRISPR-Cas9 gene editing technology can significantly improve the preparation efficiency of CAR-T cells. It genetically modifies a normal immune T cell and, when introducing the CAR sequence, removes the endogenous αβ T cell receptor genes and the human leukocyte antigen class I (HLA I) coding genes from the T cell, to prevent host-versus-graft reactions when used in different patients.

Improving the function of CAR-T cells. CRISPR-Cas9 technology can also improve the function of CAR-T cells by knocking out genes encoding signaling molecules or T cell inhibitory receptors. CAR-T cell therapy has remarkable anti-tumor efficacy, but can only specifically recognize receptors on the tumor cell surface, whereas tumor-specific T cell receptor (TCR-T) cells can, through genetic modification, express specific receptors that recognize antigen peptides presented by class I major histocompatibility molecules on the tumor cell surface, thereby recognizing intracellular specific molecules. However, the endogenous TCR present in recipient T cells may compete with the modified TCR. Therefore, CRISPR-Cas9 gene editing technology is used to prepare CAR-T cells deficient in TCR, HLA class I molecules and PD1, reducing their alloreactivity without causing graft-versus-host disease, and improving in vivo anti-tumor efficacy.

It can also improve the efficacy of CAR-T cells by knocking out genes of immune co-inhibitory pathways or signaling molecules (such as CTLA4 and PD1). Scientists have effectively used CRISPR-Cas9 technology to gene-edit CAR-T cells, obtaining multi-gene knockout CAR-T cells, preventing the influence of immune modulators on T cell activation and proliferation, successfully improving the defense of CAR-T cells against immune cells, and better exerting anti-tumor efficacy.

A CRISPR clinical trial approved by the Recombinant DNA Advisory Committee under the NIH (National Institutes of Health) and led by Professor Carl June. In this trial, the researchers will use CRISPR-Cas9 to knock out the gene encoding PD-1 and the endogenous T cell receptor genes in CAR-T cells targeting melanoma; after knocking out PD-1, the killing ability of the CAR-T cells becomes stronger.

In December 2017, the team of Wang Haoyi of the Chinese Academy of Sciences published an article in Frontiers of Medicine titled CRISPR-Cas9 mediated LAG-3 disruption in CAR-T cells. The research team established a method for efficiently knocking out the LAG-3 gene in T cells or CAR-T cells using the CRISPR-Cas9 system, and found that LAG-3-knockout CAR-T cells could maintain antigen-specific cytokine release and anti-tumor function in vitro and in vivo.

Researchers at Memorial Sloan Kettering Cancer Center in the United States used CRISPR-Cas9 technology to deliver the CAR gene to specific sites in the T cell genome, constructing more potent CAR-T cells in this way. These CAR-T cells are less prone to exhaustion and can continuously exert anti-tumor effects for a long time.

Scientists at the Washington University School of Medicine in St. Louis used the gene editing technology CRISPR to modify human T cells, removing the expression of CD7 and the T cell receptor alpha chain (TRAC). This is a CAR-T cell therapy (UCART7) targeting CD7+ T cell malignancies that avoids "fratricide". It attacks cancerous T cells while protecting normal T cells. The research results showed that mice in the treatment group receiving gene-edited CD7-targeting T cells had a median survival of 65 days, while mice in the control group had a median survival of only 31 days. The results were published in Leukemia, a sub-journal of the authoritative academic journal Nature.

The application of CRISPR technology in the genetic modification of CAR-T cells has broad prospects. But there are currently two limiting issues: the methods for introducing CRISPR into T cells cannot make ordinary cells proliferate normally, limiting the cell culture and enrichment process. Among the introduction methods, the non-integrating transfection method is safe but inefficient, while the integrating transfection method is more efficient but its safety cannot be guaranteed. The CRISPR editing system has off-target phenomena; once off-target occurs, it will cause changes in non-target genes or damage to regulatory elements, with irreversible consequences. Therefore, precise editing and efficient delivery of CAR-T cells are the main research directions for gene editing technology in the future.

Application in immune checkpoints

PD-1 and CTLA-4 are now very well-known immune checkpoints that can significantly inhibit T cell activity, making it easier for tumor cells to escape the body's immune mechanisms and leading to poor T cell treatment outcomes. At present, in order to better exert the efficacy of T cell tumor therapy, the use of blocking antibodies of immune checkpoint inhibitors is a common and effective method to block the inhibition of T cell immune responses by immune modulators.

Among them, using CRISPR-Cas9 gene editing technology to knock out genes involved in immune negative regulation can also achieve the same anti-tumor effect, which is a brand-new therapeutic approach. Using electroporation to introduce Cas9 and sgRNA into T cells and knock out the PD-1 gene reduces its expression; in long-term in vitro culture, no effect of PD-1 on T cell activation was found, allowing T cells to better exert their anti-tumor efficacy. This new blocking therapy is often used in combination with adoptive T cell immunotherapy to enhance the activity of immune cells.

An article published by Professor Carl June, a pioneer in the CAR-T field, in Clinical Cancer Research confirmed that in in vitro and animal model studies, after knocking out the two immune exclusion-related genes TCR and B2M using CRISPR technology, the alloreactivity of T cells decreased without causing GVHD.

In addition, Chinese and British scientists also found that knocking out TRAC (T cell receptor alpha constant chain) with CRISPR can construct universal CAR-T. At the same time, these advances indicate that using targeted gene editing technology to knock out immune checkpoints is already a very promising strategy for improving CAR-T treatment of solid tumors.

Application in antibody-targeted therapy

Tumor cells express antigens on their surface that differ from normal cells, and these antigens can serve as targets recognized by monoclonal antibodies; binding by the antibodies causes tumor cell death, thereby achieving the anti-tumor goal. At present, the FDA has approved many antibody drugs, such as anti-HER2 antibodies, anti-CD20 antibodies and anti-VEGF antibodies.

Identifying potential specific targets on the surface of tumor cells. CRISPR-Cas9 gene editing technology can be used to identify potential specific targets on the surface of tumor cells and further develop antibody-targeted therapy applications. Steinhart et al. used CRISPR-Cas9 technology to conduct a genome-wide screen among pancreatic ductal tumor cells with RNF43 mutations and found that the Frizzled-5 receptor plays a key role in the growth of pancreatic tumor cells. Antibodies that specifically bind Frizzled-5 can significantly inhibit the growth of cancer cells in tumor-bearing mice.

Recently, the research team of Chen Sidi at Yale University developed a CRISPR system for screening genes on CD8+ T cells related to tumor immunotherapy, and found key genes related to tumor infiltration and the cytotoxic killing exerted by CD8+ T cells. They discovered that a gene named DHX37 can suppress the response of CD8 killer T cells to mouse tumors; after knocking out this gene in CD8+ T cells, the cells have stronger tumor-killing ability in vivo.

Antibody preparation. In addition, CRISPR-Cas9 can not only identify specific targets on the surface of tumor cells, but also plays a role in antibody preparation. Based on the amino acid composition of the heavy chain constant regions of antibodies, the corresponding antibodies can be divided into five classes: IgG, IgA, IgM, IgD and IgE. These five classes differ in activating the complement system, activating effector cells and killing target cells, and also play different roles in antibody-targeted therapy of tumors. Therefore, antibody class switching diversifies antibodies and enables more applications in targeted therapy. B cell-specific cytidine deaminase (AID) participates in the process of antibody class switching; the most important step in Ig gene class switching is the double-strand break of DNA, which is generally initiated by B cell-specific enzymes such as recombination activating gene proteins 1/2 (RAG 1/2) and AID.

Researchers used CRISPR-Cas9 technology to edit human and mouse Ig genes, using CRISPR-Cas9-mediated efficient DNA breaks in the heavy chain constant region genes of IgM+ mouse B cells, hybridoma cells and human B cells, thereby inducing class switch recombination and achieving antibody class switching from IgM to IgG to IgA. Through CRISPR/Cas9 technology, the genes of the immunoglobulin heavy chain constant region of hybridoma cells or B cells can be targeted-edited to obtain antibodies of specific classes, achieving the anti-tumor goal.

The antigen-binding fragment (Fab) of antibodies has a small molecular weight and more easily penetrates tumor tissue, playing a key role in antibody-targeted therapy of tumors. Therefore, research has been conducted to obtain hybridoma cells that secrete only antibody Fab fragments; by using CRISPR-Cas9 technology to delete the Fc segment gene of mouse hybridoma cells, antibody Fab fragments can be obtained more efficiently and simply, and are easy to conjugate with other drugs to penetrate tumor tissue, with more significant anti-tumor effects.

Conclusion

Due to off-target effects and low intracellular delivery efficiency in the operation of CRISPR-Cas9 technology, its clinical application is limited. How to effectively reduce the incidence of off-target effects while not affecting the simplicity and efficiency of CRISPR-Cas9, and improve its transfection efficiency into immune cells to achieve the ideal anti-tumor immunotherapy effect, will also be the direction for scientists to continue their efforts.

But it is undeniable that CRISPR-Cas9 gene editing technology plays an increasingly important role in tumor immunotherapy. With further improvement of this technology, it will be more widely applied in gene research, while promoting clinical anti-tumor immunotherapy. [Source: Sina Medical News] References: ScienceNet: Targeted gene editing to knock out immune checkpoints, a new strategy for tumor immunotherapy; Frontiers of Medicine: What will targeted gene editing to knock out immune checkpoints bring to tumor immunotherapy?; Medical Health Trends: The past, present and latest progress of CRISPR gene editing technology; Zhang Yu, CRISPR/Cas9 gene editing technology and its application in tumor immunotherapy.