TEAMWORK MAKES THE DREAM WORK: COMBINING CAR-T AND CRISPR/CAS9 TO TREAT CANCER?

Reading time: 5 minutes

Yousra Iftequar

You know what they say, “It takes two flints to make a fire.” Researchers have exactly put that into action and have made an effort to combine these two revolutionary techniques.

WHAT IS CAR T-CELL IMMUNOTHERAPY?

Immunotherapy utilizing the body’s immune system has emerged as a promising new approach to treat cancer, offering more durable and efficient treatment compared to traditional methods like chemotherapy, radiotherapy, and surgery. T-cell therapy, a specific type of immunotherapy, harnesses the body’s own T cells to target cancer cells directly. T cells, abundant in lymphatic tissues and circulating in the bloodstream, play a crucial role in detecting and eliminating infected cells through their T cell receptors (TCRs).

CAR T-cell therapy involves harvesting the body’s immune cells, activating and expanding them in a laboratory setting, and then reintroducing them intravenously to the patient.This innovative approach uses genetically modified autologous or allogeneic T cells (autologous T cells are taken from the patient’s own body, while allogeneic T cells are extracted from a donor other than the patient) as a therapeutic agent to enhance the patient’s immune system to effectively target and eliminate cancer cells.

Unlike monoclonal antibody treatments, which are widely in use, they specifically bind to targets to stimulate the immune response, T cells can multiply and directly eradicate cancer cells. Furthermore, a major advantage is that T cells possess immunological memory, enabling prolonged anti-cancer activity within the patient’s body compared to traditional medications.

WHAT IS CRISPR/CAS9 TECHNIQUE?

CRISPR/Cas9 technology originates from the bacterial and archaeal type II immune systems, which defend against intruders such as viruses (bacteriophages), plasmids, and other foreign nucleic acids. CRISPR/Cas9 is a powerful tool that scientists use to edit genes. Imagine genes are like instruction manuals that tell our cells what to do. Sometimes, these instructions can have mistakes that cause diseases. CRISPR/Cas9 works a bit like a cut-and-paste tool for genes.

Here’s how it works: CRISPR is like a GPS that guides a molecular scissors called Cas9 to a specific spot on our DNA—the code that makes up our genes. Once there, Cas9 cuts the DNA. Scientists can then either remove the cut-out part, add in new DNA, or make specific changes to fix errors. Think of it like fixing a typo in a book—it helps researchers correct genetic mistakes that could cause diseases or improve how cells work. This technology is super precise and has opened up new possibilities for treating genetic disorders and studying how genes affect our health.

USING CAR-T CELLS WITH CRISPR/CAS9 TECHNIQUE:

Conventional CAR T-cells in most clinical trials are derived from a patient’s own autologous T cells. However, producing these autologous CAR T-cells is time-consuming and costly, and it can be challenging to collect sufficient high-quality T cells from patients with severe diseases, limiting their availability. These limitations have spurred the development of universal, or off-the-shelf CAR T-cells, which are genetically modified allogeneic T cells from healthy donors. These universal CAR T-cells could potentially simplify the manufacturing process and be available to a larger number of patients.

To create universal CAR T-cells, two primary challenges must be overcome: graft-versus-host disease (GVHD, is a condition that can occur after an allogeneic stem cell transplant, where the donor’s T cells attack the recipient’s tissues because they recognize them as foreign) and the recipient’s rejection of the infused allogeneic T cells. The αβ T-cell receptors (TCRs) on allogeneic T cells can recognize recipient alloantigens, causing GVHD. Additionally, the human leukocyte antigen (HLA) molecules on these allogeneic CAR T-cells are often recognized as foreign by the recipient’s immune system, leading to rapid rejection.

To address these challenges, researchers have explored methods to silence or disrupt both TCRs and HLA molecules in allogeneic T cells. Multiple studies have demonstrated that using CRISPR/Cas9 to knock out the TCR beta chain and beta-2-microglobulin (B2M), a key component of the HLA-I molecule, can produce universal CAR T-cells. These cells have been shown to function effectively both in vitro and in vivo without causing GVHD.

However, the knock-out of B2M and the subsequent production of HLA-I negative CAR T-cells introduce a new issue: these HLA-I negative cells become targets for natural killer (NK) cells, which can lead to their rejection. Solutions to this problem include engineering the T cells to express HLA-E or using anti-NK cell depletion antibodies.

CONCLUSION:

In summary, studies using CRISPR/Cas9 to modify CAR T-cells demonstrate that this technology is  a highly efficient method for generating genetically modified CAR T-cells, with a targeting efficiency of about 90% for single gene disruptions. While the efficiency decreases with multiple gene targets due to competition among gRNAs for Cas9, the effector function of the CAR T-cells remains unimpaired, and a pure population of genetically modified T-cells can be achieved through enrichment. The combination of CRISPR/Cas9 with CAR T-cell therapy offers a promising approach for effective cancer treatment, and several companies are investing in improving CAR T-cell therapy using this technology.

REFERENCES: 

          1. Mollanoori, H., Shahraki, H., Rahmati, Y., & Teimourian, S. (2018).CRISPR/Cas9 and CAR-T cell, collaboration of two revolutionary technologies in cancer immunotherapy, an instruction for successful cancer treatment. Human Immunology, 79(12), 876–882.  https://doi.org/10.1016/j.humimm.2018.09.007

           2. Fard, G. B., Ahmadi, M. H., Gholamin, M., Amirfakhrian, R., Teimourian, E. S., Karimi, M. A., &      Bafghi, M. H. (2023). CRISPR‐Cas9 technology: As an efficient genome modification tool in the cancer diagnosis and treatment. Biotechnology and Bioengineering, 121(2), 472–488.  https://doi.org/10.1002/bit.28603

           3. Feins, S., Kong, W., Williams, E. F., Milone, M. C., & Fraietta, J. A. (2019). An introduction to chimeric antigen receptor (CAR) T‐cell immunotherapy for human cancer. American Journal of Hematology, 94(S1). https://doi.org/10.1002/ajh.25418