CRISPR-engineered T cells in patients with refractory cancer

Cancer is the second leading cause of death in the United States, taking half a million lives each year. CAR T technology has successfully harnessed the human immune system to produce awe-inspiring cancer remission rates. CRISPR-mediated genome engineering has enabled new developments in CAR T cells to bypass logistical, technical, and immunological roadblocks, making the therapy more successful and widely accessible.

By combining CAR-T therapy with CRISPR-Cas9 gene editing technology, scientists are now hopeful that they will be able to cure cancer for good.

An Overview of CAR-T Cell Therapy

For those who aren’t familiar with cancer treatments and immunotherapy, CAR-T can be a daunting topic. Let’s get started with the basics of CAR-T cells and how they can be used to fight cancer. 

What are CAR-T Cells?

CAR-T cells are human immune cells, known as T lymphocytes, that have been genetically engineered to express a chimeric antigen receptor (CAR), an artificial receptor that can recognize specific types of cancer. Genetically altering T cells to express a CAR enables the immune system to target and destroy cancer cells.

The CAR T receptor is composed of five distinct components which work together to lyse cancer cells. The single-chain variable fragment (scFv) antibody recognizes a CD19 tumor-associated antigen on the surface of a tumor cell. This fragment is fused to linker and transmembrane domains which in turn are bound to a costimulatory domain that modulates the immune response to a cancer cell. Finally, a CD3 signaling domain activates the immune response within the T cell upon antigen binding, serving to lyse the offending tumor cell.

What is CAR-T Cell Therapy and How Does it Work?

CAR-T cell therapy is a form of immunotherapy, a method of altering the natural immune system to make it more effective at recognizing and destroying cancer cells. The human immune system is naturally primed to fight off tumor cells, and T cells are the front line of the body’s natural defense system against cancer. 

T cells express receptors on their surface that recognize and bind antigens produced by invading pathogens and rogue cancer cells in the body. In addition to directly killing infected or cancerous cells, T cells also “store” an immunological memory of past diseases to prime the body for faster and more effective immune responses when disease-causing agents are detected again in the future. However, sometimes T cells don’t recognize/destroy cancer cells, especially solid tumors

CAR T cell therapy essentially makes T cells better at doing their job, and can be either autologous (from the patient) or allogeneic (from a healthy donor). In autologous CAR T, treating clinicians will identify the antigen produced by particular cancer a patient is suffering from. After harvesting T cells from the patients, they will engineer the cells to express a CAR on their surface membrane that will recognize and bind that specific antigen. This modified “living drug” population of cells is then transfused back into the patient,  identifying and destroying tumor tissue or cancerous cells in the blood.

In allogeneic CAR-T, the cells are harvested from a healthy donor and engineered to express the specific CAR required before being transfused into the patient. This approach is used because it is typically easier to obtain a high number of T cells from a healthy donor rather than a patient who is already fighting cancer. However, allogeneic CAR-T is associated with higher safety risks, immune rejection, and graft-versus-host disease (GVHD). 

Advantages of CAR-T Cell Therapy

Conventional cancer treatments, like radiation therapy or chemotherapy, cannot discriminate between healthy cells and cancerous cells. This means that the treatment destroys both types of cells, resulting in severe side effects. In contrast, CAR-T therapy uses a patient’s own immune system to fight cancer. 

This is an example of ‘personalized medicine’ or ‘precision medicine’ wherein treatments are tailored to the individual patient based on their condition and genetic predisposition. CAR-T is a targeted therapy, meaning that only cancer cells will be killed, not healthy cells, meaning it has fewer side effects. Once transfused back into patients, the cells also proliferate and continue to fight cancer over long periods of time, making it a long-term option without the need for multiple or continuing treatments. 

Obstacles to Successful CAR-T Cell Therapy

CAR T therapies greatly outpace previous methods of cancer treatment and represent a massive jump forward for curative cancer care. However, CAR T therapy still has a long way to go. One of these roadblocks to its widespread use is CAR-T cell dysfunction, including both exhaustion and senescence of the cells, which make them less effective. 

Insufficient quantity and quality of patient-harvested T cells present another obstacle. CAR-T cells can also experience uncontrolled proliferation when transfused into patients, resulting in potentially life-threatening inflammation and toxicity. As we mentioned earlier, allogeneic CAR-T carries the risk of immune rejection and associated issues, hindering its clinical application.

Another limiting factor is that each step of harvesting, engineering, expanding, and transfusing cells tailored to each patient takes time, meaning that patients must wait long periods before treatment can be administered. This process also comes with staunch requirements for specialized facilities, scientific, and logistical support. These restrictions, combined with the fact that there are few patients in the US who qualify for this therapy, create a substantial financial barrier to treatment.

What You Need to Know About CRISPR and CAR-T Cells

The development of CRISPR-Cas9 gene editing technology has been a major breakthrough for the CAR-T field. Let’s explore why CRISPR is so important in this field of research and how it can be used to improve patient outcomes.

What is the Difference Between CRISPR and CAR-T?

The difference between CRISPR and CAR-T cell therapy is that CRISPR is a gene editing technology, while CAR-T cells are cells that have been created using gene editing technology. Until CRISPR was invented, CAR-T cells were generated using other genome engineering technologies. 

Originally, the CAR was transduced into T cells via viral vectors, like lentiviruses. Because viral vectors work via random integration of the target, this process can result in such undesired effects as clonal expansion, oncogenic transformation, variegated transgene expression, and transcriptional silencing. 

More recently, other gene editing methods, including transcription activator-like effector nucleases (TALENs) and zinc-finger nucleases (ZFNs), have also been used. However, none of these prior technologies possess the precision or simplicity of CRISPR for generating CAR-T cell therapies – CRISPR-Cas9 allows for the insertion of the CAR at precise target regions of the genome with relative ease and few off-target edits.

Benefits of Using CRISPR for CAR-T Cell Therapies

CRISPR gene editing can be used to improve both the safety and efficacy of CAR-T cell therapies. CRISPR is a much more precise genome engineering tool than previously existing gene editing technologies, meaning that it has fewer off-target effects which can be detrimental to patients. It also has the ability to generate multiple gene edits in T cells, a feat that has historically been challenging. 

One of the ways CRISPR can be used is to precisely insert the CAR into the correct position in the genome of the T cells so that it is expressed at sufficient levels. It can also be used to correct genetic defects on autologous T cells, some of which hinder the ability of the cells to successfully target and destroy cancer cells. Additionally, CRISPR edits can make CAR-T cells even more efficient at killing cancer cells.

Another key application of CRISPR in CAR-T therapy is that it can be used to generate universal, or “off-the-shelf” CAR-T cells. The concept of universal allogeneic CAR-T products bypasses the need to harvest, edit, expand, and transfuse T cells from every individual patient, saving time and resources. 

By editing allogeneic (healthy donor) T cells to express the CAR via knock-in and then knocking out the genes which are responsible for recognition of these non-self cells by the immune system, the cells can successfully be transfused into patients without the risk of immune rejection. These CRISPR-edited designer cells have the potential to be produced en masse, then cryopreserved and stored in hospitals or facilities around the country, eliminating the need for transportation of living cells back and forth from manufacturing facilities for every patient.

CAR-T Therapies and CRISPR are Fighting Cancer and Revolutionizing Medicine

CRISPR-edited CAR-T cells are certainly on track to form safe and effective treatments for a range of different cancers, and universal CAR-T products are edging closer to clinical trials. Many studies are being published in this rapidly expanding field of research, using different methods and techniques.

While CAR T cells have shown considerable promise in B cell malignancies, targeting other blood cancers as well as solid tumors has proven more elusive. One of the limiting factors of using CAR-T cells to fight solid tumors is the immunosuppressive tumor microenvironment (TME). A key 2020 study used CRISPR to enhance the tumor-killing ability of CAR-T cells by inhibiting the transforming growth factor-beta (TGF-ꞵ) gene. This simple edit allows CAR-T cells to persist and continue killing tumor cells over longer time periods.

CRISPR can also be used to disrupt PD-1, a receptor that binds the ligand PD-L1 and leads to inhibition of T cell function, as shown in a 2019 study published in Cancer Immunology, Immunotheray. By knocking out or limiting the expression of PD-1 on the surface of CAR-T cells with CRISPR, the cells show stronger responses to PD-L1-expressing cancer cells, with increased tumor-killing activity and prevention of cancer relapse.  

Whole-genome screens using CRISPR are also helping to make CAR-T cell therapies more effective. CAR-T has previously shown little efficacy against glioblastoma (GBM), however a 2021 study used whole-genome CRISPR screens of both CAR-T cells and patient-derived GBM stem cells to identify molecular markers that determine the GBM-killing ability of CAR-T cells. These results will help increase the ability of CAR-T cell therapies to kill these ‘difficult’ cancer cells. 

A huge breakthrough in 2021 was the generation of a universal, “off-the-shelf” allogeneic CAR-T therapy using CRISPR. The study, published in Nature Biomedical Engineering, used a step-by-step multigene knockout approach to eliminate three different genes that are responsible for the recognition of allogeneic cells by the host immune system. 

The resulting CAR-T cells were able to evade the host immune response while retaining their tumor-killing capacity and showing long-term survival. These universal cells can be used to treat a variety of cancers in potentially any patient, and researchers are currently gathering data to seek approval for clinical trials.

6 Resources to Learn More About CAR-T and CRISPR

There are plenty of resources available to help you learn more about CRISPR-edited CAR-T cells and how they’re currently being used. Here’s a list of our top papers, videos, websites, and blogs, to help you get your head around the vast amount of CRISPR and CAR-T information out there. 

  1. CAR T Therapy: Treating Cancer with Engineered Cells

From Synthego’s CRISPR Cuts Podcast, this interview with CAR-T researcher Dr. Avery Posey discusses how CAR-T works, improvements to CAR-T using CRISPR, and the latest in clinical trials.

  1. CAR T Cells: Timeline of Progress

For a detailed look at how CAR-T cell therapy developed, including all the significant breakthroughs, you can check out this great timeline from the Memorial Sloan Kettering Cancer Center.

  1. Applications and Explorations of CRISPR/Cas9 in CAR T-Cell Therapy

This 2020 review published in Briefings in Functional Genomics describes how CRISPR-Cas9 has advanced CAR-T therapy, including the creation of universal allogeneic CAR-T cells.

  1. Updates in CAR-T Cell Therapies

This YouTube video from the National Cancer Institute features Carl June, an expert in CAR-T cell therapy, discussing key updates in the field.

  1. Use of CRISPR-Cas9 in CAR-T Cell Therapy

This short video explains the application of CRISPR in creating better CAR-T cell therapies, including universal CAR-T products.

  1. A deep insight into CRISPR/Cas9 application in CAR-T cell-based tumor immunotherapies

This review article published in 2021 answers all your questions about CRISPR-edited CAR-T cells, specifically relating to the treatment of solid tumors and universal products.

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