Abstract
This article will explore the use of CRISPR Cas9 technology to create models of tumors to improve understanding of which genes are responsible for tumor initiation and progression, and to develop therapies that target those genes. There are already different types of immunotherapies, such as engineered TCR therapy and CAR T cell therapy, which are being used to treat cancer, and CRISPR has the potential to improve these therapies so that they are more precise, efficient and affordable. This article will also discuss the challenges that need to be overcome to make CRISPR-based cancer therapies available in clinics and to develop future cancer therapies using this technology.
Introduction
CRISPR-Cas9 is a very precise and efficient gene editing technology that is used to create mutations at a specific location in a gene to completely remove (silence) or overexpress a gene, or to insert synthetic sequences in a gene to change its function. In the lab, scientists produce a short RNA segment with a ‘guide’ sequence, which binds to a particular sequence in the DNA and to an enzyme called Cas9. The RNA recognises the desired DNA sequence and the Cas9 enzyme cuts the DNA at that specific location. After the DNA has been cut, the cell’s own DNA repair mechanism is used to remove segments of DNA or to replace a segment of DNA with a synthetic DNA sequence (Genetics Home Reference, 2020).
Currently, most cancer treatments include surgery, chemotherapy and radiotherapy, or a combination of them. Although these treatments are effective, they also attack healthy body cells, resulting in serious side effects. This article will explore how CRISPR technology can be used to improve existing immunotherapies and to create new cancer therapies which are more efficient and have minor side effects. This article will also discuss the importance of CRISPR, which allows researchers to create models of tumours so that they can better understand the mechanisms by which cancer develops and the genetic mutations involved in order to develop treatment which targets cancer cells more accurately.
Using CRISPR to model patient tumors
With the knowledge from the Cancer Genome Atlas, a program which identifies genetic mutations present in specific cancers by sequencing patient tumors, and the use of CRISPR technology, scientists can now create models of patient cancers in vitro (in the lab) by editing certain genes in cell cultures. Using these models to study the impact of drugs on the specific cancer allows effective drugs to be identified quicker because several drugs can be tested at the same time and many of the same types of models can be produced to test the drugs on (Synthego, 2020).
Using CRISPR screens to find new cancer drug targets
To date, scientists only knew about a small number of targetable genes that initiate tumors and encourage their growth, which is one of the reasons why cancer recurrence is common after treatment. Researchers are now using CRISPR in genetic screens to study the effect of introducing genetic mutations. This allows them to look for new genetic mutations related to cancer and ,therefore, to develop new drugs to target those genes (Synthego, 2020).
An example of such a genetic screen is CRISPRa, in which sequences of gene edits are assembled in a barcoded library and then put into chosen tumor cells. The tumor cells are left to grow, and those that live are sequenced and matched with their barcode. The barcodes associated with the cells that survived enhance the growth of cells and are potentially cancer-causing. Using this screen will allow researchers to narrow down the number of genes which may cause cancer, so they have a more manageable number of genes to study in greater depth and develop drugs targeting those genes faster. (Synthego, 2020).
In a study at MIT, professor David Sabatini and his team used CRISPR to find the gene mutations cells need to become cancerous and which would disable cancer if repaired. They studied four distinct blood-based cancer lines, finding many new genes which appeared to be necessary for the cancerous cells to flourish and could be the target of new chemotherapy drugs. This is just one of many examples demonstrating how CRISPR provides a cheaper and faster method of finding gene targets and developing cancer treatments. In other studies, researchers used genome-wide knockout screens, in which every potentially cancer-causing gene is removed from cells in a culture, but only one gene is removed from each cell. This allowed them to find genetic factors responsible for the capability of cancer cells to metastasize (spread to other parts of the body) (Doudna and Sternberg, 2017). It is particularly important to understand how cancer metastasizes because once this happens in the patient, it becomes much harder to treat the cancer. However, if metastasization can be prevented, cancer will be easier to target as it remains in one place and will allow the patient to have a much greater likelihood of recovering. Some people opt for medical tourism to get treatment from a better medical expert and for a better price.
Using CRISPR for cancer immunotherapy
Cancer immunotherapy is a new treatment in which the patient’s own immune system is used to destroy cancer cells. The T cells (lymphocyte cells that attack pathogens and cancer cells in the body) are slightly changed so that they can identify the molecular markers of cancer cells and initiate an immune response to destroy cancer cells. This is called adoptive cell transfer (ACT), also known as cellular immunotherapy, in which T cells are genetically engineered to attack a patient’s unique tumor cells. A new gene which produces a receptor that targets the molecular markers of tumor cells is added to the T cells. However, there is already a receptor gene in T cells and having several of these genes can disrupt the cell. Using CRISPR, researchers are now able to remove the original receptor gene so that they can then add the new cancer specific receptor gene (Doudna and Sternberg, 2017). There are many types of adoptive cell transfer including engineered TCR therapy and CAR T cell therapy. Thus far, these therapies used viral integration to edit genes in T cells, but now with CRISPR technology allowing for more precise gene editing, these therapies are targeting cancer cells more accurately (Cancer Research Institute, 2020). 
Engineered TCR therapy is used when patients do not have T cells which have recognized their tumor. T cells are harvested from patients and a synthetic T cell receptor which targets specific tumor antigens is added to them using viral integration or CRISPR. This treatment can be personalized for individual patients as a specific T cell and an optimal target can be chosen for each patient’s tumor (Cancer Research Institute, 2020).
In CAR T cell therapy, the patient’s T cells are harvested and a receptor known as CAR (chimeric antigen receptor), which targets antigens on cancer cells, is added to the cells using viral integration or CRISPR. These modified CAR T cells are returned to the patient where they target and destroy cancer cells (Synthego, 2020). An important advantage of this therapy is that CAR T cells can attach to cancer cells even if their antigens are not presented on the surface, which can render more cancer cells vulnerable to their attacks (Cancer Research Institute, 2020). Currently, there are two CAR T cell therapies, developed using viral integration, which were approved in 2017. Yescarta is used to treat large B-cell non Hodgkin lymphomas (a cancer of the immune system) in adults, with over 80% of patients resulting in a complete or partial response during clinical trials. Kymriah is used to treat advanced acute lymphoblastic leukaemia in children and adults younger than age 25, with 80% of patients going into remission during clinical trials (Cleveland Clinic, 2020). 
In 2019, the first clinical trial showed that using CRISPR in immunotherapy procedures was safe. The treatment was given to three patients. Two of them were suffering from multiple myeloma (a type of blood cancer) and the other was suffering from sarcoma (a connective-tissue cancer). Researchers first used CRISPR to remove three genes in the patient’s T cells’ DNA. Two of those genes coded for structure on the cell membrane which stopped the T cells from attaching to tumors. The other genes coded for the protein PD-1 which cancer cells flip to prevent being attacked by immune cells. The researchers then used a special virus to add a receptor, which helps the cells to target and destroy tumor cells, into the T cells’ DNA and return the cells into the patients. The edited T cells survived and successfully multiplied in the patients’ bodies. Moreover, the patients did not suffer from any serious side effects. Three months after receiving treatment, one of the patient’s cancer remained stable, the second patient continued to worsen, and the third patient’s reaction could not be evaluated because they had been treated too recently (Lanese, 2019). Although these results may not seem remarkable, the trial was designed to assess the safety of this technology and not to fully treat the patients. The promising results of this first trial show that the treatment is safe and allow the next stages to take place with more patients to further determine the safety of this treatment and how efficient it is at destroying cancer.
In the near future, it is easy to imagine that cancer-causing mutations in genes of patients will be corrected using CRISPR as a cancer therapy. Personalized therapy will also certainly become a standard treatment for cancer, as CRISPR will allow a patient’s genome to be quickly screened to find the individual’s genotype-specific alterations. New advances in this technology, such as combining Cas9 and guide RNA libraries, will help to find the genes in which genetic mutations have resulted in disrupted signaling pathways, which may initiate cancer or encourage its growth. These gene screening will help to develop novel therapies targeting the genes which are found to cause cancer (Tian et al, 2019). At the moment, the CAR T cell therapy Kymriah costs $475,000 per treatment (Tian et al, 2019), restricting the availability of this drug to only a small percentage of society. However, using cheap CRISPR technologies to manufacture different types of immunotherapies will help lower the cost, making them accessible to more patients.
Although CRISPR offers many opportunities for new cancer treatments, this technology comes with challenges, especially concerning its safety and how it is delivered to the patient. It will be important to improve the effectiveness of gene targeting and to make sure that CRISPR can accurately edit the genome. Furthermore, the Cas9 protein is sourced from bacteria and might trigger an immune response which would reduce its gene-editing efficacy (Tian et al, 2019).
Conclusion
CRISPR Cas9 is a novel and quickly developing technology which has revolutionized cancer research. It offers the tools to model cancers so we can understand their mechanism and develop treatments which would have been unthinkable of only a couple of years ago. This new CRISPR technology offers better precision to fix cancer-causing mutations compared to previously used gene-editing technologies such as ZFNs and TALENs. It also has the potential to improve existing immunotherapies, especially CAR T cell therapy, and to develop new cancer therapies. It will be fascinating to see the results of clinical trials over the coming years and the development of these new cancer therapies.
References
1. Genetics Home Reference. 2020. What are genome editing and CRISPR-Cas9? Accessed April 13, 2020. https://ghr.nlm.nih.gov/primer/genomicresearch/genomeediting.
2. Synthego. 2020. CRISPR Cancer Research. Accessed April 13, 2020. https://www.synthego.com/crispr-cancer
3. Doudna, Jennifer, and Samuel Sternberg. 2017. A Crack in Creation. London: Vintage.
4. Cancer Research Institute. 2020. How Cellular Immunotherapies Are Changing the Outlook for Cancer Patients. Accessed April 13, 2020. https://www.cancerresearch.org/immunotherapy/treatment-types/adoptive-cell-therapy.
5. Cleveland Clinic. 2020. CAR T-Cell Therapy. January. Accessed April 13, 2020. https://my.clevelandclinic.org/health/treatments/17726-car-t-cell-therapy/risks–benefits?view=print.
6. Lanese, Nicoletta. 2019. Doctors Are Trying to Use CRISPR to Fight Cancer. The 1st Trial Suggests It\’s Safe. November. Accessed April 13, 2020. https://www.livescience.com/crispr-to-fight-cancer.html.
7. Tian, X., Gu, T., Patel, S. et al. 2019. CRISPR/Cas9 – An evolving biological tool kit for cancer biology and oncology. Accessed April 13, 2020. https://www.nature.com/articles/s41698-019-0080-7
Figure References
Figure 1: Available from https://www.cancerresearch.org/immunotherapy/treatment-types/adoptive-cell-therapy
Figure 2: Available from https://www.genengnews.com/insights/ethical-and-biosafety-oversight-of-new-car-t-cell-products/
Biography
Name: Andrada Durantel
Email: [email protected]
Birthday: 2003
School: St Paul\’s Girls\’ School
School Address: Brook Green, Hammersmith, London W6 7BS
Teacher Email: [email protected]
Country: United Kingdom
Biography: Andrada Durantel is a Year 12 student studying Biology, Chemistry, Maths and Further Maths for A-levels in at St Paul\’s Girls\’ School in London. She enjoys drawing, swimming and reading in her free time.
Article Summary: This article explores how a novel technology is being used to treat cancer.
How they found us: Teacher/School
How they started the article:
Andrada Durantel is a Year 12 student studying Biology, Chemistry, Maths and Further Maths for A-levels in at St Paul\’s Girls\’ School in London. She enjoys drawing, swimming and reading in her free time.