Treatments and Therapies on the Horizon
Cancer affects millions of people worldwide, yet researchers have been working for decades to find effective treatments. New drugs, chemotherapy, and radiation techniques have been used over the years to treat patients, but none of these are devoid of side effects. The discovery of CRISPR as a powerful gene editing tool ushers hope with its promising potential in cell and gene-based therapies, such as CAR-T therapy. Here we will discuss the basics of cancer and how CRISPR is transforming cancer medicine.
Cancer is a disease in which abnormal cells divide uncontrollably and, if malignant, can invade other tissues of the body. There are several types of cancer, commonly categorized by the affected tissue, including breast cancer, lung cancer, skin cancer, etc., or by the type of cell it originates from: carcinoma, lymphoma, leukemia, etc.
In addition to carcinomas, cancer can also exist in the form of sarcomas, leukemia or lymphomas:
While there are a number of different cancer types, few genetic mutations are common between them. These genetic mutations can either turn on a gene (oncogene), or turn off a gene (tumor suppressor) leading to uncontrolled cell growth. Commonly mutated genes between different cancer types are TP53, APC, BRCA1/2, and Rb, while commonly mutated oncogenes include mutations in growth-promoting pathways such as HER2, EGFR, RAS, and MYC.
Scientists use specific laboratory assays to measure specific characteristics of cancer, as they typically display abnormal cell growth or inhibition of cell death and the ability to grow in an anchorage-independent manner (i.e., no extracellular matrix). While cell growth and viability tests often involve fluorescent/colorimetric assays, anchorage-independent growth and invasion potential of cells are tested by their colony forming ability in soft agar.
Disease models are essential for understanding cancer because they provide a controlled environment to study the complex biological mechanisms driving tumor development, progression, and response to treatments. By mimicking the behavior of cancer in living organisms or cell cultures, these models allow researchers to investigate genetic mutations, cellular interactions, and environmental influences that contribute to the disease. They also facilitate the testing of new therapies and personalized treatment approaches, accelerating the discovery of more effective cancer treatments.
One commonly used model to study cancer is using immortalized cell lines. Immortalized cancer cell lines, obtained from cell culture banks like ATCC, are generated from tumors that can grow indefinitely in two-dimensional cultures under specific conditions. Researchers often use cancer cell lines to study cancer biology and to test potential cancer treatments due to their ease of use and scalability.
While cancer cell lines are generated from specific tumor types, the physiological relevance to cancer is currently up for debate. In this regard, primary cells are increasingly used as a model system to understand cancer. Primary cells are isolated from tissues of organisms, and thus closely represent the actual organism than cell lines, which may show altered behavior from infinite propagation.
Three-dimensional organoid cultures, which involve embedding cells into an extracellular matrix, are also being explored as model systems to more fully recapitulate physiologically-relevant events in tumor formation and progression (1). Tumorigenic potential of cancer cell lines can be assessed using animal models (for example, injecting tumor cells into mice).
Depending on the tumor type, there are a number of options for cancer treatments, all targeting the cancer cells to stop growing. Common treatment strategies include surgical removal of the tumor, radiation, chemotherapy, and targeted therapies. Newer treatments are currently being explored with initial success including immunotherapy and cell-based therapies, such as CAR-T therapy.
Surgical resection is the physical removal of the tumor by surgery. While chemotherapy and radiation are sometimes thought to be the same, radiation therapy uses high-energy wavelengths to kill cancer cells, while chemotherapy uses specific drugs aimed at interfering with DNA replication to kill cancer cells.
Targeted therapies are a very specific form of therapy for certain patients, if a specific genetic mutation involved in the disease condition is identified. In cases where targeted therapies are an option, genetic testing of tumors are performed to assess if the patient is eligible to receive it.
One example is the FDA approval of the use of Trastuzumab/Herceptin for HER2 positive breast cancer and gastric cancer patients. Trastuzuamab is a monoclonal antibody that blocks HER2 signaling and therefore stops those cells from growing out of control.
Another type of cell-based immunotherapy to treat cancer is CAR-T therapy. In CAR-T therapy, the patient’s immune cells (T cells) are extracted, and the receptors on the extracted cells are altered to express the chimeric antigen receptor (CAR) that is known to attack cancer cells. Those modified cells are reintroduced into the patient where CAR-T cells go find and kill the dangerous cancer cells. Clinical trials in the CAR-T therapy of certain blood cancers showed high success rates and sparked the movement of using CAR-T therapy in the clinic (2). In 2017, the FDA approved two new CAR-T therapies for specific cancers.
"We are seeing a cancer drug being made from a person's own cells that did not cause disease in that person before, and did not cause toxicity in that person before. So, if our modifications don't go on to cause toxicity—and that's something that the field definitely has to work on and be very conscious about—then we are actually improving on the safety profile of cancer therapies by using immunotherapies."Avery Posey, leading CAR-T cell researcher at the University of Pennsylvania.
Companies like oNKo-innate are also making major strides to combat cancer as they focus on developing cytokine and CAR-T therapies. We sat down with Imran House, Ph.D. and Junyun Lai, Ph.D. from the oNKo-innate team to discuss their latest developed therapies in the immuno-oncology space.
Since its discovery in 2012 as a gene editing tool, CRISPR-Cas9 has been used by many scientists to further our understanding of cancer biology. CRISPR has enabled scientists to edit genetic sequences in a very specific and efficient way, such as introducing specific site mutations, silencing or overexpressing genes of interest, or adding large sequences to drastically change the function of that gene.
With CRISPR technology, in parallel with the widespread genetic sequencing of patient tumors by many consortiums such as the Cancer Genome Atlas, scientists are more educated of which genetic alterations occur in specific tumor types. With this knowledge, researchers are now modeling patient cancers more efficiently by editing specific genes using CRISPR-Cas9 in vitro, providing large-scale biomass whereby functional and drug studies can be performed. This scalability identifies drug targets faster, because more drugs can be screened simultaneously.
While there are common mutations that turn on oncogenes and turn off tumor suppressor genes, the number of targetable genetic drivers of tumor initiation and progression is low, making cancer an extremely difficult disease to fully treat without recurrence. To attempt to narrow down the list of potential cancer-causing genes, scientists are using CRISPR screens to identify new and relevant cancer targets.
Large-scale genetic or small molecule screens allow scientists to test the effect of changing a large number of genetic mutations or small molecules, to begin to identify new cancer-associating genetic mutations or potential drug therapies.
In genetic screens using CRISPRa or CRISPRi, a barcoded CRISPR gRNA library is assembled with the nucleases of choice and injected into tumor cells of interest. These cells are then allowed to grow in vitro, and cells that survive over time are then sequenced and aligned to their barcode. Depending on the goals of the over-expression or inhibition experiments, scientists can then assess the genetic target correlations with cancer. Ideally, scientists will obtain a smaller, more manageable number of genes of interest that can be followed up individually. Similar to the genetic screens, researchers also use small molecule screens to identify new potential drugs for cancer treatment.
As described in a previous section, scientists can engineer the patient’s own T-cell receptors to express CAR antigen that recognizes cancer cells in the patient using cutting-edge nucleases and CRISPR CAR-T Guide RNA. Once engineered in the lab, doctors can transplant the modified CAR T-cells back into the patient, whereby they can attack the cancer cells. As CRISPR is known for its precision in gene editing, scientists have been using CRISPR to edit T cells with high efficiency. One study found that CRISPR engineered CAR T-cells are more potent against tumors (4). Thus CRISPR precision editing is a promising tool for future cancer cell therapies.
CRISPR-Cas9 has been proven to be a powerful tool in studying cancer biology and therapy development. However, bringing scientific breakthroughs from early discovery to the clinic has been met with many hurdles. The first clinical trial using CRISPR for cancer therapy was reported in 2016 in China. Since then, additional trials have begun, mainly using CRISPR to edit immune cells (T cells) to treat cancer via immunotherapy.
While CRISPR-based cell immunotherapies are on the horizon, researchers are also using this tool to understand basic gene function in cancer. For instance, researchers at the National Cancer Institute at National Institutes of Health used CRISPR to understand the mechanism of how olaparib, an FDA approved drug for certain cancer types, increased natural killer cell-mediated lysis (5).
In order to elucidate its mechanism, the researchers screened hundreds of genes in a prostate cancer cell line. The results narrowed down their search to TRAIL-R2, a gene coding for a death surface-receptor. The team used high-efficiency TRAIL-R2 knockout cells to verify the role of this gene in NK-mediated lysis.
A study from UCSF used CAR-T therapy in a virus-free system (6), bringing it one step closer to the clinic. The use of viruses to introduce DNA into cells, which was common in conventional molecular biology experiments, is amenable to patients.
The research team reported a new way to engineer CAR-T therapy using CRISPR without relying on viral integration of CRISPR gRNA and Cas9. This study brings CRISPR closer to the clinic, addressing one of the main hurdles of cell-based therapies for cancer treatment. Companies such as Ligandal are working on specialized CRISPR delivery platforms to alleviate this bottleneck in gene therapy applications.
In addition to using CRISPR-Cas9 to generate new clinical therapies, researchers are also using CRISPR in the lab to model cancers in order to better understand their behavior. Researchers at Stanford University used CRISPR to introduce marked mutations into the lungs of mice, and tracked their growth over time (7). Results from their studies revealed mutations primarily in tumor suppressor genes drive tumor growth in a specific setting, meaning they require the presence of other mutations or genetic events to have an oncogenic effect. These results provide researchers with greater detail on the complexity and genetic crosstalk within lung tumors, and are guiding future studies of cancer development and therapies.
CRISPR has revolutionized cancer research, offering unprecedented precision in gene editing to better understand cancer development and progression. It has significantly contributed to the advancements of CAR-T therapy, where modified immune cells target cancer cells more effectively. This ultimately has increased the demand for best-in-class CRISPR solutions that enable scientists to discover new therapies they can develop and take into the clinic. These breakthroughs hold tremendous potential for personalized treatments and bring us closer to curing the various different types of cancer.
Additionally, the exploration of alternative nucleases, like hfCas12Max and eSpOT-ON (either as protein or mRNA format), expands the toolkit available to scientists as they develop these life-saving treatments. These alternative nucleases could provide more tailored and efficient approaches, paving the way for novel therapies that can target cancer with higher accuracy and fewer side effects, ensuring a broader impact in the fight against this complex disease.
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