CRISPR Technology for DNA Editing Might Raise Cancer Risk, Israeli Scientists Warn

The gene-editing tech is considered a promising method for treating many diseases, but Tel Aviv University researchers say it sometimes destroys genetic material – as happens in cancer

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A researcher performs a CRISPR/Cas9 process at the Max-Delbrueck-Centre for Molecular Medicine in Berlin, Germany.
A researcher performs a CRISPR/Cas9 process at the Max-Delbrueck-Centre for Molecular Medicine in Berlin, Germany.Credit: Gregor Fischer / DPA
Gid'on Lev
Gid'on Lev
Gid'on Lev
Gid'on Lev

It was a groundbreaking development a decade ago: CRISPR gene-editing technology that allows the snipping of DNA to remove undesired sequences or add desired ones. It has since been used to treat a variety of diseases, winning its inventors, Jennifer Doudna and Emmanuelle Charpentier, a Nobel Prize in Chemistry two years ago.

But Tel Aviv University researchers are highlighting the risks of CRISPR, which stands for clustered regularly interspaced short palindromic repeats. The scientists examined how the technology affects the immune system’s white blood cells – T cells – and found that some of the patient's cells had chromosomal truncations – a loss of DNA fragments, a characteristic of cancer.

The first trial approved for using CRISPR to treat people was done by researchers under Prof. Carl June at the University of Pennsylvania. The scientists removed T cells from a healthy donor and changed the so-called cell receptor on them to better identify cancer cells.

The researchers also used CRISPR to destroy the original cell receptor so that the engineered T cells wouldn’t recognize and mistakenly attack cells of the recipient and another molecule that cancer cells use to exhaust T cells. The engineered cells were injected into cancer patients whose cancers did not respond to any other treatment.

The results were published in 2020 in the journal Science: The engineered cells survived in the patient’s body for a long period and homed in on the cancer cells, even though they didn't destroy the growth entirely.

The general consensus in the field was that after CRISPR's excising of undesired parts of DNA, the cell carries out repair. In the new study, the researchers conducted a test to determine if indeed this repair mechanism works perfectly or that maybe repair doesn't always occur, and when it does, it’s not always complete.

From left: Dr. Uri Ben-David, Dr. Adi Barzel and Dr. Asaf madiCredit: Tel Aviv University

To examine the technology that presents this risk, the scientists reconstructed the trial conducted at the University of Pennsylvania. They used CRISPR to cut the genome of T cells in exactly the same places where June and his colleagues did: at chromosomes 2, 7 and 14. (Each human cells has 23 pairs of chromosomes.)

They then analyzed thousands of cells and found that up to 10 percent of the chromosomes that were cut did not repair themselves.

The study was conducted under the leadership of Tel Aviv University's Dr. Adi Barzel, in conjunction with the city's Sourasky Medical Center and Tel Aviv University's Dr. Asaf Madi and Dr. Uri Ben-David. The findings were published in the journal Nature Biotechnology.

As Madi told Haaretz, T cells play a vital role in activating the immune system and killing cancer cells. “These cells have to identify cancer cells, and they do so via specific receptors on the T cells. In addition, they have molecules on them called co-inhibitory receptors that act like brakes on the T cells,” he says.

CRISPR cuts and removes the DNA sequence at desired points. The natural mechanism of DNA repair in a cell is what's fusing the cuts together and keeping the chromosome intact, but sometimes the cell fails to execute the repair, and large parts of the chromosome are lost. That creates a very serious situation.

Dr. Uri Ben-David

“Normally, these molecules prevent an over-activation of these cells, which can result in autoimmune situations where the T cells attack body tissue. Cancer exploits this natural mechanism and sends signals to these co-inhibitory receptors. In doing so, it prevents the immune system's cells from harming it.”

With CRISPR, T cells can be made to better recognize cancer cells and prevent the recognition of normal cells. Furthermore, CRISPR can be used to remove the molecules that act as brakes on T cells, allowing the cells to exert their full killing potential.

Following the use of CRISPR, a mechanism in the cell repairs the cut DNA, but sometimes the cell fails to be repaired and might even lose large parts of the chromosome. This is serious because of the association with diseases including cancer.

In reenacting the research at the University of Pennsylvania, the Tel Aviv University scientists – aided by the students Alessio Nahmad and Ella Goldschmidt, and research assistant Eli Reuveni – sought to investigate CRISPR’s safety in general, not just in treating cancer.

“CRISPR only cuts and removes the DNA sequence at desired points. The natural mechanism of DNA repair in a cell is what's fusing the cuts together and keeping the chromosome intact,” Ben-David says.

Jennifer Doudna and Emmanuelle Charpentier, won a Nobel Prize in Chemistry two years ago.Credit: Alexander Heinl / AP

“But sometimes the cell fails to execute the repair, and after this failure large parts of the chromosome – or even the entire chromosome – are lost. That creates a very serious situation because of aneuploidy – a change in the number of chromosomes.”

Ben-David says aneuploidy occurs in 90 percent of solid tumors; it's the most frequent genetic change in cancer, more so than DNA mutations.

“In healthy cells, it never happens. There are always 46 chromosomes,” he says. “If in the process of genome editing via CRISPR, aneuploidy cells are generated and injected into the patient, this could be a serious problem. Until now, this problem hadn't been examined in depth.”

The researchers found that some of the cells where CRISPR was used suffered a significant loss of genetic material. When the three chromosomes were cut at the same time, 9 percent of the cells in chromosome 14, 10 percent in chromosome 7 and 3 percent in chromosome 2 did not repair the damage.

These differences stem from the location of the gene that was cut from the chromosome. When the gene is closer to the middle of the chromosome (a region called the centromere), the cell’s failure to fuse the cut sequence will result in the loss of the entire part beyond the point of the cut.

In chromosome 14, the splitting is near the centromere; in chromosome 2 the splitting is closer to the end of the chromosome, so when there's a failure to fuse, only a small portion of the chromosome is cleaved, containing few genes.

The researchers thus sought alternate ways to consider their hypothesis.

In particular, they examined – via advanced RNA sequencing at the cell level – whether several of the genes on the chromosome that had been cut were able to generate RNA as a proxy of their intact DNA state. On the chromosome, the researchers found numerous cells lacking such RNA expression from the point of the cut onward.

The scientists concluded that in these cells the entire chromosome section was lost. “We used three different methods to validate the significance of our findings in this controversial hypothesis,” Barzel says.

As Ayal Hendel of Bar-Ilan University near Tel Aviv puts it, “When you get into clinical trials on people, you want to evaluate the accuracy of the genetic editing methods in the best way possible, and the article presents an important new finding.”

He says CRISPR technology is constantly improving. “When you conduct a trial on people there's always a risk-benefit ratio. Thanks to the article by these three researchers, we have more subtle tools for appraising the risk,” Hendel says.

He says dozens of clinical trials using CRISPR are already being conducted. The U.S. Food and Drug Administration is expected to approve the first CRISPR treatments next year.

Illustration of scientists analyzing DNA helix and editing genome within organisms using CRISPR technology.Credit: elenabsl/Shutterstock
Illustration of scientists analyzing DNA helix and editing genome within organisms using CRISPR technology.Credit: Shutterstock

Still, the Tel Aviv University scientists recommend caution in using the technology; they're proposing less-dangerous methods for certain procedures. Similarly, they recommend further research for cases where CRISPR is the only option.

“There's a consensus that you shouldn't inject aneuploidic cells into people. Now we have to see how we can reduce the risk of injecting these sorts of cells,” Ben-David says.

“For instance, once the cells are manipulated using CRISPR, the damaged ones should be sorted out, and only those that have undergone complete fusion should be injected to patients. There are steps that can be taken to reduce the risk.”

Barzel adds: “The findings show that this technology can be dangerous, but there are situations where there is no choice but to use it. When it's used it has to be done in ways that reduce the risk.

“For example, there are CRISPR-based methods where the manipulation of a DNA segment is more controlled. When possible, I recommend this. Still, there are medical applications where there is no choice but to use the current approach – for example, when you want to insert a gene into the genome.”

Barzel says he set up a company that uses cleavings in chromosomes. “I'm playing on both sides of the field – using technology and also acknowledging its dangers and shining a light on them,” he says.

“It looks like there's a contradiction here, but that's science: We're not choosing a side but rather taking an issue and scrutinizing it from all sides, positive and negative, and seeking answers.”

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