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A new paper points to a previously unknown hurdle for scientists racing to develop therapies using the revolutionary genome-editing tool CRISPR-Cas9: the human immune system.

In a study posted Friday on the preprint site bioRxiv, researchers reported that many people have existing immune proteins and cells primed to target the Cas9 proteins included in CRISPR complexes. That means those patients might be immune to CRISPR-based therapies or vulnerable to dangerous side effects — the latter being especially concerning as CRISPR treatments move closer to clinical trials.

But researchers not involved with the study said its findings, if substantiated, could be worked around. (Papers are posted to bioRxiv before being peer-reviewed.) Many of the first planned CRISPR clinical trials, for example, involve removing cells from patients, fixing their DNA, and then returning them to patients. In that case, it’s possible that there will be few or no CRISPR proteins remaining for the immune system to detect.


They also noted that scientists are already studying other types of CRISPR that use different proteins, which could stave off the immune responses.

“At the end of the day, I’m not that concerned about it,” said Daniel Anderson of the Massachusetts Institute of Technology, who has studied the delivery of CRISPR therapies and who was not involved with the new study. “But we want to do some experiments to learn more.”


The new study should not put the brakes on developing CRISPR therapies, agreed Dr. Matthew Porteus of Stanford, a senior author of the paper and who is himself at work on a CRISPR-based therapy for sickle cell disease. But he said he and his colleagues investigated the immune issues because he felt they were being overlooked as the excitement around CRISPR grew.

“Like any new technology, you want to identify potential problems and engineer solutions for them,” Porteus said. “And I think that’s where we’re at. This is an issue that should be addressed.”

(Porteus and Anderson are both scientific founders of CRISPR Therapeutics, one of the most prominent companies exploring CRISPR-based therapies.)

Immune survey

CRISPR has gained fame in recent years as researchers have deployed it to correct an array of disease-causing mutations in cells in the lab and in animal models, with hopes that the same results can be achieved in people. There are different types of CRISPR systems, but the most well known is dubbed CRISPR-Cas9; it includes Cas9 proteins that cut DNA so that it can be edited. Cas9 proteins come from bacteria.

For the study, the researchers decided to check for immune signals against two of the most common types of Cas9 proteins used, those from the bacteria S. aureus (called SaCas9) and those from S. pyogenes (called SpCas9). In their samples of blood from 22 newborns and 12 adults, the scientists found that 79 percent of donors had immune proteins, called antibodies, against SaCas9, and 65 percent had antibodies against SpCas9.

The researchers then searched for immune cells called T cells. They discovered that about half of the donors had T cells that specifically targeted SaCas9, so that if the immune cells detected that protein on the surface of a cell, they would rally a response to try to destroy it. The researchers did not find anti-SpCas9 T cells, though they said the cells might still have been present.

It’s not surprising so many of the donors had antibodies and T cells against the Cas9 proteins, experts said. That simply means that those people had been exposed to the bacteria containing the proteins in the past, and other studies have found that, at any given time, 40 percent of people are “colonized” by S. aureus and 20 percent of schoolchildren have S. pyogenes. The bacteria only sometimes cause disease.

But what then does that previous exposure mean for our receptiveness to CRISPR therapies?

A lot remains unclear, Porteus said. It’s not known how severe the immune response would be, and whether it would trigger a dangerous inflammatory attack or just render the treatment useless.

Experts also said that perhaps the immune responses could be avoided. If the CRISPR complex does its editing after the cells are removed from the patient — what’s called ex vivo — or in a place like the eye that is isolated from the immune system, then the antibodies and T cells might not detect any Cas9 proteins. Even in in vivo therapies — in which CRISPR complexes would be ferried into cells in a patient’s body — much depends on what kind of delivery system is used and whether the Cas9 proteins become expressed on the outside of the cells in which the editing is taking place.

Porteus said he and his team decided to post the paper on bioRxiv because they wanted CRISPR researchers to start thinking now about possible immune system challenges. The team has also submitted the paper to a journal for peer review and publication.

Possible workarounds

As a cautionary tale about the importance of asking these questions now, Porteus pointed to what happened with gene therapy in 1999. In that case, a patient in a trial died after an immune system attack, likely because he had preexisting antibodies against a virus used as part of the therapy. The death led to years lost in gene therapy development, experts say. (Patients who have preexisting antibodies to viruses used in gene therapies are now generally excluded from trials.)

“I would hate to see the field have a major setback because we didn’t address this potential issue,” Porteus said. “We should learn from that.”

Roland Herzog, a gene therapy expert at the University of Florida, agreed that the hype around CRISPR meant that possible immune issues were not being given enough credence.

“I suspect that the field has not been aware of it sufficiently,” he said. “It’s not a show stopper,” he added about the paper, “but the field needs to know about this, that it’s a potential problem that they need to work around or fix.”

One possible fix is simply using a different protein — or enzyme — in the CRISPR complex, one that doesn’t come from such common bacteria. If people haven’t been exposed to the bacterial protein previously, then they won’t have specific antibodies or T cells ready to attack.

“New Cas editing enzymes are being described all the time from bacterial species that are not human pathogens (and so there would be no chance to develop the pre-existing antibodies),” Jacob Corn, of the University of California, Berkeley, who was not involved with the new paper, wrote in an email. “I also know some people have already been working on making Cas enzymes that would be invisible to the immune system.”

He added: “The field moves very fast!”

CRISPR is a powerful gene-editing tool with transformative potential. Feng Zhang, a scientist at the Broad Institute, explains how it works. Dom Smith, Matthew Orr, Hyacinth Empinado/STAT

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