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Illustration of a laptop with gene sequencing on the screen.
Illustration by Jorn Kaspuhl

The Gene Editors

U of T scientists are using a powerful new technology to alter DNA and possibly cure disease

Fourteen-year-old Gavriel Rosenfeld of London, England, thinks it’s “freaky” that cells from his body are being studied in a U of T lab. His parents, however, are thrilled that their son’s cells are the focus of intensive, cutting-edge research that could make a huge difference in the life of Gavriel and the lives of thousands of children and youth around the world.

Gavriel has Duchenne muscular dystrophy, one of the biggest genetic killers of boys worldwide. Gavriel inherited a mutation in his genes that causes his muscles to progressively weaken. Two years ago he lost the ability to walk. Soon he will no longer be able to feed himself, roll over at night or hug his parents.

But U of T researchers, using a new gene-editing technology called CRISPR/Cas9 on Gavriel’s cells in a lab dish, succeeded in eliminating the mutation that causes a duplication in a portion of his DNA. They are now on the verge of repeating the success in animal studies. A treatment for Gavriel can’t come quickly enough for his mother, Kerry, who with her husband runs a charity to find and fund the best research on the disease. Excited by CRISPR/Cas9’s promise, she says, “We are at a critical point where we realistically have the opportunity to save this generation of boys.”

CRISPR/Cas9, or CRISPR for short, isn’t a trade name. It’s an acronym for – here it comes – clustered regularly interspaced short palindromic repeats, the particular, naturally occurring DNA pattern on which the system is based. Essentially, CRISPR refers to a technique that allows scientists to quickly, precisely and inexpensively edit the genes in the cells of any living organism, whether a microbe, a plant, an animal or a human. (See: “How CRISPR Works.”)

The implications are enormous. CRISPR has the potential to change the way we treat genetic diseases such as muscular dystrophy or Huntington’s disease, help us better understand cancer and make more efficient drugs. It could also help us create better biofuels, gradually wipe out the mosquitoes that carry malaria or the Zika virus, and raise healthier livestock and better crops. CRISPR/Cas9-engineered foods, likely available in the near future, might include allergen-free peanuts, drought-resistant grains and higher-yielding plants.

Gene editing in itself isn’t a new technique, but previous methods, which were restricted to highly specialized labs, were cumbersome and time-consuming – somewhat like having a painfully slow computer that you needed to rewire for every new task. By contrast, CRISPR is more like easily programmable software. Editing a gene can now be as straightforward and accurate as using the find-and-replace function in a word processing program to zero in on a specific phrase in a document, then delete it, tweak it or replace it.

Developed as a gene-editing tool only four years ago, CRISPR has rapidly made its way into labs worldwide. Researchers have called CRISPR revolutionary, jaw-dropping, a game changer, and while scientists are famously reluctant to use the word “breakthrough,” in 2015 the prestigious journal Science boldly named CRISPR the Breakthrough of the Year. Scientists have already reversed mutations that cause blindness and killed HIV in cells taken from an infected patient. They have also altered pig genes to potentially grow organs for transplanting into humans and turned off the genes in five cancer cell lines. So far, much of the research is happening only in mice, monkeys and lab dishes, but advances are coming quickly. Creating lab mice with specific genetic profiles used to take 18 months; with CRISPR, it takes four or five, which accelerates the pace of research exponentially (and saves many animals). Clinical trials on humans are likely to start in the U.S. next year, with therapies only a few years away. (See: “The Ethics of Gene Editing”)

“I have been mesmerized by this technology ever since I first read about it,” says Ronald Cohn, professor and chair of U of T’s department of pediatrics, and the lead researcher in the groundbreaking study using Gavriel’s cells. “CRISPR has already revolutionized how we do science, and it could revolutionize how we practice medicine.”

Treating Duchenne muscular dystrophy

Of all the types of muscular dystrophy, Duchenne is the most serious. It causes immobility at an early age and, as the heart and lung muscles weaken, leads to premature death. But Cohn, a clinician-scientist who next month will become the chief of pediatrics at the Hospital for Sick Children in Toronto, believes gene-editing therapies for some Duchenne patients could be only three to five years away. Last fall, using CRISPR on Gavriel’s own cells in a lab dish, Cohn’s team succeeded in cutting out the mutation and restoring the normal gene function, complete with normal dystrophin, the essential protein that Duchenne patients lack.

Cohn’s team has since repeated the success using the cells of several patients. He’s now working on recreating the duplication in mice – “we’re almost there,” he says – after which drug therapies will be investigated. Cohn says it’s neither possible nor necessary to fix 100 per cent of the cells in order to make a patient significantly better. “In fact, the threshold of correcting muscle cells in a patient with Duchenne muscular dystrophy might be as low as 10 to 15 per cent,” he says. He predicts that even such a small change could sufficiently delay a patient’s muscle weakening so he would not lose mobility until much later in life, if ever.

The technical challenges are small, the regulatory ones somewhat larger, Cohn says. Since patients with genetic diseases often have very individual mutations, the usual one-size-fits-all regulatory framework involving clinical trials on thousands of patients won’t be possible. “A lot of these therapies will be incredibly individual, so we’ll have to change the paradigm on the regulatory side,” he says. He has already begun high-level conversations with Health Canada and hopes to do the same soon with the U.S. Food and Drug Administration.

To encourage pharmaceutical companies to develop individualized drugs, industries will need incentives to make a drug that may be administered only once to a small number of patients. Cohn has already talked with two drug companies who recognize this may be the future of genetic medicine and who are eager to pursue it. They believe the current model of drug development is unsustainable. Developing a new medication today costs at least $2 billion, and many drugs never make it to market because clinical trials can’t recruit enough volunteers, or the drug doesn’t work in large enough numbers. Genetics-based precision medicine could better pinpoint which patients are likely to respond to a certain treatment, which means clinical trials could be shorter and smaller. “It’s going to require all of us to think differently about how we can make this happen for patients,” says Cohn. “I have no doubt that we will.”

Meanwhile, he’s racing against the clock to help Gavriel and many other patients with genetic diseases. “There’s so much excitement among the people in my laboratory because we feel like we can really make a difference.”

A new way to understand cancer

The mapping of the human genome in 2003 showed us there are about 20,000 genes in a human cell. What it didn’t show us is which genes are essential for a normal cell to grow and divide. Or which genes are essential to make a cancer cell grow and divide. Knowing this could help us understand which genes to target with specific drugs in order to kill only the cancer cells, while leaving healthy tissue intact.

One of the best ways for researchers to learn how genes function is to switch them off and watch what happens. Last fall, a team led by Jason Moffat, professor of molecular genetics at the Donnelly Centre for Cellular and Biomolecular Research, did exactly that. Remarkably, in a single experiment, Moffat and his team used CRISPR to switch off, one by one, almost 90 per cent of the entire human genome, or more than 17,000 genes. Of these, they discovered that only about 10 per cent are essential for the survival of a cell; the rest are presumed to have more minor functions.

Focusing on cancer cells, Moffat’s team was able to switch off the genes in five different cancer lines, including brain, ovarian, retinal and two types of colorectal cancer. “The big question is, will CRISPR actually solve cancer?” asks Moffat. “And the answer is, it might help us come to a very detailed understanding of cancer.” Some blood cancers with very defined, consistent mutations in the genome might be fixable through direct CRISPR gene editing, he says. Other cancers are trickier.

“Most cancers have their genomes completely messed up, with each cell having a very different genome,” explains Moffat, who is also Canada Research Chair in the functional genomics of cancer. But while the genomes are very diverse, the proteins produced by those genomes are often more consistent. “So instead of targeting each genome, we’re trying to figure out how to target those proteins.” Attacking those targets may involve either new or existing drugs. For example, Moffat and his team found that metformin, a diabetes drug now in clinical trials for cancer, was effective against brain cancer cells and one type of colorectal cancer cell but useless against other cancers. They also found that two antibiotics – chloramphenicol and linezolid – killed another type of colorectal cancer cell. His lab has recently developed new biologicals – drugs based on natural sources such as cells – to target pancreatic cancer. Moffat is working with several start-ups to move drug development forward.

“CRISPR’s not going to cure cancer,” Moffat says, “but we’re pretty excited by CRISPR’s potential to give us a much faster way to get a comprehensive picture of what’s going on in many different cancers.”

Potential tool against superbugs

When we think of antibiotics, we generally picture a drug that can kill bacteria. But that’s not the only kind of antibiotic.

Equally deadly to bacteria is a bacteriophage – literally, “bacteria eater.” Bacteriophages, or phages for short, are viruses that can hijack a bacterium’s DNA and take over. These phages, the most common organisms on Earth, have been called natural antibiotics.

In the ongoing war between bacteria and viruses, bacteria have evolved their own CRISPR defense system to protect against invading phages. But Alan Davidson, a professor in the departments of molecular genetics and biochemistry, found that phages are fighting back. In 2012, Davidson’s lab was the first in the world to identify anti-CRISPRs: proteins that can bypass a bacterium’s defenses. “We discovered these genes that turn off the CRISPR system,” Davidson says. “No one had ever seen such things before, so that was a very exciting discovery.”

Why all the interest in anti-CRISPRs? With antibiotic-resistant diseases on the rise, phages that kill bacteria could potentially be used in place of conventional antibiotics. Davidson is particularly interested in phage therapy to treat lung infections in people with cystic fibrosis, the most common fatal genetic disease among children and youth in Canada. Patients are vulnerable to deadly infections caused by Pseudomonas aeruginosa, a ubiquitous environmental bacterium that is increasingly resistant to antibiotics. While phage therapy has been used in parts of Eastern Europe, Davidson says rigorous studies have yet to be done. He thinks they should be. Using a virus to fight a bacterial infection comes with its own risks – what if the phage, which can carry virulent genes, makes the patient worse? But Davidson says studies in mice are very encouraging.

Since it’s becoming increasingly difficult to find antibiotics that work against highly resistant bacteria, Davidson says, “It’s very important to have these alternative approaches at the ready when all other approaches have been exhausted. In the longer term we’ll get better at developing these things, and they may take over as the main treatment.”

So far, several phage-based therapies have been approved for use in food in the U.S., including a spray formulation to kill listeria, a food-borne bacterium, on deli meats.

Davidson says he didn’t set out to work on CRISPR at all, until his basic research led him to the surprising discovery of anti-CRISPRs. “It’s fantastic how in basic research you can start working on something just because it’s interesting and cool – not research tied to a particular problem – and you can discover something very unexpected that may have exciting implications for science.”

Marcia Kaye (, of Aurora, Ontario, is an award-winning writer.

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