CRISPR gene-editing has been used to improve muscle function in dogs with a Duchenne muscular dystrophy (DMD)-like condition – and eventually the technique might lead to a treatment for humans too. The study represents the first use of CRISPR gene-editing in a living large animal.
People with DMD have a genetic mutation that makes them unable to produce dystrophin, a protein that maintains muscle structure and function. The condition can result in heart or lung failure. In 2010, Richard Piercy at the Royal Veterinary College in London, and his colleagues identified the same mutation in a Cavalier King Charles Spaniel that was brought into the veterinary hospital showing muscle weakness.
“It was right in the middle of the spot where we see it in humans. That’s when we realised it was potentially important for developing treatments,” he says. They found relatives of that dog and bred them with beagles, three of which have now been used to test a potential treatment for DMD.
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Today there is only one approved DMD treatment to elevate dystrophin levels, and in clinical trials it had very modest positive effects: people taking the drug could produce dystrophin but only at about 0.4 per cent the levels seen in healthy individuals. The new potential DMD treatment has been able to restore dystrophin expression up to 92 per cent in the beagles’ heart tissue, 58 per cent in the diaphragm, and 64 per cent in the biceps.
CRISPR test
This method relies on CRISPR Cas9, a genome editing enzyme that cuts a cell’s DNA in a specific spot, guided there by RNA that acts as a kind of GPS honing signal.
“When we first read about CRISPR, we decided to put it to the test in the most challenging of all muscle diseases. It worked in cells from patients, then we tested it in mice, and then we asked if it could work in a larger animal,” says Eric Olson at the University of Texas Southwestern Medical Center. “The results are dramatic, but again this is in a dog, not a person.”
It’s important to use dogs for this kind of work, Piercy says, because unlike mice, they can demonstrate physical signs of the disease – and signs of improvement if the treatment works.
The team injected a virus carrying CRISPR and its RNA molecular guide into the skeletal muscle and heart tissue of two of the one-month old beagles with the DMD mutation – the first time, says Olson, that CRISPR has been used in live animals of such a large size. Six weeks later, they analysed the dogs’ muscles and sequenced their genome and compared them to one dog with the same mutation, and one healthy dog.
Jaw-dropping
“We looked through the microscope and it was jaw-dropping,” says Olson. “Virtually all of the muscle fibres showed high levels of dystrophin underlying every membrane. We were exuberant.”
The dystrophin levels they measured may not be from all cells in the muscle fibres, so there could still be fibre loss and muscle weakening, says Melissa Spencer at the University of California, Los Angeles. “If you have a muscle cell with 10 nuclei and you only correct two of the ten, eventually you probably will lose that fibre,” she says. Because of that, it may still be necessary to administer follow-up doses, which this method doesn’t allow.
Spencer says the results are a great first step towards a treatment, but she cautions that they require more safety testing. “The Cas9 protein expresses for a long time, so the immune system could attack it. We need a better assessment of the immune response, and we also need to be sure CRISPR isn’t cutting off-target,” Spencer says.
Recently, concerns have been raised that CRISPR does indeed cut DNA in places it isn’t supposed to. Olson says they didn’t see evidence that this was happening in the dogs, and blood tests were normal after the treatment. Piercy adds that he hopes the treatment could be applicable to dogs, as well.
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