Fighting fire with fire, researchers working with dogs have fixed a genetic glitch that causes Duchenne muscular dystrophy (DMD) by further damaging the DNA. The unusual approach, using the genome editor CRISPR, allowed a mutated gene to again make a key muscle protein. The feat—achieved for the first time in a large animal—raises hopes that such genetic surgery could one day prevent or treat this crippling and deadly disease in people. An estimated 300,000 boys around the world are currently affected by DMD.
The study monitored just four dogs for less than 2 months; more animal experiments must be done to show safety and efficacy before human trials can begin. Even so, "I can't help but feel tremendously excited," says Jennifer Doudna of the University of California, Berkeley, who heard the results last week at a CRISPR meeting she helped organize. "This is really an indication of where the field is heading, to deliver gene-edited molecules to the tissues that need them and have a therapeutic benefit. Obviously, we're not there yet, but that's the dream."
The study, which also appears online this week in Science, was led by molecular biologist Eric Olson of the University of Texas (UT) Southwestern Medical Center in Dallas, whose team earlier had similar results in mice. "We wanted to put this to the ultimate test and see if we could do it in a large animal," Olson says. The positive findings—CRISPR quickly restored the protein dystrophin in critical body muscles, including the heart—"brought tears to the eyes and were jaw-dropping," he says.
The study offers little evidence that dogs regained muscle function, however, and that, coupled with the short duration of the study and the small number of animals studied, left some scientists less enthusiastic. One researcher in the tight-knit DMD field who asked not to be named wonders whether the study was rushed to help draw investment in Exonics Therapeutics, a Boston-based company Olson launched last year to develop the potential treatment.
Olson says his team worked quickly not because of corporate ambitions, but rather to prove the concept before expanding to longer, more thorough dog experiments that ultimately are needed to launch human trials. The few animals initially studied, he adds, reflects sensitivities about experimenting with dogs. "We're very mindful of ethical concerns and have done our best to keep our use of dogs to an absolute minimum."
The dystrophin gene, the largest in the human body, contains 79 separate coding regions, or exons, that work together to create a protein that has 3500 amino acids. That much DNA offers a lot of opportunities for mutations that can cause DMD. But only one functional copy of the gene is needed, and because it sits on the X chromosome, girls have a backup copy. Boys with their one copy disabled develop walking problems early in life and die on average in their mid-20s from heart and respiratory failure.
About 13% of boys with DMD have mutations in a region between exon 45 and 50, which bumps exon 51 "out of frame" and throws a wrench into the cellular machinery that reads the gene's instructions, stopping production of dystrophin. In 2009, a team led by Richard Piercy at the Royal Veterinary College in London identified a spaniel with signs of DMD that had a spontaneous mutation deleting exon 50, which similarly moves exon 51 out of frame. They later bred a relative of that dog with beagles, which have long been used in biomedical research, to create a colony with DMD symptoms.
Together with Piercy's group, Olson and colleagues designed CRISPR's molecular scissors to make a cut at the beginning of exon 51 in the diseased beagles. The team hoped that when the cell tried to repair the slice, it would accidentally introduce errors to exon 51, leading its proteinmaking machinery to skip the exon altogether and produce a shortened but still functional dystrophin. (A newly approved drug for DMD, eteplirsen, promotes such exon-skipping as well, but its efficacy remains hotly debated.)
Another challenge was to alter billions of muscle cells throughout a living animal. So the team enlisted a helper: a harmless adeno-associated virus that preferentially infects skeletal muscle and heart tissue. Two 1-month-old dogs received intramuscular injections of the virus, engineered to carry CRISPR's molecular components. Six weeks later, those muscles were making dystrophin again. Those results led the researchers to give an intravenous infusion to two more dogs, also 1 month old, to see whether the CRISPR-carrying viruses could add the genome editor to muscles throughout the body. By 8 weeks, Olson told the meeting, dystrophin levels climbed to relatively high levels in several muscles, reaching 58% of normal in the diaphragm and 92% in the heart. But because the dogs were euthanized, Olson could show little evidence that they had avoided DMD symptoms, save for a dramatic video of a treated dog walking and jumping normally.
"There are a lot of questions that have to be addressed," acknowledges Leonela Amoasii, who works in Olson's lab at UT Southwestern and is director of gene editing at Exonics. Skeletal muscle is constantly being replaced, so the treatment would have to reach its stem cells to avoid the need for repeated injections. Longer studies will be needed to make sure that the CRISPR treatment does not introduce cancer-causing mutations. Even if it safely restores the ability to make dystrophin, the treatment likely will only help boys who receive it early in life because the muscle damage is irreversible. And ultimately the treatment would have to target many other DMD-related mutations to help most boys with the disease. "We have to make sure that we dot all the i's and cross all the t's because the implications for both DMD and CRISPR therapy are immense," Olson says.