If Chengzu Long hadn’t been quite so unlucky, he might never have attempted to study and treat Duchenne muscular dystrophy. As a PhD student in Eric Olson’s lab at the University of Texas Southwestern Medical Center, Long had spent years knocking out genes in mice to try to identify their role in muscle development and disease, only to find that each of the resulting knockouts had no discernible differences from wildtype individuals.
In the fall of 2013, with only about a year left until his planned graduation, Long decided to take a different approach: rather than generate yet another knockout mouse that might again lack a phenotype, he would use the new CRISPR-Cas9 gene-editing technique to correct a disease-causing mutation, such as those that cause muscle wasting in Duchenne muscular dystrophy. “I thought to myself, I’m really good at making mice without a phenotype, so maybe we can use CRISPR to cure an existing mutation,” says Long, now an assistant professor at New York University.
Like many muscle disorders, or myopathies, Duchenne is caused by mutations in a single gene—the X chromosome’s DMD gene, which encodes the protein dystrophin. Without a functional dystrophin protein, Duchenne patients gradually lose their mobility as their muscles degenerate. Most die by their early 30s from breathing complications or heart failure. “Duchenne is devastating,” says Olson, whose lab has worked on muscle development and disease for 30 years. “Right now there [are] 300,000 boys in the world with Duchenne, so it’s a large patient population, and there’s a desperation for a really transformative therapy.”
Because the genetics of the disease are well understood, researchers could theoretically replace the mutated version of DMD with a healthy copy to cure the disease. Unfortunately, the gene for dystrophin is massive, with 2.6 million base pairs. As a result, it’s not feasible to insert the entire gene, or even just the 11,000 coding base pairs (introns excluded), into a viral vector that could deliver the therapeutic package to the muscle. “Gene editing therefore was a great opportunity to correct the endogenous gene rather than trying to deliver” a nonmutated version of it, says Charles Gersbach, a biomedical engineer at Duke University.
By the end of 2013, multiple labs had successfully used gene editing to rescue the dystrophin protein in vitro, using cells from patients. So Long decided to try his luck at using CRISPR-Cas9 to edit the dystrophin gene in vivo. He injected the CRISPR system into the zygotes of mdx mice, which carry a single mutation in the gene for dystrophin. He then implanted the zygotes into female mice, and confirmed in their 10-day-old progeny that CRISPR-Cas9 had successfully corrected the mutation that causes Duchenne symptoms. When the corrected mice were a month old, Long tested their muscle function and found that it had also improved compared with mice carrying the uncorrected mutation. “We published one of the first in vivo rescues of phenotype [using CRISPR] in an animal model,” he says.