DMD is a muscle wasting disease caused by mutations in the dystrophin gene, which is involved in muscle development. It is a progressive disease that usually causes death in early adulthood, with serious complications that include heart or respiratory-related problems. In humans, it mostly affects boys, about 1 in ever 3,500 or 5,000 male children. In September 2016, the U.S. Food and Drug Administration (FDA) approved Sarepta Therapeutics’ Exondys 51 to treat DMD after a lengthy and dramatic approval process.
Exondys 51 works with a technique called exon skipping. An exon is part of a gene that codes for a protein. Throughout the exons are introns, which are sometimes called “junk DNA.” During the process of protein production, introns are cut out and discarded. The dystrophin gene is the largest gene in humans, with 79 exons. In DMD, an exon or exons are deleted, which interferes with the production of the dystrophin gene.
Exon skipping causes the machinery in the cell involved in protein production to “skip over” an exon. This allows the cells to produce a functional, although incomplete, dystrophin gene.
Because of its size, the dystrophin gene is not generally viewed as a candidate for “traditional” gene therapy, because the entire gene is too large for the viral vectors usually involved in gene therapy.
About a decade ago, veterinarians in the U.K. at the Royal Veterinary College discovered a family of King Charles Spaniels whose male puppies sometimes developed a form of DMD. They began breeding these dogs with beagles to form a «canine colony» that they hoped would someday lead to a cure.
What the team at UT Southwestern did, led by Eric Olson, was use CRISPR gene editing technology to modify the DNA of four puppies, which reverse the molecular defect that caused DMD.
Wired writes, “DMD isn’t an obvious candidate for CRISPR’s find-and-replace function; the dystrophin gene is the largest in the human genome, and there are thousands of different mutations that can all result in the disease. But Olson found a way to target an error-prone hot spot on exon 51, which he figured could, with a single slice, benefit approximately 13 percent of DMD patients.”
Olson and his group had successfully performed CRISPR work in mice and human heart cells. They worked with the Royal Veterinary College and its beagles to see if the technique would work in larger mammals. Wired describes, “The researchers first packed the instructions for the CRISPR gene-editing components into a virus with an affinity for muscle cells. Then they injected millions of copies of that virus into four one-month-old dogs—two got the shot directly in the lower leg, and two received an intravenous infusion. After eight weeks, CRISPR had restored dystrophin levels in the second group to more than 50 percent of normal in the legs, and more than 90 percent in the heart.”
Although a fair distance from being tested in humans, it does establish a proof-of-concept for single-cut gene editing in dystrophic muscle. Olson stated, “Children with DMD often die either because their heart loses the strength to pump, or their diaphragm becomes too weak to breathe. This encouraging level of dystrophin expression would hopefully prevent that from happening.”
The laboratory now plans to conduct longer-term studies to evaluate whether the dystrophin levels are stable and to evaluate any potential side effects. Potential side effects and whether the changes are permanent are a major controversy in CRISPR use in humans, with the jury still out.
Olson is the founder and chief scientific adviser of a biotech company, Exonics Therapeutics, which licensed the technology from UT Southwestern. Exonics assisted in funding the dog study.
On a research level, Olson’s team found that the technique had restored about 50 percent of the normal levels of dystrophin in the legs and more than 90 percent in the heart. Generally, DMD experts estimate that a 15 percent restoration of normal levels, in people, at least, would have a significant, even possibly curative result. Olson and his group were looking out for the possible worst side effects, such as anaphylaxis, liver toxicity, and inflammation, but they saw no adverse effects.
Because it didn’t fall under the typical scientific language and parameters of the study, this didn’t go into the published paper, but Olson told Wired, “They showed obvious signs of behavioral improvement—running, jumping—it was quite dramatic.”