An international team of researchers has successfully treated dogs with the canine form of Duchenne muscular dystrophy (DMD), a rapidly progressing and ultimately fatal muscle disease that afflicts one out of every 3,600 boys. The researchers used a novel technique called exon skipping to restore partial function to the gene involved in Duchenne. The study, published in Annals of Neurology, gives hope that a similar approach could work in humans.
DMD is caused by an aberration in the gene that encodes dystrophin, an important structural protein in muscle cells. Patients with DMD are unable to produce functional dystrophin, which leads to holes in the outer membranes of their muscle cells. Eventually, their muscles degenerate faster than they can be rebuilt, and few patients survive beyond their early 30s.
Unlike traditional gene therapy, which attempts to replace a mutated gene with a functional copy, exon skipping relies on a variation of a technique called antisense, in which short synthetic DNA or RNA molecules are designed to bind to a region of DNA or RNA and block its function. Companies are developing antisense therapies for cancer, diabetes, heart disease, and autoimmune diseases, among others.
The approach grew out of studies comparing DMD to a milder form of disease called Becker muscular dystrophy (BMD). Both diseases arise when patients are missing portions of the dystrophin gene’s exons, the areas of DNA that code for protein. Paradoxically, some BMD patients are missing much larger pieces of the gene yet are far healthier than patients with DMD. Several years ago, scientists found that the difference is not in how much of the gene is missing, but in how those missing portions affect the remaining gene sequence. Most Duchenne patients have frameshift mutations, which interfere with the cell’s reading of three-letter DNA code. These deletions shift the remaining DNA sequence into different triplet groupings, rendering the gene unreadable. In Becker patients, the remaining DNA can still be read normally, allowing them to produce a smaller but still functional version of dystrophin.
Eric Hoffman, a lead author of the study at Children’s National Medical Center, in Washington, DC, says that scientists realized they might help DMD patients by creating a “patch” that blocks transcription of a portion of the gene in a way that puts the remaining code back into sequence–essentially recreating the milder Becker muscular dystrophy.
Hoffman’s team worked with Shin’ichi Takeda at the National Center of Neurology and Psychiatry, in Japan, and gave three beagles with a naturally occurring canine form of DMD weekly or biweekly intravenous injections of a cocktail of three different antisense molecules. After several weeks of treatment, the dogs showed noticeable improvements in muscle function tests and symptoms, and their cells produced dystrophin at an average of 26 percent of normal levels. Hoffman says that these levels are similar to those found in human patients with BMD, and “the clinical improvement was about what you’d expect based on what we know about human patients.”
A similar approach was tested in rodents and in four humans in 2007, but only as a local injection into muscle cells. This study used a newer type of antisense molecule called a morpholino that binds to DNA but isn’t recognized as foreign DNA and degraded in the body, making it possible to deliver the treatment intravenously. Hoffman says this is the first time that researchers have successfully delivered an antisense therapy systemically to alleviate DMD in a larger animal.
“I’m really encouraged by this,” says Richard Moxley, a neurologist at the University of Rochester, adding that the study provides important evidence in favor of the idea of shifting the clinical severity of DMD to mimic a lesser disease. Moxley says that the ability to deliver the treatment in a systemic injection is also important for its practical use. However, he says that several questions remain about how well the treatment will translate into humans. The antisense patches did not always work in the dogs as was predicted by studies in individual cells, suggesting that there are still unknown details about how they interfere with transcription.
In addition, because different Duchenne patients are missing different pieces of the dystrophin gene, many would require a combination of different antisense molecules to see benefits similar to those seen in the dogs. Hoffman says that this complication will pose a regulatory challenge and present a test for developing a personalized approach to medicine. He believes that an initial drug could at least target the most common mutation and help about 15 percent of DMD patients.