Mouse Studies Bode Well for Exon Skipping in DMD

MDA grantees Qi Long Lu at Carolinas Medical Center in Charlotte, N.C., and Stephen Wilton at the University of Western Australia in Perth, were on a research team that recently achieved encouraging results with a strategy known as exon skipping to repair the mutated dystrophin gene in mice with a disease resembling Duchenne muscular dystrophy (DMD).

The investigators, who announced their results in the February issue of Nature Medicine, achieved widespread production of the dystrophin protein, which is absent in DMD-affected muscles, and improvement in muscle function. Without using any viruses, they injected a compound called a morpholino antisense oligonucleotide (AON) into a vein.

The morpholino AON causes cells to remove (skip) the mutation in the dystrophin gene and to splice together the surrounding genetic instructions. Although the type of mutation present in the dystrophin-deficient mice is a “premature stop” mutation, other types of mutations seen in DMD patients can also be targeted using exon skipping for correction.

AONs act at the RNA stage of protein synthesis. RNA is the genetic message made from DNA. This message undergoes processing from a rough draft stage to a final set of instructions for protein synthesis. The processing normally removes some sections, known as introns, and keeps others, known as exons. Therapeutic AONs cause cells to treat mutated exons as if they were introns.

The investigators say three consecutive intravenous injections of a specific AON resulted in dystrophin production that was about 30 percent of normal levels in the muscles of the abdomen, upper legs and rib areas of the mice. Lower levels were present in the front lower leg muscles and diaphragm, and no dystrophin was detected in the heart.

After seven weekly injections, the mice produced up to 50 percent of the normal level of dystrophin in some leg muscles, and 10 percent to 20 percent of the normal level in their rib, abdominal and other leg muscles. Unfortunately, dystrophin remained absent in the heart.

The dystrophin-producing muscle cells showed restoration of a normal protein cluster at the cell membrane and other signs of cellular normalization. When lower leg muscles were tested, they showed greater force generation than did the same muscles in untreated dystrophin-deficient mice, although it wasn’t as great as that of normal mice.

“Our results show that morpholino-mediated antisense therapy can achieve and maintain therapeutic levels of dystrophin throughout the body musculature and provide a realistic option for the treatment of the majority of DMD,” the authors say in their publication.

Asked how the majority of DMD could be treated using AONs, Qi Lu said, “This strategy will be effective for boys with DMD mutations that occur in the functionally noncritical region of the dystrophin gene. This is the situation for the majority of DMD-affected boys. By analyzing the structure of the dystrophin gene and the types of mutations, it has been estimated that no more than 20 antisense oligos are required to treat the majority of the patients in whom this would be applicable.”

Lu cautioned that much still needs to be done to improve the efficiency of the dystrophin production, especially in the heart muscle, and that the potential of the new AON strategy for DMD remains to be tested in clinical trials. Trials in DMD patients are now on the drawing board in the United Kingdom. (See www.clinicaltrials.gov, enter “Duchenne muscular dystrophy” into the search box, and select the antisense oligonucletides trial.)