ARTICLE IN BRIEF
In a dog model of Duchenne muscular dystrophy, systemic delivery of an antisense therapy restored functional dystrophin to muscle membranes throughout the dogs' bodies, and stabilized or reversed the decline in mobility.
New results from a treatment trial in a canine model of Duchenne muscular dystrophy (DMD) offer one of the first, best hopes for restoration of dystrophin in humans, according to the study's lead researcher, Eric Hoffman, PhD, director of the Research Center for Genetic Medicine at the Children's National Medical Center in Washington, DC.
In a paper published online March 13 in advance of the print Annals of Neurology, Dr. Hoffman and colleagues reported that systemic delivery of an antisense therapy restored functional dystrophin to muscle membranes throughout the dogs' bodies, and stabilized or reversed the decline in mobility.
Antisense molecules bind to specific target sequences on DNA or RNA, rendering the target sequence unable to perform its normal genetic function. Dr. Hoffman and colleagues used an antisense molecule called a morpholino, similar in structure to RNA and DNA, with the usual four bases along a backbone. But the morpholino backbone is resistant to degradation by the cell enzymes that destroy foreign nucleotide chains, and doesn't provoke an immune reaction, two critical flaws in previous strategies for altering gene expression in DMD.
Viral gene therapy has relied on introducing a new dystrophin gene using a viral vector that may be immunogenic. Unless they are injected locally, DNA-or RNA-based antisense molecules may be degraded before reaching the target, but local injection is not practical when every muscle in the body is affected. In contrast, Dr. Hoffman's group delivered the morpholino systemically, with an intravenous injection, and reached muscles throughout the body.
The antisense morpholino they used was designed to target the messenger RNA from the mutant dystrophin gene. As in humans, the mutations in the dog introduce a stop signal that halts protein production, producing a truncated and useless dystrophin protein. Previous work in mice had shown that, by choosing the right morpholino sequence, it is possible to cause the RNA processing machinery to skip over part of the transcript, ultimately leading to a shortened dystrophin protein.
Shortened dystrophin is still functional, Dr. Hoffman pointed out, because it is found in patients with Becker muscular dystrophy. Mutations in that disorder introduce deletions of as much as half the protein, but they do not introduce stop signals, and they spare the two ends. Becker MD is quite variable clinically, with some patients remaining physically active well into old age. In contrast, boys with DMD lose the ability to walk in childhood.
The dystrophin protein is like a Tinker Toy, he said, whose long center is built from many repeating units. Many of these units can be deleted without severe effect, as long as the two ends remain to anchor the muscle cell cytoskeleton to the surface membrane. And it is in the gene's exons for that long central region where most mutations occur; thus, skipping over these exons in the RNA transcript is a rational strategy for ameliorating Duchenne muscular dystrophy.
The DMD dog has been an important preclinical model for two reasons. Lack of dystrophin causes muscle damage, and that damage is more severe in dogs (and humans) than it is in mice.
If we only needed to cure DMD in mice, we'd be done, Dr. Hoffman said. Second, the dog is even more severely affected than humans are, with high mortality just after birth. Improvement in the dog, then, is a better test of a therapeutic potential than any result in the mouse. The dogs in this study were bred and treated in Tokyo, at a facility underwritten by the Japanese government for support of muscular dystrophy research. The infrastructure is unparalleled there, Dr. Hoffman said. Our collaboration with them is what made the whole study possible.
The muscle damage caused by DMD is actually important to the success of the therapy. Normal contractions rip tiny holes in the muscle membrane, through which the morpholino can enter the cell. Mutant dystrophin makes the walls unstable, Dr. Hoffman said. That's where we have an advantage here.
Figure. DR. PAULA CL...Image Tools
The location and type of mutation in the dog meant that three different exons had to be skipped. The mutation itself, at the border between exons 6 and 7, causes exon 7 to be omitted. But when exon 6 and 8 are then joined, the reading frame of the RNA is shifted, introducing a stop signal. Skipping only exon 6 with antisense would omit the mutation, but introduce a different stop signal further on. But by using a cocktail of three different sequences, the researchers caused the RNA editing machinery to skip exons 6 through 8, joining exon 5 to exon 9 and preserving an open reading frame all the way to exon 79, the end of the gene.
CLINICAL IMPROVEMENT
Three dogs were treated with the cocktail, delivered intravenously into the saphenous vein, every one to two weeks, for five to 11 weeks. All skeletal muscles showed production of new dystrophin, although the amount produced varied from trace amounts to 50 percent of normal, with an average of 26 percent in the dog receiving the largest dose. Dystrophin production was lower in the heart than elsewhere, a phenomenon also seen in mice treated similarly. Muscles also improved by several other histological measures, showing fewer signs of degeneration, less inflammation, and more normal localization of the binders of dystrophin in the muscle membrane. Serum creatine kinase, a sign of muscle damage, was reduced after treatment. There were no signs of toxicity, inflammation, or organ dysfunction.
Two months after treatment, all three of the dogs ran faster than at baseline, in contrast to untreated littermates, which all ran slower. Other clinical signs stabilized or improved, including gait disturbance, drooling, dysphagia, and muscle atrophy.
Dr. Hoffman is cautiously optimistic about the implication of morpholinos for treating boys with DMD. While keeping in mind that the trial was very small and was not blinded, he said, It is one of the first, best hopes for these kids.
Results from a clinical trial of intramuscular morpholinos in DMD were announced in January, showing safety and the ability to restore dystrophin in humans. A clinical trial of systemic delivery is now underway in England.
A key hurdle remains, though: We have to bring the costs down, Dr. Hoffman said. Each dog received $200,000 worth of drug, for less than three months of treatment. Strategies are in place to increase both the scale of production and the potency of the molecules, which Dr. Hoffman thinks could reduce costs by a factor of ten.
CHALLENGES
It's a really exciting study, said Paula Clemens, MD, a gene therapy researcher and professor of neurology at the University of Pittsburgh Medical Center. Both the systemic delivery and the skipping of multiple exons were important aspects of the trial. Many patients will require multiple exons to be skipped, since their mutations, like the canine's, cannot be overcome by targeting just one. You wouldn't know without trying whether it would be effective, or whether there would be unpredicted molecular interactions that would interfere, she said.
A major advantage to the morpholino approach is its lack of immunity, she agreed, but on the other hand, it's more like a drug - it's not a one-time treatment. We don't know how frequently it will need to be readministered.
Regulatory approval may also be challenging, she noted, because the drug cannot ethically be tested in healthy humans, since skipping those same exons when there is no mutation would likely introduce a stop signal. If it's effective, you generate a disease, she said. This will need to be explored with the FDA, to determine how to adequately test safety. But the prospects for early morpholino trials in the US are good, she said: It's coming.
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