LONDON - The search is on for a drug to boost production of a natural protein that may compensate for the loss of the muscle protein dystrophin in muscular dystrophy. Researchers in the U.K. have found that the protein utrophin, which is very similar to dystrophin in structure, can correct the muscle abnormalities present in mice deficient in both utrophin and dystrophin.
The finding raises hope that if a compound can be found which can up-regulate natural production of utrophin, it may be possible to treat muscular dystrophy with a pill. OSI Pharmaceuticals Inc., of Uniondale, N.Y., already is conducting a screen of 150,000 novel compounds in the hope of identifying a suitable drug.
If the search is successful, such a breakthrough would mean avoiding the difficulties scientists have encountered in trying to develop gene therapy for this disease. The therapy, if it is to work, will involve delivering a good copy of the dystrophin gene to every single muscle in the body. Nevertheless, those wanting to persevere with this approach will find the mouse generated for these experiments highly useful in evaluating delivery systems for potential gene therapies.
Kay Davies, professor of genetics at the genetics unit of the department of biochemistry at the University of Oxford, in the U.K., and her team report their results in a paper in the May edition of Nature Genetics. The joint first authors of the paper, titled “Skeletal muscle-specific expression of a utrophin transgene rescues utrophin-dystrophin deficient mice,“ are Jill Rafael and Jonathon Tinsley.
Tinsley, senior research fellow, told BioWorld International, “What this paper shows is that if you express a utrophin transgene in the skeletal muscles of mice which have no utrophin and no dystrophin, the mice are essentially cured of their symptoms. This supports the idea that utrophin plays a role in normal muscular dystrophy - that the severity of the disease may rely on how much utrophin can be made.“
Muscular dystrophy affects one in every 3,500 boys born. It is caused by a defect in the gene coding for the protein dystrophin. In its most severe form, Duchenne muscular dystrophy, the defective gene fails to produce functional dystrophin, although in milder cases the protein is partially functional.
Dystrophin is a key component of muscle, forming part of the protein complex which anchors the cytoskeleton of the muscle fiber to the extracellular matrix. Without it, the muscle fiber degenerates.
In 1989, researchers led by Davies identified the gene for a protein closely related to dystrophin, which they called utrophin. Dystrophin is found in muscle, where it is spread throughout the inner membranes, and to a lesser extent in the brain. By contrast, utrophin is found in all tissues of the body. In muscle, it appears only in two kinds of specialized junctions: those between nerves and muscles and those between muscles and tendons.
“We felt that the utrophin protein was so similar to the dystrophin protein that they are functionally interchangeable,“ Tinsley said. “We also hypothesized that if you could come up with a way of altering the expression of utrophin in muscles so that it was localized throughout the fiber, then in patients with Duchenne muscular dystrophy it could replace the dystrophin and basically cure the disease.“
To prove this, Davies and her colleagues first generated a transgenic mouse that lacked dystrophin. Their starting point was the mouse model for muscular dystrophy, which is unable to produce dystrophin. These mice have a slightly shorter life span than would be expected. Although severe abnormalities of the muscle are visible on microscopy, they do not show symptoms comparable to those experienced by humans with muscular dystrophy.
Davies, Tinsley and their colleagues investigated what happened when a utrophin transgene was expressed in the dystrophin-deficient mouse, using a promoter that would ensure the gene was expressed throughout the muscle fiber, rather than in just the specialized junctions where utrophin is normally found.
“This experiment showed,“ Tinsley said, “that if you can get utrophin localized to the membrane then it cures these dystrophin-deficient mice.“
Because the researchers were interested in the function of utrophin, they also generated a mouse in which the utrophin gene was knocked out. These mice, they found, appeared to be normal, although they did have slight abnormalities of the neuromuscular junction.
The next question was what would happen when the dystrophin-deficient mice were crossed with the utrophin-deficient mice. The team found that offspring which were deficient in both proteins were clearly identifiable: These animals' growth and movement slowed by about four weeks of age, and they died between six and 18 weeks of age. Oddly, their muscle pathology did not appear to be very different from that of the dystrophin-deficient mice.
“It appeared that, without utrophin, the disease did become a lot more severe, even though the muscle pathology did not appear to be worse,“ Tinsley said.
The team's next experiment, as reported in the Nature Genetics paper, was to express a utrophin transgene in the mice deficient in both utrophin and dystrophin. Again, they used the promoter, which ensured the transgene was expressed only in skeletal muscle, and throughout the muscle fibers.
“The skeletal muscle weakness in these mice was essentially cured,“ Tinsley told BioWorld International. “This finding suggests that we should be looking at muscle to see what the true functions of these two proteins are. Amounts of utrophin have never before been associated with the severity of muscular dystrophy, but these mice suggest that we should look at this factor.“
One important question raised by this experiment is whether it is important to deliver dystrophin to the hearts of those affected by muscular dystrophy, or whether it is sufficient to target only the skeletal muscles. The mice treated with the utrophin transgene in the team's most recently reported experiment had neither utrophin nor dystrophin in their hearts, yet were apparently normal.
Davies and her colleagues are embarking on several further experiments which they hope will answer outstanding questions about the therapeutic strategy they are pursuing.
“First, we need to know how much utrophin you need to be able to get to the muscle in order to have a significant effect,“ Tinsley said. “Second, we need to find out what happens when you get a lot of utrophin in tissues other than muscle, such as kidney or lung. Third, because in many cases muscular dystrophy is diagnosed only at two to three years of age, we want to know whether utrophin can have its beneficial effect on muscle which has already succumbed to the disease.“ *