Genetic Flaw May Yield to Transplants : Unprecedented Tests Hold Hope for Cure of Muscular Dystrophy
Physicians at Stanford University and four other institutions around the world are gearing up to perform an unprecedented transplantation procedure that holds the promise of achieving a feat that has tantalized molecular biologists since the inception of genetic engineering: correcting an inborn genetic defect in humans.
Within the next month or two, surgeons in at least one of the groups are expected to withdraw a sample of healthy muscle tissue from adult volunteers, grow it in a test tube briefly to increase the number of cells present, and then inject millions of healthy cells along the path of a deteriorating muscle in a young victim of Duchenne muscular dystrophy, a fatal, degenerative disease for which there is now no effective therapy.
The researchers hope that the injected cells will fuse with the child’s own wasted muscle cells and donate the gene for a protein that is missing in victims of muscular dystrophy, thereby allowing the deteriorated cells to regenerate and regain their function.
Every Muscle Affected
If the preliminary attempts are successful, massive transplants of cells would be required for effective therapy because virtually every muscle in the body is affected by the disease. In particular, it would be necessary to perform the procedure on muscles of the heart and the diaphragm to enable the muscular dystrophy victims’ hearts to keep beating and lungs to keep breathing.
The proposed experiments have drawn a great deal of attention as well as some controversy because of the haste with which researchers are proceeding as well as the uniqueness of the proposed therapy.
So far, the procedure has been attempted only in a limited number of mice and, because of the unusual nature of the disease in those animals, the researchers have had difficulty demonstrating that the procedure will be effective at restoring muscle function.
A few researchers are thus calling for a much larger series of experiments in other animals before the procedure is attempted in humans, but many physicians argue that the procedure is so promising and the prospects for muscular dystrophy patients so gloomy that the risk is justified.
The experiments are relevant to the whole field of birth defects because they represent the first case in which knowledge obtained about a genetic defect through the use of new genetic engineering techniques has actually been put to use. Molecular biologists have continually promised that the discovery of the genetic defect responsible for a disease will lead to new forms of therapy, but this case represents the first instance in which that promise may be fulfilled.
Remarkable Speed
And this case is all the more remarkable because of the speed with which new developments have occurred. The defective gene that produces Duchenne muscular dystrophy was discovered only three years ago and the nature of the biochemical defect produced by the gene was revealed only late in 1987.
But most of the physicians involved in the proposed tests are not thinking as much about the larger implications of their proposals as about the small but very real gains they may be able to achieve, such as the continued use of a particular muscle by a single patient. “Having the prolonged use of a single muscle, such as the thumb, can mean a lot to (muscular dystrophy victims),” according to developmental biologist Helen Blau of the Stanford University School of Medicine.
In addition to Stanford, others planning such tests are the University of Tennessee, the Montreal Neurological Institute, Tufts University in Massachusetts and British researchers.
About one in every 3,500 boys born in the United States has the genetic defect that causes Duchenne muscular dystrophy. Females can be carriers of the defective gene, but they rarely contract the disorder themselves.
The babies appear normal at birth, but develop a progressive weakening of the muscles that usually places them in a wheelchair by the age of 11. Most die in their late teens or early 20s, when the muscles that operate the heart and lungs cease functioning.
Only three years ago, pediatrician Louis J. Kunkel and his colleagues at the Harvard Medical School reported that they had discovered the location of the gene that is defective in Duchenne muscular dystrophy.
In a follow-up to that discovery, Kunkel and his colleagues reported in December, 1987, that the gene is the blueprint for a protein that they named dystrophin. Dystrophin makes up part of the structure of the muscle cells, but is present in only tiny quantities, accounting for only 0.002% of the protein in the muscle cells. Kunkel showed that the protein is located in the region of the muscle cell where an electrical impulse from the brain is converted into a chemical signal that causes the muscle to contract.
In Duchenne muscular dystrophy patients, the gene for dystrophin is defective, and little or none of the protein is produced, which causes the cells to malfunction and die. Its presence in such small quantities explains why researchers did not notice its absence in muscular dystrophy patients before the gene was discovered.
Next month, Genica Pharmaceuticals Corp. of Worcester, Mass., will begin marketing a test for dystrophin that will allow physicians to determine whether dystrophin is present in muscle cells. The test will allow doctors to diagnose the disease in infants who might have inherited the disease before symptoms occur, and will assist them in distinguishing among several different types of muscular dystrophy.
The fact that dystrophin is a structural protein at first proved daunting to researchers. “It doesn’t flow around in the the bloodstream like an enzyme or go in and out of cells like a hormone,” said neurologist George Karpati of the Montreal Neurological Institute. “It has to fit into the cellular structure like a beam under a roof. That is a tall order.”
Unusual Cells
But not an impossible one, it turned out, because muscle cells have very unusual properties. Unlike every other cell in the body, a muscle fiber, which is actually one cell, has hundreds to thousands of nuclei, each with its own--but identical--genetic information. Muscle tissue also contains immature cells, called myoblasts, that can fuse with existing muscle fibers, insert their own nuclei, and cause the muscles to regenerate.
In muscular dystrophy victims, unfortunately, the myoblasts have the same genetic defect as the fibers themselves, and the muscles quickly use up all their regenerative capacity.
Earlier this year, two groups of researchers--Karpati and cell biologist Paul Holland in Montreal, and Kunkel and Terry Partridge of the Charing Cross and Westminster Medical School in London--independently made a crucial discovery.
They removed myoblasts from healthy fetal mice and injected them into the muscles of so-called mdx mice, which also have a defective dystrophin gene. The researchers observed that the injected myoblasts fused with the muscle cells of the mdx mice and the cells then began producing dystrophin.
But the mdx mouse is an imperfect model for human muscular dystrophy. Although their muscle cells lack dystrophin and begin degenerating at the age of about three weeks, for some as yet unknown reason, their muscle cells simultaneously begin regenerating. In fact, the mice remain healthy and eventually become even stronger than normal mice.
Encouraging Signs
Nonetheless, researchers were elated to see the mdx cells producing dystrophin. “The animal studies to date are very encouraging . . . and suggest that human transplantation is feasible,” said neurologist Theodore Munsat of Tufts University. “We’re as excited as we can be about the prospects,” added Lawrence Z. Stern, acting scientific director of the Muscular Dystrophy Assn., which sponsored most of the preliminary work and will sponsor the human trials.
The possibility that human trials would be successful became even more likely earlier this month when neurologist Peter J. Law of the University of Tennessee at Memphis reported his work with the “DyDy-J” mouse, which is not missing dystrophin, but which undergoes degeneration of its muscles and usually dies by the age of 9 months.
When Law injected myoblasts from healthy mice into these animals, he found that their muscles did not degenerate as much as normal and that many of the mice lived at least 19 months.
“The beauty of myoblast transfer therapy is that it doesn’t matter which gene or protein is missing,” Law said. “You are basically replenishing all the normal genes back into the dystrophic cell and allowing the normal genes to express themselves.”
These discoveries have set the stage for trials of the therapy in humans. Some researchers argue that more experiments should be conducted in animals, particularly in a new breed of dog developed at Cornell University that is lacking dystrophin and that shows muscle degeneration.
But Stern contended that “there are many questions about the therapy that can only be answered in human trials. If we are talking about limited experimental trials to test primarily safety issues, I think those . . . probably should proceed at this time.”
If there are any side effects, they most likely would involve muscle damage resulting from rejection of the transplant.
Law will probably be the first to make an attempt, since he has already received permission from his institutional review board to conduct the experiments. Sometime later this summer, he will inject about 8 million myoblasts from a muscular dystrophy patient’s father or brother at eight sites along a short muscle on the top of the right foot that causes one of the toes to rise.
This muscle, called the extensor digitorum brevis, is known to begin degenerating at about the age of 6, so Law will be able to compare the injected muscle to the corresponding muscle in the left foot to determine if the transplant is effective. But the muscle is not crucial to the patient’s functioning, so the patient will not be harmed if the experiment goes awry and the muscle is inadvertently damaged.
Law will use the immunosuppressant cyclosporine a to prevent the injected cells from being rejected, but his work in animals suggests that cyclosporine will not be required for more than about three months. Fortunately, muscle cells do not provoke a strong immune reaction.
If the experiment is successful in a small group of children, Law added, he will begin testing larger amounts of myoblasts in larger numbers of children.
Blau, Munsat, Karpati and one British researcher are planning similar experiments sometime within the next year, although they will focus on different muscles, such as the bicep or muscles in the hand. Their procedures will otherwise be virtually identical to Law’s.
Many Volunteers
Stern says parents of more than 4,000 muscular dystrophy victims have already called or written and offered their children as subjects for the studies--a measure of the desperation caused by the lack of effective therapy for the disorder.
Despite the apparent haste with which all the researchers are moving, many, like Stanford’s Blau, are urging caution. Blau, who will probably not make an attempt this year, noted that “the purity of the myoblasts is critical. They cannot be allowed to become contaminated while they are being cultured because contaminants stimulate a strong immune response.”
Karpati, who like Law will probably undertake the procedure within a couple of months, added that the issue of how much immunosuppression must be used and for how long has also not been settled. He will try varying amounts and varying lengths of time among the first 10 children he treats.
Blau and Munsat are also planning to perform more animal studies before they try the procedure in humans. “I would like to caution other people who may not be moving so prudently,” Blau said. “I would hate for the field to be pushed backwards by people who move forward too quickly.”
