LONDON - It may one day be possible to use an intravenous infusion of cells from a patient's own bone marrow in delivering gene therapy to muscles wasted by muscular dystrophy, said a team of Italian researchers who have found that bone marrow cells can migrate to damaged muscle and help to repair it.

Although the scientists warn that many more experiments need to be carried out in order to test their hypothesis, they believe they may have found a way to overcome the multiple difficulties that have beset previous attempts to deliver gene therapy to the muscles of patients with muscle wasting diseases.

Fulvio Mavilio, codirector of the Human Gene Therapy Programme at the San Raffaele-Telethon Institute for Gene Therapy, in Milan, Italy, told BioWorld International that a new treatment is in sight, based on the findings.

“We envisage in future the use of the patient's own bone marrow cells, which can be taken out, genetically engineered so that they carry a good copy of the defective gene, and then returned to the patient's bloodstream, where they would migrate to the damaged muscle and help to repair it,“ Mavilio said.

Mavilio, with Giuliana Ferrari and colleagues in Milan, collaborated with a team led by Guilio Cossu of the University of Rome to show that cells from the bone marrow could reach damaged muscles and help to repair them. They report their results in the March 6 issue of Science, in a paper titled “Muscle regeneration by bone marrow-derived myogenic progenitors.“

The group has applied for a patent in Italy on the use of genetically engineered bone marrow for muscle replacement and expects to extend the patent worldwide. Terence Partridge, head of the Muscle Cell Biology Group at the Medical Research Council's Clinical Sciences Centre - which is part of Imperial College School of Science, Medicine and Technology, of London - said the cells apparently move and transform themselves.

Current Gene Therapy Can Cause Damage

The cells apparently “have migrated out of the bone marrow, through the blood and into the muscle and then turned into muscle cells,“ Partridge said.

“This is biologically interesting as well as being potentially useful for therapy, because the big problem with therapies which involve delivering cells to the muscle is in getting those cells to the places you want to get them to.“

One in every 3,500 males is affected by muscular dystrophy. The disease is caused by a defect in the X-chromosome gene which codes for the protein dystrophin. Although the defective gene may be carried by women, a high proportion of cases of muscular dystrophy are due to new germ-line mutations, thus limiting the opportunities for carrier screening and prenatal diagnosis.

In the most severe form of the disease, known as Duchenne muscular dystrophy, the defect in the gene coding for dystrophin is so severe that no functional dystrophin can be made, although in milder cases the protein may be simply slightly abnormal.

Dystrophin is a key component of muscle. It forms part of the protein complex that anchors the cytoskeleton of the muscle fiber to the extracellular matrix. Without it, the muscle fiber degenerates.

The disease is frequently diagnosed only when a child attempts to walk. In the case of Duchenne muscular dystrophy, the child may need to be in a wheelchair by the age of 10 or 12 and, because the muscles of the heart and lungs are eventually affected, death occurs at about age 20.

Attempts to treat muscular dystrophy have therefore centered on efforts to deliver a good copy of the dystrophin gene to the wasting muscles. These have met with little success. Researchers have tried using donated cells that are capable of becoming muscle cells, but these are difficult to culture in sufficient numbers. Also, neither cells nor viral vectors genetically engineered to contain the normal dystrophin gene can penetrate efficiently into muscles from the blood circulation.

The only way of delivering gene therapy, therefore, has been to inject cells, DNA or viral vectors at multiple sites into the muscle. This inevitably causes further damage - apart from pain and discomfort.

Mouse Experiments Use IacZ Gene

To address these problems, Mavilio and his colleagues investigated other types of cells that might be capable of becoming muscle cells. They knew that satellite cells, which surround muscle fibers, help skeletal muscles to grow and to repair damage. But after a muscle injury, the number of satellite cells locally is much smaller than the number of muscle cell precursors - suggesting that some precursors migrate from elsewhere.

“We started with the idea of injecting cells such as fibroblasts, which can be obtained from a small skin biopsy, and encouraging these cells to become muscle cells,“ Mavilio said.

As part of this study, the group injected various kinds of cells into the muscles of mice, including fibroblasts from the bone marrow and a range of control cell populations. One of their controls consisted of bone marrow cells from which the fibroblasts had been removed.

“To our surprise,“ said Mavilio, “we found that, in the non-fibroblast portion of the bone marrow, there were cells which could become muscle cells, without us forcing them in any way.“

Since this observation, team members have spent two years attempting to scientifically prove their discovery. Having been able to distinguish between the cells of the recipient animal and those of the donor animal, they worked with transgenic mice which had been developed for another purpose by Margaret Buckingham and colleagues at the Pasteur Institute, of Paris.

The mice carry the lacZ gene, which codes for the enzyme beta-galactosidase under the control of a promoter, which turns the gene on only in muscle cells. Conveniently, when appropriately stained, nuclei are blue in cells in which the gene is manufacturing beta-galactosidase.

Mavilio and his group took bone marrow from the mice with the lacZ gene and transplanted it into mice in which the bone marrow had been destroyed by irradiation. They then induced muscle damage in the transplant recipients, with a local injection of snake venom toxin. Damage of this kind is normally repaired within two to three weeks.

“We reasoned that if there are cells in the bone marrow that can sense muscle degeneration and can access the bloodstream to the regenerating muscle and participate in muscle regeneration, then we would be able to trace them, because these cells would have blue nuclei because of activation of the transgene,“ Mavilio told BioWorld International.

That is exactly what happened.

“We looked at sections of regenerating muscle in the recipient animals and found that in all transplanted animals there were a small number of blue nuclei,“ he said. “These could only have come from the donated bone marrow cells.“

Mavilio predicted that this finding could represent a “potential breakthrough for therapy“ because the cells concerned can be delivered through the circulation. Although only small numbers of bone marrow cells migrated into the muscle in this way, Mavilio suggested that the contribution of these cells in patients with muscular dystrophy could be much more significant because they would not have to compete with healthy satellite cells to carry out the repair work.

Partridge cautioned against too much optimism at this stage. “This finding is potentially important because the only real hope of dealing with muscle fibers is if you can reach them via the vascular system, and this work says that you can,“ he said.

“The down side is that this is a very rare event - not many cells get through,“ Partridge went on. “If you use less sensitive markers than Mavilio and his group used, you can't see the effect. But if you could boost what is happening in some way, then you might well be able to get some benefit from it.“

The next goal for Mavilio and his colleagues is to find out if the strategy will indeed work as a therapy. They are working with a natural mutant mouse, the MDX mouse, which - like patients with Duchenne muscular dystrophy - lacks the gene for dystrophin. After destroying the bone marrow of these mice, the researchers are transplanting bone marrow from mice which are genetically identical except for the dystrophin gene.

“We are now waiting to see if there will be spontaneous repair of muscle from cells coming from the donated bone marrow,“ Mavilio said. *