By the time Dongsheng Duan finished high school in 1981 in Gansu Province, China, the country was emerging from the repression and anti-intellectualism of the Cultural Revolution of the 1960s and ‘70s. (Duan calls the area “the Missouri of China” because of its mid-China location, its dry climate, and its lack of sophistication relative to Beijing and Shanghai.) Things were loosening up, and Duan was able to enroll at West China University of Medical Sciences in Sichuan Province — not quite his first choice, but close.

“I went to medical school, because that was my parents’ wish,” he says. “My top choice was to study biology.”

After medical school, Duan was sent to the Chinese Academy of Medical Sciences in Beijing to do government-sponsored research on male contraceptives. In 1989, he began applying to graduate programs in biology in the United States.

“I think that’s the time when I became less lucky than I used to be,” Duan says. “I got several offers from universities, but that was the year that there were demonstrations in Tiananmen Square, and everything was frozen.”

“I think that’s the time when I became less lucky than I used to be,” Duan says. “I got several offers from universities, but that was the year that there were demonstrations in Tiananmen Square, and everything was frozen.”

It would be 1993 before Duan could manage to leave the country. By the following year, he was in Philadelphia, at the University of Pennsylvania, where he would meet Katherine High, a pediatrician working on gene therapy for hemophilia.

“I did a rotation in her lab,” Duan says. “That’s when I got exposed to gene therapy. That was very hot and very new, cutting edge. I was so attracted to that, so I decided to work on gene transfer for my graduate work.”

Duan began work on development of the adeno-associated virus (AAV) as a gene transporter. He later pursued this strategy with a gene to treat the lung disease cystic fibrosis and, starting in 1999, Duchenne muscular dystrophy, with MDA grantee Jeffrey Chamberlain.

Chamberlain had miniaturized the gene for dystrophin, the protein needed in Duchenne MD, and he wanted to see if it would fit inside an AAV shell for delivery to muscles.

“Jeff had this microgene, and he was looking for somebody to help him to test it in AAV in the mouse,” Duan, who had relocated to the University of Iowa in 1997, recalls. “He wanted some people to help him, so he approached my mentor, John Engelhardt, in the fall of 1999. From then on, my interest was in muscular dystrophy.”

Almost immediately, Duan found a niche for himself. “Jeff was working on skeletal muscle, and Xiao Xiao [see “Proving a Principle”] had published his work on skeletal muscle, too. I realized that nobody was working on cardiomyopathy, and I realized that the heart is also an important organ that needs to be treated if you eventually want to cure patients.”

In 2003, about a year after he moved to the University of Missouri, Duan, Chamberlain and others conducted an MDA-funded study showing that gene transfer was feasible and effective in dystrophin-deficient mice, using Chamberlain’s microdystrophin genes encased in AAV transporters and injected into the heart.

The miniaturized dystrophin genes diffused through the heart tissue, where they resulted in the synthesis of enough dystrophin to make a difference, even 10 months later. The dystrophin protein, though miniaturized, normalized the appearance of the heart muscle cell membranes and was sufficient to keep out a dye that leaks into damaged cells.

The heart, Duan says, is different from skeletal muscle, and gene therapy for it has to adapt accordingly.

“In skeletal muscle,” Duan says, “we don’t contract all the muscles every day, every minute, every second. But the heart has to be contracting at all times, so physiologically, its work is more demanding. Second, gene transfer in the heart will be more difficult because the heart is a moving organ.”

Duan says his group is looking at transferring other genes that might be useful in treating MD-associated cardiomyopathy with a more generic approach. “We can deliver some genes which will be good for a lot of different situations.”

He admits his team doesn’t have all the answers, although they’re pretty sure they’re on the right track. “We’re still trying to figure out the best questions,” he says.

The laboratory of Xiao Xiao at the University of Pittsburgh is an integral part of the Paul D. Wellstone Muscular Dystrophy Cooperative Research Center, which is jointly supported by MDA and the National Institutes of Health.

Like Dongsheng Duan (see “The Heart Is Different”), Xiao is from China and wanted to move to the United States. But unlike Duan, he already knew he wanted to work on gene therapy in 1986, when he first emigrated.

In 1988, when Xiao joined the Biological Sciences Department at Pittsburgh, he had the good fortune to work with Jude Samulski, who was developing adeno-associated virus (AAV) transporters for delivering therapeutic genes to muscle tissue, where it was hoped they might benefit a variety of diseases (see “Bridge Over Troubled Waters,” January-February 2005).

These days, Xiao is perhaps best known for his role in developing the gene therapy construct for an MDA-supported safety trial in boys with Duchenne MD, now under way at Columbus (Ohio) Children’s Hospital. He focused on dystrophin because DMD affects a large number of children.

But since the late 1990s, Xiao has also been interested in hamsters with cardiomyopathy caused by a mutation in the delta-sarcoglycan gene. The cardiomyopathic Syrian hamster has been studied in laboratories since the 1960s, but its gene defect was unknown until 1997.

Genes for sarcoglycan proteins — whose absence causes human limb-girdle MD — are much smaller than dystrophin genes, small enough to fit easily inside AAV transporters, which remain front runners for gene transfer to muscle tissue.

“The gene can be very comfortably placed into an AAV, and the animal model [Syrian hamster] is available,” Xiao says.

Last fall, Xiao’s group showed that, months after an injection of delta-sarcoglycan genes into a vein or into the abdomen, treated delta-sarcoglycan-deficient hamsters were alive, had good heart function and could run on a treadmill. None of this was true for their untreated counterparts.

His group used a type 8 adeno-associated viral shell, which was extremely effective in delivering the genes to the heart, as well as to the diaphragm and the rest of the body’s muscles. (Since then, he’s found that type 9 works even better.)

Xiao also used a synthetic, instead of a natural, promoter (molecular “on switch”) to drive protein production from the new genes. The natural promoter that was previously used has to be truncated to fit inside an AAV shell, and somehow the truncation interferes with its ability to get into the heart or diaphragm.

The synthetic promoter is about half the size of even the truncated natural one, he says, and “it turns out that this promoter is strong in the heart and also in the diaphragm.”

Even though very few people need this particular gene, Xiao’s success helps prove the principle that gene transfer to the heart is both feasible and effective, he says.

For one thing, cardiomyopathic Syrian hamsters have severe cardiomyopathy, as well as skeletal muscular dystrophy, while dystrophin-deficient mice don’t. That gives these hamsters a definite advantage as a model of MD-associated heart disease.

“Many Becker and DMD patients die of heart failure,” Xiao says, “but cardiomyopathy is not a profound problem in mdx [dystrophin-deficient] mice. You have to look very hard to find cardiomyopathy in the mdx. You have to let them grow older, and you have to subject them to a stress, like an adrenaline injection, to make their hearts work extra hard, before you can observe anything.”

While Xiao’s group pushes ahead with the clinical trial of gene transfer into skeletal muscles, they’re not forgetting about cardiac muscle. But, Xiao says, “we have to do more preclinical studies first. The heart is a more vital organ than skeletal muscle, so we have to be very, very careful.”

Molecular research in cardiology was “a bit slow coming to the table,” says John Ross Jr., a cardiologist, research professor and MDA grantee at the University of California at San Diego.

In fact, he says, in 1988, 20 years after he first came to UCSD to head the medical school’s Cardiovascular Division, molecular cardiology was still in its infancy, and Ross wanted to see it mature.

That year, he brought Kenneth Chien, a cardiologist and assistant professor of medicine at the University of Texas Southwestern in Dallas, to the department. “He was a young researcher who was an unknown at that time,” Ross says, “but I thought he would be a good bet to set up the program.”

Chien was particularly interested in the molecular pathways underlying dilated cardiomyopathy, a form of heart muscle disease common in muscular dystrophy, where the heart’s main pumping chamber sags, dilates and loses pumping capacity.

“My guess was that if we could find pathways that were important for this particular form of heart failure, they would hold for many varieties of heart failure,” says Chien, who was an MDA grantee until recently.

He thought the pathways would have to do with the way heart cells handle calcium. “Calcium triggers muscle contraction,” he says, “and there’s a whole cycle that regulates calcium coming in and out of the heart and skeletal muscle.”

In 1997, when it was learned that mutations in the gene for delta-sarcoglycan caused a known hamster cardiomyopathy (see “Proving a Principle”), Ross decided to work on sarcoglycan-based gene therapy for cardiac disease.

But two years later, Chien, Ross and their colleagues found that deleting a single gene that carries instructions for the protein phospholamban could completely prevent heart failure in mice genetically destined to develop the condition.

Under resting conditions, the phospholamban protein acts as a brake on the heart’s rate and force-generating capacity. But when the body senses a need for greater cardiac output, phosphate groups are tacked onto it, and the braking action ceases.

Ross wanted to pursue replacing the delta-sarcoglycan gene, while Chien became increasingly interested in blocking phospholamban.

Working with colleagues Masahiko Hoshijima and others, Chien created a phospholamban gene that gave rise to a protein just like the original phospholamban with one exception: At position 16 in its structure, the new phospholamban had a glutamic acid molecule instead of a serine molecule. This small substitution, they hoped, would keep phospholamban from acting as a brake on heart activity.

Ross’s group designed a system they hoped would deliver the altered phospho-lamban genes to a beating heart.

In 2002, the Ross and Chien labs treated delta-sarcoglycan-deficient hamsters with genes for what they now called S16E phospholamban. The S16E genes, injected into blood vessels supplying the heart and encased in adeno-associated viral shells, partially prevented the expected cardiomyopathy and heart failure.

Two years later, they showed that when rats that already had significant heart damage were given S16E phospholamban genes, they got much better than untreated rats. The altered phospholamban, it seemed, competed with the natural phospholamban in the rodents, blocking much of the natural protein’s ability to apply the brakes, and in effect, pressing on the accelerator.

Chien says he doesn’t see blocking phospholamban as a treatment for early heart damage. “This is something you bring in later,” he says, comparing it to the various drugs used at different stages in cancer. (Drugs that reduce the heart’s workload are used earlier.)

Chien, who had the original MDA grant to study phospholamban, moved to Harvard Medical School in Boston last summer. Ross remains at UCSD, where he and Hoshijima have taken over the phospholamban project and are following up on delivering delta-sarcoglycan genes.

Their next step, Ross says, will be to give delta-sarcoglycan-deficient hamsters with established heart disease a combination of delta-sarcoglycan and S16E phospholamban gene therapy and compare the results with those from delivery of delta-sarcoglycan alone.

If gene transfer technology moves ahead and is accepted by the Food and Drug Administration, he says, “I think a lot of the dystrophies, both skeletal and heart muscle components, are potentially treatable.”

Soichiro Yasuda came to the University of Michigan in 2002, soon after the university had established its Center for Integrative Genomics, under the direction of physiologist Joseph Metzger.

Yasuda was less than a year out of a doctoral program in Japan when he joined the Metzger laboratory, but he’d already co-authored two studies describing new ways to measure tension in heart muscle cells.

“I attached a pair of carbon fibers on both ends of a cardiac cell and stretched it with various extensions,” Yasuda explains. When he came from Tokyo to Ann Arbor (Mich.), he brought the system with him.

“I knew that muscular dystrophy involves cardiomyopathy and heart failure when I was working in Japan as a physician,” Yasuda says, “but that was just technical knowledge, without concrete experience.

“When I started my project at the University of Michigan, using dystrophin-deficient mouse heart cells, I got interested in how dystrophin deficiency causes cardiomyopathy.

John Quinlan

“I thought that understanding basic cardiac contractile function at the level of living cells would help me develop new gene therapies for heart disease,” he says.

But, as it happened, gene therapy wasn’t the path that Yasuda followed.

Yasuda, who received an MDA grant this year to study cardiomyopathy in MD, learned that experiments had shown that a synthetic compound called poloxamer 188 (p188) could prevent cell membrane damage in rats exposed to electric shocks and was also somewhat effective in normalizing cell surfaces in the blood disease sickle cell anemia.

He began thinking about p188’s possible use in muscular dystrophy. (Small holes in cell membranes appear in MD-related damage.)

MDA grantee John Quinlan at the University of Cincinnati tested the effects of the compound on skeletal muscle cells in dystrophin-deficient mice. Unfortunately, he didn’t find it protective; but heart muscle, scientists in the Metzger lab thought, might be different.

“The heart is probably more sensitive to small tears than skeletal muscle is, because of differences in how these two tissues handle the calcium coming through these tears,” says DeWayne Townsend, a postdoctoral research associate working with Yasuda.

Townsend also notes that skeletal and cardiac muscles contract differently, and may have different-size tears.

“Poloxamer 188 is like a molecular bandage,” he says. “If there’s a small tear, it can be helpful; but if there’s a large tear, p188 may not be able to seal the hole.

The heart may have a lot more of these small tears, whereas in skeletal muscle, you get these big tears in the membrane.”

Yasuda developed a technique to stretch single heart cells lacking dystrophin, and the researchers found they were less compliant than normal heart cells and more susceptible to damage when stretched.

The research team then put some p188 into a solution with heart cells for about 20 minutes and found, somewhat to their surprise, that it protected them from stress-related tears.

They decided to move on to cardiac stress testing in mice, and last summer, they showed success there as well.

When dystrophin-deficient mice received intravenous p188 before they got infusions of dobutamine, a drug that makes the heart work extremely hard, they withstood the challenge and survived, while several of the mice that didn’t get any p188 developed acute heart failure.

Toxic effects don’t seem to be a problem, Yasuda says, because the compound apparently remains in cell membranes rather than entering cells, at least at the doses and exposure periods his team is using.

He cautions, however, that effective, long-term p188 treatment, as things now stand, would require frequent IV infusions, which may limit its usefulness in a chronic disease like muscular dystrophy.

“We showed the therapeutic effects of p188 in rescuing dystrophin-deficient hearts under acutely stressed conditions,” Yasuda explains, adding that “it may be effective in blocking acute worsening of heart function in DMD patients.”

Townsend says that figuring out a way to administer p188 “in a more user-friendly manner” is high on the list of his group’s goals.