Designing a Better Gene

Article Highlights:
  • Dongsheng Duan at the University of Missouri-Columbia has found that many of the miniaturized versions of the dystrophin gene used in gene therapy experiments do not contain certain sections that stick to neuronal nitric oxide synthase (nNOS) and are therefore unlikely to provide full therapeutic benefit.
  • Neuronal nitric oxide synthase is necessary for providing muscle fibers with increased blood flow during exercise.
  • The findings have implications for gene therapy for Duchenne and Becker MD.
by Margaret Wahl on March 31, 2011 - 11:40am

QUEST Vol. 18, No. 2

Dongsheng Duan's interest in gene therapy to treat diseases goes back a long way, although his initial focus wasn't muscular dystrophy.

"I came to the muscle field by accident," says Duan, an MDA research grantee and professor of molecular microbiology and immunology at the University of Missouri at Columbia. "It was not something I intended to do when I was young."

After earning a doctorate in pathology at the University of Pennsylvania in 1997, Duan joined the laboratory of John Engelhardt at the University of Iowa for his postdoctoral training. The lab was focused on gene therapy for cystic fibrosis, a genetic lung disease.

By the early 2000s, Duan was looking for a different field in which to apply his gene therapy expertise. He became interested in muscular dystrophy and in 2002, moved to the University of Missouri, where academic veterinarian Joe Kornegay had established a colony of dogs with a DMD-like disease. (Kornegay, a current and former MDA grantee, is now director of the National Center for Canine Models of DMD at the University of North Carolina at Chapel Hill.)

Almost as soon as he started working in Missouri, Duan identified a gap in muscular dystrophy research that he wanted to help fill.

What happens between gene mutation and disease symptoms?

Since the late 1980s, scientists had known that mutations in the gene for the muscle protein dystrophin were the root cause of DMD and Becker muscular dystrophy (BMD). But, Duan noticed that the steps between dystrophin mutations and the symptoms of DMD or BMD were not clearly defined.

"Every time I opened a journal article, I read, 'Duchenne muscular dystrophy is caused by mutations in the dystrophin gene,'" he notes. "But from the mutation to the disease, there seemed to be a gap. The pathways were not clearly defined." To define them, Duan decided to look back in scientific history.

When his search took him all the way back to the early 1970s, Duan found that some experts at the time had reported impaired blood flow — ischemia — as a possible factor in DMD.

Most of that work had been forgotten by the 1990s, after the dystrophin gene had been identified and the majority of scientists had shifted their focus to the protein's crucial role in protecting the muscle-fiber membrane. Duan wondered whether dystrophin's absence could account for the ischemia noted by those researchers many years earlier.

"When I moved through history into 1995, there were papers showing that one of dystrophin's roles is to recruit neuronal nitric oxide synthase [nNOS] to the muscle-fiber membrane," he says. The nNOS enzyme makes nitric oxide (NO), a protein that opens up (dilates) blood vessels. Although it’s called "neuronal," it's also found in muscle fibers. Could it be, Duan reasoned, that lack of dystrophin could lead to lack of nNOS, lack of NO, and ultimately, lack of blood-vessel dilation with exercise?

Things really clicked for Duan when he read a 1998 paper showing that dystrophin-deficient mdx mice, which, like humans with DMD, lack nNOS at the muscle-fiber membrane, have impaired blood flow in exercising muscles. (For more, see Enhancing Blood Flow to Exercising Muscles.) Maybe some of the damage in muscles affected by DMD and BMD was because of ischemia after all.

"This kind of all added up," Duan recalls. "When I went back and looked at my own samples, which we had collected from dystrophic dogs and dystrophic mice, what was really striking to me was that not every muscle fiber is injured, even though they all lack dystrophin. You get a kind of focal lesion. It's not like the entire muscle is damaged. That's similar to a situation where you have ischemia, where you get locations where there wasn't enough perfusion of blood. I thought all this probably had something to do with the function of nNOS."

Utilizing minidystrophin genes

Dystrophin, a very large gene, has to be miniaturized to be given as gene therapy. When it's miniaturized, sections of its DNA are removed, resulting in production of a less-than-full-length dystrophin protein molecule.

Duan suspected — and later showed — that the miniaturized dystrophin genes (known as minigenes and microgenes) that researchers had developed since the 1990s did not contain the sections of DNA that would allow the dystrophin protein to stick to nNOS. Therefore, he speculated, they might only be fixing some of the problems that stem from dystrophin deficiency.

"We made two types of transgenic mice," he recalls. "Both expressed minidystrophin genes. They had identical structures except one had sections called R16 and R17, which carry the code for the nNOS-binding part of the protein. The other one did not have those sections. We looked at muscle function to see if there was a difference between them. If nNOS meant something, I reasoned, we should see a difference."

The experiments found that the mice with a minidystrophin gene missing sections R16 and R17 could not restore NOS to the muscle-fiber membrane and showed reduced blood flow and ischemic damage in their muscles, while the mice with the slightly longer gene that contained these sections had normal blood flow and no ischemic damage.

In later experiments, Duan and his co-workers put the two types of mice on a treadmill. "On the first day, both groups performed the same," he says. "But after that, muscle function in the group with the smaller gene and no nNOS binding started going down. By the time we got to day six or eight, we saw a big difference. We looked at sections of the muscle to find out if ischemic damage was occurring in the mice that did not restore nNOS, and we found it.

"That clearly provided evidence for saying that if you don't have nNOS at the membrane, there is going to be ischemic damage to the muscle fiber. That's definitely going to affect function, such as walking ability."

Duan says the findings have important implications for therapies being developed for DMD and BMD and perhaps even for other forms of muscular dystrophy, noting that nNOS may be mislocalized in some other muscular dystrophies as well.

"If you have genes that restore nNOS and some that don't restore nNOS to the membrane, you want to choose the ones that restore nNOS," he says.

In addition, he notes, "you may want to find other ways, such as vasodilating medications, to help patients open their blood vessels when they exercise. As we develop therapies that help people with muscular dystrophy become healthier and lead more normal lives, they'll want to exercise."

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