Gene Therapy Updates
by Margaret Wahl

The muscular dystrophy community holds its breath waiting for the results of a gene therapy clinical trial in Duchenne muscular dystrophy (DMD), planned for this fall at Ohio State University Medical Center in Columbus, even as still newer strategies to treat the disorder are in development. Which gene, which vector and which overall strategy will win the race to treat DMD still remain as questions.

The Ohio State trial, which doesn't yet have Food and Drug Administration approval, is designed mostly to test the safety of the gene transfer (see "Human Trials Set for DMD," Quest, Vol. 5, No. 1). As things now stand, the researchers will use a dystrophin gene that's expected to give rise to a full, normal-length version of dystrophin, the protein that's missing in the muscles of boys with DMD. They'll use a vector (gene delivery vehicle) known as the "gutted adenovirus," which was developed in the laboratory of molecular geneticist and longtime MDA grantee Jeffrey Chamberlain at the University of Michigan in Ann Arbor. Neurologist and MDA clinic director and research grantee Jerry Mendell is the clinician in the trial.

Chamberlain is fairly optimistic about the first test of this vector and gene construct in humans, but he's cautious too. "We're continuously making changes and updating our vectors depending on what we see," he says. "We're going to evaluate what's involved with FDA approval and institutional review board approval and how quickly we can bring the latest vector on line. At any point, we want to use the best thing we have, but if we make a new vector and it's going to take nine months to get it approved and it's already August, we won't wait for that; we'll start with what we have in hand." Adverse immunologic responses against either the shell of the adenovirus used to deliver the gene or to the dystrophin protein itself pose the most serious threats to safety and effectiveness of the gene transfer, Chamberlain says. (Since boys with DMD make little or no dystrophin, there's always been a concern that their immune systems might reject new dystrophin.)

Chamberlain says he hopes people aren't impatient with the FDA for requiring at least several more months to grant approval for the trial, because he says, "We need that much time anyway to get everything ready, and everything is going on in parallel with the approval process."

diagram illustrating protein clusters in muscle fiber
Clusters of proteins are found around the periphery of each muscle fiber, embedded in the muscle fiber membrane. These proteins probably keep the fiber intact during muscle contractions and may also carry signals between the inside and outside of the fiber. A lack of dystrophin leads to DMD, while loss of any of the four sarcoglycans leads to the somewhat less severe disorder known as limb-girdle MD. These clusters are linked to the inside of the fiber by dystrophin along most of their border. However, utrophin plays this linkage role at the neuromuscular (nerve-muscle) junction. Recent experiments have shown that utrophin could probably do the same thing that dystrophin does all around the border if it could be delivered to these areas.


The University of Michigan gene and vector have center stage at the moment, but Chamberlain and others emphasize that there are plenty of alternative strategies in development or already in the wings. "No matter what we use now, these are clearly not the ultimate constructs that will be used to treat this disease," Chamberlain says.

Near the top of the list of alternatives are gene transfer and gene regulation strategies that involve the protein utrophin instead of dystrophin. Chamberlain recently went to England to work with MDA grantee Kay Davies at the University of Oxford on utrophin-related gene therapy strategies. Davies discovered utrophin in 1989, and since then she and many other MDA-supported researchers have been studying this protein.


Studies in mice suggest utrophin could work as well as dystrophin if it could be delivered to muscle or if its production in muscle could be increased. Utrophin is almost exactly like dystrophin, and its potential as a replacement for dystrophin has been apparent for a long time. Its great advantage over dystrophin, which might seem the most logical choice as a therapy for DMD, is that boys with the disorder already make utrophin, so their immune systems would almost certainly accept the protein and not see it as foreign.

Several MDA-supported mouse experiments have lent credence to utrophin's ability to replace dystrophin. Davies developed and tested mice bred with extra utrophin genes but lacking dystrophin in 1996 and expanded these experiments last year. The studies, published in the Nov. 28, 1996, issue of Nature and the November 1997 issue of Nature Medicine, showed that these mice had muscles that looked normal in every way, with the utrophin protein tucked into the right place in the muscle cell just the way dystrophin would be. Of equal importance, the mice actually behaved more like normal mice, including generating greater force in their muscles to escape having their tails gently pinched. Their muscles also regulated their calcium levels better than those of the mice without extra utrophin.

Other MDA-backed experiments, published in the Aug. 22, 1997, issue of Cell, showed that mice born lacking both utrophin and dystrophin were far more severely affected with muscular dystrophy than were mice that lacked only dystrophin. These experiments were interpreted to mean that utrophin has a compensating effect in dystrophin-deficient muscles.

In fact, production of utrophin is significantly increased in the muscles of dystrophin-deficient mice and in boys with DMD, indicating that there's a natural attempt by dystrophin-deficient muscles to compensate for dystrophin deficiency. The technical word for this increase is "upregulation." This natural upregulation doesn't seem to increase utrophin enough to protect dystrophin-deficient muscles from degenerating, but a therapy that increased utrophin still more would probably provide adequate compensation, researchers say.


MDA grantee George Karpati, a neurologist and neuroscientist at the Montreal Neurological Institute at McGill University in Montreal, recently performed gene transfer experiments with Davies and others to introduce utrophin genes into dystrophin-deficient mice.

The researchers inserted a utrophin gene into an adenoviral vector and then injected the vector into leg muscles of the mice. They found that abundant utrophin inserted itself properly into the muscle cells and that all the surrounding proteins that normally associate with dystrophin (see illustration, above) were also in the right place. They published their results in the January issue of Biochemical and Biophysical Research Communications.

"It looks like the surrogate is at least as good as the real McCoy, dystrophin," Karpati said. "In fact, utrophin may ultimately be the therapeutic choice for gene therapy of DMD."


But gene transfer isn't the only way to get extra utrophin into boys with DMD. Utrophin upregulation by manipulating utrophin genes that are already in muscle fibers (endogenous utrophin genes) is an equally promising therapeutic avenue.

Utrophin is coded for by a gene on chromosome 6 -- a gene that's completely normal in boys with DMD -- and many copies of the utrophin gene are present in all muscle cells.

MDA grantee Bernard Jasmin, a cell and molecular biologist at the University of Ottawa in Ontario, Canada, has been working with Davies and others to understand how the utrophin gene is normally regulated and how its regulation could be changed to both increase the amount and expand the location of utrophin in muscle cells and fibers. (Mature muscle cells are called fibers.)

Since the early 1990s, when utrophin studies began, it has become clear that utrophin and dystrophin share some important similarities, but they also have some differences.

The locations of utrophin and dystrophin make a crucial difference when thinking about utrophin as a substitute for dystrophin. Although both utrophin and dystrophin are found near the muscle fiber membrane, dystrophin is found all around the fiber, while utrophin is found in only one small section -- where nerve meets muscle, the neuromuscular junction. Interestingly, muscle fibers have the potential to produce utrophin all over their surfaces. They do it during fetal life in both humans and mice. But after the fetus is fully developed and the muscles and neuromuscular junctions are fully formed, something changes. It seems a switch is thrown somewhere, causing dystrophin to replace utrophin everywhere except at the neuromuscular junction, where utrophin remains along with dystrophin.

What boys with DMD need is utrophin all around their muscle fibers, not just in one place. How to get it there is the question of the day, one that several MDA teams are working hard to answer.

Jasmin's group is working on the area of the utrophin gene that serves as an "on switch," called a "promoter." The promoter is the area of the gene that responds to signals and allows the cell to start making the protein coded for by the gene. Jasmin has found an area inside the on switch, called an N box, that he says influences where utrophin will be found in the cell after it's made. The N box, a DNA sequence, increases production of utrophin at the neuromuscular junction. It doesn't, however, seem to stop utrophin production elsewhere in the fiber, which is what the researchers were hoping it would do. (If it had, then turning off the N box might have been a good way to get utrophin production re-established in the whole fiber.)

"The most straightforward interpretation would have been that the N box is a silencer for utrophin around the fiber, as it is with some other genes; but, based on our data, it does not appear to be so," Jasmin says. "I still think the N box is very important, but there are other areas of the gene that are also important. I think eventually we'll be able to lift the repression [downregulation] of the utrophin gene, so that it's expressed all over the fiber." Jasmin published recent papers about utrophin gene regulation in the March 28, 1997, and the Jan. 9, 1998, issues of the Journal of Biological Chemistry.

Another aspect of his work deals with the effect of a protein known as agrin, which is normally present in muscle and nerve and which Jasmin has now found increases utrophin production. Jasmin emphasizes that he doesn't think agrin would be practical as a drug for many reasons; he also says it's probably not agrin itself that directly turns on the utrophin gene, but rather that agrin is part of a chemical cascade that turns on utrophin. However, identifying what parts of the agrin molecule are specifically involved in this cascade may be an important piece of the utrophin regulation puzzle.

Jasmin says upregulating utrophin production at the gene is only part of the story. "What's also important is all the cell biology associated with utrophin," he notes. "The stability of the protein, where the protein is located in the cell, and everything that happens to it after it's produced from the gene is also crucial."

As to whether or not a utrophin upregulation strategy is a practical avenue toward DMD treatment, Jasmin says, "I think it's very practical, once we know a little more about how this gene is regulated and how the expression of utrophin is controlled at other stages. I think then we'll be able to design a specific intervention at one specific regulatory stage."