Racing toward Gene Therapy: After years of dashed hopes, researchers are poised to start clinical trials for muscular dystrophy

Neurology Now
January/February 2006
Volume 2(1)
p 22–30
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When 4-year-old Ashanthi DeSilva became the first person to undergo gene therapy in 1990, many believed the next frontier in medicine had arrived. By replacing damaged genes with healthy ones, gene therapy held the promise of overcoming a wide range of previously incurable diseases that have an inherited component, from cancer to muscular dystrophy.

But it is only now, more than 15 years after the revolutionary procedure, that researchers are gearing up to perform human studies of gene therapy for Duchenne muscular dystrophy, an inherited muscle-wasting disease. Given that the genetic defect that causes Duchenne — the most common lethal inherited disease in children — was identified 20 years ago, what took so long?

It certainly wasn't that first 1990 experiment: that was widely recognized as a success. Ashanthi DeSilva suffered from an extremely rare and deadly inherited disorder called adenosine deaminase (ADA) deficiency. Lacking a functioning ADA gene, children with the disorder cannot make an enzyme that cells use to get rid of toxic byproducts, severely weakening their immune system and leaving them susceptible to even the most minor of infections. Her doctors inserted the gene for the ADA enzyme, which she was missing, into blood cells taken from her circulation. The cells, carrying the new genes, were infused back into her body. The defect was corrected, and the sick little girl got better.

The successful experiment sparked dozens of human trials designed to test gene therapy against a wide range of inherited diseases. Most of the trials followed the same basic recipe: First, researchers isolate the disease-related gene. Then they package the gene into a delivery vehicle called a vector, typically a disabled virus that cannot reproduce and cause disease but can act like a missile to deliver its genetic cargo to its target. Doctors then inject the vector into the body, usually into tissue affected by the illness, such as muscle cells in the case of Duchenne. The vector unloads its genetic payload, which then begins producing the missing protein and restores the cells to normal. The patient gets better.

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Figure. Two scientists are using two different methods in pursuit of the same goal: gene therapy as a cure for the most common lethal inherited disorder in childhood.

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That was in theory. Unfortunately, things didn't always turn out that way. Very few of the trials were successful. Hyperbole trumped reality. Hopes were dashed.

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Then came an even more serious setback: In 1999, Jesse Gelsinger, a teenager with a rare metabolic disorder, died after receiving an experimental gene therapy. “Clinical trials stopped; there was a lot of backlash,” says Richard Jude Samulski, Ph.D., a gene therapy specialist at the University of North Carolina–Chapel Hill. “This slowed everything down dramatically.”

Figure. A new gene is packaged into a delivery vehicle called a vector, which is used to introduce the modified DNA into a human cell. If the treatment is successful, the new gene will make a corrected protein.

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Things were to get worse before they got better. In early 2003, the Food and Drug Admin istration suspended 30 more trials after two children in a French gene therapy study developed leukemia. Soon afterward, a gene therapy trial for hemo philia was stopped after one participant showed signs of liver damage.

But Dr. Samulski was not deterred. In fact, he says the setbacks gave basic researchers like him time to explore better and safer ways of getting new genes into cells. “Many of the early problems could be traced to the delivery system,” he says. The first-generation gene delivery systems — typically disabled viruses known as adenoviruses or retroviruses — often caused toxic effects, he explains.

The researchers' perseverance paid off. Last year, the Muscular Dystrophy Association (MDA) awarded grants to three teams of American researchers, including one led by Dr. Samulski, to help fund human trials of gene therapy for Duchenne muscular dystrophy.

The race is on.

“It's a really exciting time,” says Paula Clemens, M.D., a gene therapy expert at the University of Pittsburgh who is not involved in the trials. “It's still very early, but the researchers are working on approaches that could correct the underlying problem, the mutated gene. If it works, we may someday be able to get the corrected genes into muscle cells early enough to prevent damage.”

Duchenne muscular dystrophy is a perfect candidate disease for gene therapy. It causes progressive weakening of all the muscles in the body, and there is no cure or effective long-term treatment. It's fairly common: One in 3,500 males born worldwide develops Duchenne, and it doesn't discriminate by race or ethnicity. Most relevant, it is caused by a known genetic defect — one that keeps the body from making a critical protein called dystrophin that keeps the muscles structurally strong.

“This is the protein responsible for the integrity of the muscle cell membrane,” explains Dr. Samulski. In people with the flawed gene, the membrane becomes leaky, causing an inflammatory reaction as white blood cells rush in to repair the damage. The result: a vicious cycle in which muscle degenerates and the immune system tries to clean up the damage, causing more inflammation and further muscle weakness.

Learning from past mistakes in the field, all three teams of MDA-backed researchers believe they have created new vectors that will be safer and more effective than the old ones. And they have new ways of delivering the gene in what they believe will be sufficient quantities to stop the disease in its tracks.

Each team has a slightly different approach, but all have to overcome the same hurdles.

First is the sheer size of the faulty gene. The dystrophin gene is so huge — two to seven times bigger than typical genes — that packaging it into a vector is problematic, says Dr. Samulski. Then, they must pick the most efficient and safest way to deliver the genetic cargo back into muscle.

To overcome the first hurdle, Samulski and another team led by Jeffrey S. Chamberlain, Ph.D., are working with miniaturized versions of the dystrophin gene.

To create a mini-gene, Dr. Chamberlain has identified the parts of the protein that are critical for activity and chopped out the rest. The cut-and-paste version they now have is about one-third the size of the gargantuan gene, says Dr. Chamberlain, a professor of neurogenetics, biochemistry and medical genetics at the University of Washington in Seattle.

The real obstacle is getting the gene into muscle cells throughout the body, he says. To that end, his team has chosen the adeno-associated virus (AAV) as their vector due to a couple of advantages: It persists in muscle for a long time and is not rejected as foreign by the immune system — rejection that can interfere with delivery and even cause harm.

In mouse experiments published in 2004, Dr. Chamberlain and colleagues showed that giving a single injection of an AAV vector carrying a dystrophin minigene into the bloodstream produced dramatic improvement of dystrophy in muscles throughout the body. “We went directly into the bloodstream and targeted all affected muscle cells — an approach with tremendous impact,” he says. “The mice did not regain complete strength, but they regained a lot.”

In humans, too, the ultimate goal is to deliver the cargo right into the bloodstream so that the gene is delivered to all the muscle cells in the body. But in terms of clinical trials, they will start with a single intramuscular injection — a shot in the arm or leg, as with a vaccination — and treat just a limited area of the limb. “We have to make sure it is safe, not harmful, before moving on,” explains Dr. Chamberlain. Also, they need to determine if the gene makes a lot of dystrophin protein at the site of injection. His team is now working out regulatory wrinkles and hopes to start the trials within two years.

Dr. Samulski's team, too, is taking advantage of a miniaturized version of the gene for dystrophin. It's a slightly different mini-gene, discovered by his former postgraduate student Xiao Xiao, Ph.D., at the University of Pittsburgh. “People with this dystrophin mini-gene develop a milder form of Duchenne; they have muscle weakness but not as severe as the Duchenne boys,” he says. “Some people are walking around with this imperfect gene and still have a pretty good life.”

What Dr. Xiao created in the lab, he says, was a dystrophin mini-gene that would mimic the gigantic gene sufficiently enough to rescue the defect in boys with Duchenne.

Figure. Can the gene therapy breakthrough that long ago cured the 9-year-old artist of her immune system disorder finally be applied to muscular dystrophy?

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In animal tests, it worked. They put the gene into a viral vector and injected it into animals with a Duchenne-like illness. The animals not only regained their muscle strength, but also lived longer than animals that didn't get the treatment.

That still left Dr. Samulski and his team with the problem of how to best get the mini-gene into a person's body. They too started with AAV, which he says is the only known DNA virus that does not cause disease. And then they modified it into what he believes is an even more efficient delivery system.

“Think of AAV as a [delivery] truck,” he explains. “Viruses have zip codes so they will go to different parts of the body. We put a zip code on our AAV to make it go to muscle cells. Called a biological nanoparticle, or BNP, the virus was engineered to become more likely to go to muscle cells.”

Again, animal tests worked. “BNP delivered the injected mini-gene straight to the muscle,” he says. “The rodents got better and lived longer.” The researchers are now monitoring the animals for any toxic effects that might only develop over time. Pending FDA acceptance of safety data, he hopes to start human safety studies by February.

The first clinical trial will include six patients whose muscles will be injected with one or two doses of the mini-gene. “Then we'll wait one month and do a muscle biopsy,” he says. “That will tell us whether the gene started producing dystrophin and whether it was safe.”

Jon Wolff, M.D, professor of pediatrics and medical genetics at the University of Wisconsin–Madison, is using a different approach from either of the other two MDA-funded researchers.

Dr. Wolff is not using a vector at all: instead he plans to inject so-called “naked” DNA — the building blocks of genes — right into boys with Duchenne. One advantage, he says, is that full gigantic gene can be injected. The second, of course, is that no viral vector is needed — an approach he thinks will reduce the risk of toxic effects.

Even though AAV and synthetic versions of AAV seem to be safe, there is always the possibility of unwanted reactions, he explains. That's because any time the body sees a foreign invader — whether a cold virus or an AAV — it tries to mount an immune response to fight off the enemy.

“We've been testing non-viral delivery systems since 1989, as we are worried that viruses can cause unwanted immune responses that can make a person sick,” says Dr. Wolff.

In a preliminary study conducted in France, injecting naked dystrophin directly into the arm muscles of nine boys with Duchenne and Becker muscular dystrophy proved safe and was associated with an increase in dystrophin production. Dr. Wolff hopes to begin a U.S. safety trial this year.

Dr. Samulski believes the viral delivery system has advantages over using naked DNA. “It persists in the cell for much longer — six years and counting in monkeys, and perhaps forever,” he says. “So the virus could be a one-time treatment, while naked DNA has to be injected over and over.”

If any of the approaches proves safe, the researchers will move on to larger, longer studies to confirm safety and efficacy.

“This will not be a cure,” says Dr. Wolff.

“But we will be able to preserve arm and hand function and improve people's quality of life.”

Echoes Dr. Samulski, “We can't undo damage that's already done with these approaches, just preserve the function and integrity of existing muscle cells. The earlier the intervention, the better the outcome.”

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