Scientists bypass major hurdle to hemophilia gene therapy

December 03, 2000

Scientists bypass major hurdle to hemophilia gene therapy; animal studies yield high levels of clotting protein

CHAPEL HILL - For the first time, scientists at the University of North Carolina at Chapel Hill have used a gene-therapy technique in animals to continually produce very high amounts of a clotting protein similar to that lacking in people with hemophilia.

A lack of this protein, known as factor IX, occurs in hemophilia type B. If human gene therapy studies could yield sustained factor IX production in high amounts, then hemophilia patients would no longer need daily injections of the protein. The new research suggests this might someday prove feasible.

Moreover, the new findings also indicate that the gene-therapy method used in the study may be applied to hemophilia A, the more common form of the disease. And a report published Dec. 4 in the journal Molecular Therapy concludes that the approach "may be useful for the treatment of a wide variety of inherited diseases."

In animal experiments at Carolina and elsewhere in recent years, the method involved a genetically engineered virus called AAV to infect cells and thereby deliver a cloned gene into an animal's body. Previous studies used only type 2 of six known AAV serotypes, each of which differ in their protein wrapper. This time, however, the Carolina researchers tried five of the six, comparing factor IX production of AAV types 1, 3, 4 and 5 with that of type 2. The results were startling.

"Surprisingly and unexpectedly, we found the mice were producing amounts of this factor 100 to a thousand times more than we've observed before," said senior study author Dr. Christopher E. Walsh, assistant professor of medicine at the UNC-CH School of Medicine and clinical director of the university's Gene Therapy Center.

"The purpose of this experiment was to see if we could generate very high protein production long-term," he added. "These mice are now six months out and factor IX levels have remained very high with no problems."

The mice studied were genetically bred to have deficient immune system cells. They were selected because the clotting protein coded by the transferred gene was canine factor IX. In normal mice, the immune system would mount a destructive attack against the protein. In previous experiments, UNC-CH gene therapy researchers studied dogs with hemophilia B, which resembles the disease in humans. They gave recombinant AAV carrying canine factor IX to the dogs via direct intramuscular injections - similar to immunization shots, such as tetanus. In two dogs, a simple clotting test determined that the gene was working, making protein in the muscle that circulated in the blood. The animals also were at reduced risk of bleeding. However, the amount of protein produced represented 2 to 4 percent of the normal amount of factor IX in the blood both for dogs and humans.

"Our new research bypasses a major hurdle in gene therapy for hemophilia, but it also represents the beginning of a long series of experiments," Walsh said. "We know that the one particular virus we've used for years now works in mice, rabbits, dogs and primates. So since these are just slightly different variants, the assumption is they should work the same way.

"And if in fact you can generate significantly more protein, into the more therapeutic ranges, and without the immune system seeing it as foreign, then you could conceivably correct the disease."

While the UNC-CH research demonstrates that the approach works in muscle tissue, might it also work in other organs, the brain, liver, or kidney, for instance?

"My suspicion is the answer is yes," Walsh said. "By changing a few proteins on the surface, we can now direct these viruses to places where we didn't think they could go before and produce a lot of protein. You may be able to replace factor IX with fill-in-the-blank, whatever the body needs."

The gene therapy team is studying animals with active immune systems, using the AAV variants to deliver the gene for canine factor IX into dogs.

"It's the results of these studies that the U.S. Food and Drug Administration will use as a benchmark for determining whether or not clinical trials are warranted," said co-author Dr. Richard J. Samulski, professor of pharmacology and director of the Gene Therapy Center. "I think we'll generate more functional protein than is needed clinically."

Besides Walsh and Samulski, study co-authors include Gene Therapy Center postdoctoral research associates Drs. Hengjun Chao, principal author; Yaunbo Liu; Joseph Rabinowitz, and Chengwen Li. The research is funded by grants from the National Institutes of Health.

Hemophilia B is a hereditary bleeding disease that affects more than 4,000 people in the United States. The disease, which affects males, results in bleeding principally in the muscles and joints. However, internal bleeding, especially in the brain, can lead to death.

Treatment for the disease involves intravenous injections of blood products or, more recently, purified factor IX. Traditional transfusion treatment improves symptoms but fails to cure.
Note to media: Drs. Christopher Walsh and Richard J. Samulski can be reached at 919-962-3285.
School of Medicine contact: Les Lang, 919-843-9687 or

University of North Carolina Health Care

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