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

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

“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.
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