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- DURHAM, N.C. - Duke University Medical Center researchers report that they
have modified a common virus so that it can carry corrective genes to defective
cells without stimulating an immune response.
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- They believe their achievement overcomes
a major barrier to widespread use of adenovirus, a common cold virus, as
a genetic delivery vehicle.
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- Their results were published in the February
issue of the journal Human Gene Therapy. The research was supported by
grants from the National Institutes of Health, the Muscular Dystrophy Association
and the Howard Hughes Medical Institute.
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- "In our set of experiments, we were
not only able to deliver the gene to the intended site, but it also persisted
for more than two months," said Dr. Andrea Amalfitano, a Duke pediatric
geneticist who led the team. "If our approach is confirmed by further
studies, these modified adenoviruses could have great application for future
gene therapy in humans." Also on Amalfitano's research team were Duke's
Huimin Hu, Ph.D. and Delila Serra.
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- Scientists have long recognized that
the ubiquitous adenovirus offers many advantages as a "vector"
for gene therapy. It can carry practically any size gene, it can infect
virtually all cells in the body, and it can be easily mass-produced. However
until now, within two to three weeks of the virus's introduction into the
body, the immune system of recipients easily recognized the virus and its
gene payload as foreign and cleared it from the body, as well as the cells
the virus had infected.As a result of their findings, the Duke scientists
propose a "two-hit" hypothesis to explain why other adenovirus
vectors usually failed.
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- The first immune-system "hit"
after an adenovirus infection comes when the animal's immune system recognizes
the virus as foreign and attacks it, Amalfitano said. The second "hit"
comes when the immune system then recognizes the introduced gene, called
a transgene, as foreign and attacks it. Only after both hits occur does
the immune system clear the virus from the body, along with the infected
cells and the transgene.
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- Amalfitano and his colleagues designed
their "stealth" adenovirus to evade the first hit altogether.
As in previous attempts to use the adenovirus as a vector, they first deleted
from the virus the "E1" gene -- which is the first in a series
of genes the virus needs to copy itself. However, Amalfitano also deleted
a second, similar gene farther down the same pathway.
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- "This double deletion makes the
virus 'quieter' to the immune system, and allows it to deliver the transgene
unnoticed," Amalfitano said. To track the infective ability of their
altered adenovirus, they inserted into it a gene that turns blue any cell
it infects.
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- After the researchers injected this engineered
adenovirus into mice with intact immune systems, "within three days,
the modified virus had infected every single liver cell," Amalfitano
reported. Since the liver screens all blood within the body, adenovirus
infections tend to concentrate in that organ, he said. Subsequently, as
indicated by the blue marker, the modified virus persisted in the livers
of the treated animals for greater than two months. In contrast, the virus
was essentially eliminated within three weeks from the livers of animals
in a control experiment using an unmodified adenovirus vector.
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- "Our modification of the adenovirus
appears to have not stimulated an immune response," said Amalfitano,
which means the first hit never occurred. Since Amalfitano's hypothesis
holds that both hits are necessary to completely clear the virus and transgene
from the body, the modified virus and the gene persisted.
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- Amalfitano said that proving their technique's
usefulness means conducting more tests of the engineered virus using different
genes in different animal models, but he is confident that his experiments
demonstrated that appropriately modified adenovirus vectors are an effective
delivery system for gene therapy.
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- Since adenovirus easily infects every
type of cell except bone marrow cells, Amalfitano believes that adenoviruses
will play an important role in gene therapy for many diseases in the future.
The improved adenoviruses will certainly help experimental adenovirus-mediated
therapies become clinical reality, he said.
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- Amalfitano pointed out, however, that
adenovirus will likely become only one of many gene delivery agents. Scientists
have more than 250 projects currently underway to test the usefulness in
genetic therapy of adenoviruses, retroviruses, adeno-associated viruses,
and herpes virus, as well as non-viral systems to deliver genes into humans,
he said. Each of the gene-delivery systems has its own benefits and drawbacks
in such areas as capacity for carrying genes, infectability, scalability,
persistence, and stability, Amalfitano said.
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- "Gene therapy will become very much
like cancer therapy," he said. "There are many different kinds
of cancer, and each is treated with different combinations of surgery,
chemotherapy and radiation. I believe that as we perfect gene therapy,
we will use different gene therapy systems for different diseases."
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