Feb. 16, 1999 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.
They believe their achievement overcomes a major barrier to widespread use of adenovirus, a common cold virus, as a genetic delivery vehicle.
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.
"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.
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.
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.
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.
"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.
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.
"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.
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.
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.
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.
"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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