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Release: June 12, 2000

Breakthrough technologies improve prospects for gene therapy at UI center

IOWA CITY, Iowa -- Researchers at the University of Iowa have recently reported three breakthroughs in the development of gene therapies for cystic fibrosis (CF) and other genetic diseases.

Gene therapy seeks to cure genetic diseases by replacing defective or disabled genes with a corrected gene. The correct genes are transported into cells using certain viruses.

"These gene carrying vectors can be likened to a truck carrying its genetic cargo (DNA) into cells where it can correct a genetic defect," said John Engelhardt, Ph.D., associate professor of anatomy and cell biology, and internal medicine, and director of the UI Gene Therapy Core Center.

Recent research in Engelhardt's laboratory has focused on improving one of those vectors, Adeno-associated virus (also called AAV). Over the past year AAV has attracted great interest from gene therapists and was highlighted this month at the American Society for Gene Therapy meeting because of its early success in gene therapy clinical trials for Factor IX deficiency, a blood clotting disorder. One important benefit of this virus is that it has never been linked with any human disease. In addition, the genetically engineered AAV that Engelhardt is using has had all of its viral genes removed.

However, despite the great promise of AAV, a major limitation is that only relatively small disease genes can be carried. Research conducted in Engelhardt's lab has resulted in two new strategies to overcome this limitation. Both strategies proposed to expand the size of the genes that can be delivered with this vector system.

"In essence, we have created the 'extended cab' version of a pickup truck except in a miniature version at the virus level," Engelhardt said.

In the first of these strategies developed by Dongsheng Duan, Ph.D., a research scientist working with Engelhardt, two independent versions of the virus were delivered to the same cell, one containing the genetic information coding for a protein and one containing genetic material which enlists the cell's protein-making machinery and controls when and how much of that protein is made. After the two viruses simultaneously enter a cell, their genetic material "joins hands" and allows for high-level production of the therapeutic protein. This is particularly attractive for the treatment of a disease such as cystic fibrosis, where the defective gene can barely fit into one virus and leaves no room for the control elements that are required to get the therapeutic protein produced.

Engelhardt cautioned that one potential problem is that the control elements might end up in the host genome and inappropriately control the production of other proteins, causing problems. He said that further testing would be needed but at least in muscle, they have not seen this problem occur.

Ziying Yan, Ph.D., another research scientist in Engelhardt's group, reported a second breakthrough in engineering these viral vectors, described in the June 6 issue of the Proceedings of the National Academy of Sciences, which uses a similar strategy to deliver very large genes. In this case the protein-coding part of the disease gene itself is split between the two virus vectors. This strategy is unique because it could allow the use of AAV to treat diseases such as Duchenne Muscular Dystrophy where the defective gene is much too large to fit into one vector.

"This strategy has greatly enhanced the prospect of gene therapy with AAV for numerous diseases not previously approachable," Engelhardt said. "A further advantage of this approach is the potential to deliver all the genetic information necessary to tell the cell when and how much of the protein to manufacture (so called promoter regulatory information)." Often this genetic regulatory information takes up too much space in the current vector design to be included. The new strategy has found a way to avoid this space limitation.

In addition to these two related advances, a third discovery has also recently been reported by Engelhardt's lab. This finding has tremendous implications for the use of AAV virus in treating CF. UI researchers have figured out why AAV, even though it enters cells of the lung, is incapable of producing its therapeutic protein.

For gene therapy to work, the virus must deliver its genetic cargo to the nucleus of the cell where the therapeutic protein is produced. In the case of the airway cells, the virus is intercepted and rerouted for disposal. The cell tags the virus with a molecule called ubiquitin, which marks it for removal.

"In essence, this tag is the equivalent of a curbside trash pick-up sticker which reroutes the virus to regions of the cell for garbage disposals," Engelhardt said. Duan and colleagues discovered that if they interfere with this "tagging" it greatly increased the ability of the virus to get to the nucleus and produce its protein, hence allowing for successful therapy. The chemical compounds used by the researchers to block either the tagging itself or the disposal process are non-toxic to cells and hence are of great interest to researchers and companies performing gene therapy for lung diseases such as CF.

"To date, clinical trials for cystic fibrosis with this virus have met with only moderate success," Engelhardt said. "Now we feel we have found a key to let the virus in through the back door so it can express its therapeutic protein efficiently and treat disease."

Interestingly, this approach also seems to increase gene delivery to organs other than the lung. These findings appeared in the June issue of the Journal of Clinical Investigation.

Funding for this research was provided by the National Institute of Health's National Heart, Lung and Blood Institute, and the Gene Therapy Core Center is funded by the National Institute of Diabetes and Digestive and Kidney Diseases and the Cystic Fibrosis Foundation.

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