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Release: Feb. 19, 2002

UI study uses mini-proteins to repair cystic fibrosis defect in experimental cell systems

Cystic fibrosis (CF) is caused by genetic mutations in a gene that encodes the cystic fibrosis transmembrane regulator (CFTR) protein. Normal CFTR protein forms channels for chloride ions to leave cells. The controlled outflow of chloride ions maintains cells' electrolyte and fluid balance and allows bacteria to be cleared from the surface of airway cells. This action prevents infection, which is a leading cause of death in individuals with CF.

University of Iowa researchers have shown that shortened versions of the normal CFTR protein can function like the normal full-length protein in experimental models of CF disease. These findings may point the way towards strategies that could turn the potential of gene therapy for CF into a reality. The findings are reported in the Proceedings of the National Academy of Sciences online Feb. 19.

Gene therapy uses disabled viruses to deliver the correct version of a gene to cells. A shortened, functional CFTR protein would be useful for gene therapy of CF because it would allow the use of a particular disabled virus that is proving to be a safe, efficient gene therapy vector.

"We would like to be able to use adeno-associated virus (AAV) as a delivery vehicle or vector in gene therapy for CF," said Lynda S. Ostedgaard, Ph.D., UI associate research scientist in internal medicine, and lead author of the study. "This is a really nice vector. It can enter airway cells, it has a track record with other genetic diseases and, to date, its safety profile is encouraging."

However, despite its advantages as a gene therapy vector, AAV is too small to accommodate the normal CFTR gene.

"Our goal is to make a short, functional CFTR gene with a view to making it short enough to fit into AAV," said Ostedgaard.

The research team made a series of mini-CFTR genes that produce shortened protein in airway cells. Specifically, they deleted parts of the CFTR protein from a region called the R domain. The UI team used two model CF cell systems to test the ability of the shortened proteins to act like normal, full-length CFTR in airway cells. These experimental models are designed to mimic, as closely as possible, airway cells in humans with CF.

"One model uses airway cells donated from individuals who have CF. These are exactly the cells that a gene therapy treatment would target to correct the faulty protein," Ostedgaard explained. "We often get these cells from individuals with CF who undergo lung transplants. Without those people being willing to make donations, we would never be able to do these types of studies."

The second model used mice genetically engineered to lack the normal CFTR protein.

Ostedgaard and her colleagues had previously discovered that portions of the R domain could be deleted. However, they also found that a few specific residues were required for function of the chloride channel.

"Based on our earlier work, we hypothesized that if you took out much of the R domain but kept a few of the critical sites, the shortened proteins should function properly," Ostedgaard said.

In both models, the researchers found that each mini-gene made protein with all the properties and function of the normal full-length protein. In fact, two of the shortened proteins transported chloride as well as normal CFTR.

"We proved that we could actually take out a fairly significant part of the domain without affecting the protein's ability to transport chloride ions and without affecting its ability to get to the right place in the cell to start working," Ostedgaard said.

Ostedgaard added that it was satisfying and exciting when their hypotheses were confirmed by the experiments. However, she indicated that while the results were a step towards producing a working CFTR protein small enough to use in AAV, the research had not yet reached that point. Additional studies are still required to generate an AAV vector that could prove useful in CF gene therapy.

This type of research also leads to a better understanding of the structure of this important CFTR protein, which could influence development of drug therapies to treat CF.

In addition to Ostedgaard, the UI researchers involved in the study included Michael J. Welsh, M.D., the Roy J. Carver Chair in Physiology and Biophysics, professor of internal medicine and physiology and biophysics, and a Howard Hughes Medical Institute investigator; Joseph Zabner, M.D., associate professor of internal medicine; Christoph Randak, M.D., Ph.D., a postdoctoral fellow in Welsh's lab; and Daniel W. Vermeer, Tatiana Rokhlina and Philip H. Karp all research associates. Arlene A. Stecenko, M.D., associate professor of medicine at Vanderbilt University also was part of the team.

The study was funded in part by grants from the National Heart, Lung and Blood Institute (one of the National Institutes of Health) and the Cystic Fibrosis Foundation (through the UI Center for Gene Therapy of Cystic Fibrosis and Other Genetic Diseases).

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