June 1, 2000 Medical researchers trying to perfect gene therapy face a major obstacle in inserting corrected genes into cell nuclei where they can direct production of healthy proteins that the defective cellular machinery cannot make.
In the past, scientists have used weakened viruses as vectors to carry working genes into cells, but so far the body's sophisticated natural defenses against invaders -- especially in the lung -- have limited their effectiveness.
Now, University of North Carolina at Chapel Hill School of Medicine scientists have found what they think could be a big improvement. For the first time, they have targeted a special class of receptor molecules on the surfaces of airway cells. Their experiments show that when activated, a G-protein-coupled receptor can work like a revolving door allowing weakened viruses into cells, along with their attached therapeutic payload.
A report on the research appears in the June issue of Nature Biotechnology. Authors are Drs. Silvia M. Kreda, Raymond J. Pickles and Eduardo R. Lazarowski, all research associates in medicine, and Dr. Richard C. Boucher, Kenan professor of medicine.
"We linked an adenovirus vector to a small signaling molecule known as UTP that interacts with the receptor, and we obtained specific gene transfer in human airway cells," Kreda said. "This strategy is flexible and conceptually could allow us to successfully correct other cell types or use different gene therapy vectors."
Boucher, director of UNC-CH's Cystic Fibrosis Center, likened vectors to mail delivery trucks moving valuable cargo. He also called the strategy "the classic Trojan Horse approach" because it tricks the mostly impenetrable cell surfaces. Their success sets the stage for developing a system with clinical applications.
"You have to make the virus look like something else that can attach to airway cells and have the capacity to penetrate inside," he said. "Dr. Kreda's paper shows there actually are these receptors on the side of cells facing the airways and that if you can dress up viruses with UTP, you can fool the cells into opening their doors long enough for the virus to sneak in."
The scientists are now attempting to boost the vector's efficiency in entering cells, Boucher said. After that, they will try to correct defective cystic fibrosis cells in tissue culture, in mouse models of cystic fibrosis pioneered at UNC-CH and, eventually, in human volunteers.
"All of us are very keen to the notion that you have to be careful and very safe when attempting gene therapy studies in humans," he said. "It's only when people try to rush ahead and cut corners do problems arise."
Common on the airway side of cells lining the lungs, the G-protein-coupled receptor the UNC-CH team targets is a purinergic receptor known as P2Y2, Kreda said. Scientists want to correct the CFTR gene, which malfunctions in people with cystic fibrosis, so patients' lungs can maintain the proper balance of salt and water. Otherwise, lung secretions become too thick and sticky, resulting in a series of debilitating infections and premature death.
In the current study, she and colleagues did not transfer the cystic fibrosis gene but instead used "reporter" genes, which are easier to measure inside cells using fluorescent technology and enzymatic activity levels.
"This is exciting and we think very promising and important because we were able to break one of the barriers epithelial cells offer to viral vectors," Kreda said.
The National Institutes of Health and the Cystic Fibrosis Foundation supported the continuing research.
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