Gene Therapy for CF.

New research on gene replacement therapy offers the hope of a cure for cystic fibrosis (CF). This fatal disease, symptomized by coughing, wheezing, lung infections, very salty sweat, and excessive appetite with inability to gain weight, affects about 30,000 Americans.

CF manifests when a person receives a mutated copy of the CFTR gene from both parents. More than 10 million people carry one copy of the defective gene. Most CF patients die in childhood, but some live into their 30s or 40s. Advances in drug therapies and other treatments such as lung transplants have improved the quality of life of CF patients in recent years, but still do not offer a cure.

Because lung infections are responsible for most CF fatalities, research into gene therapy for treating the disease has initially focused on replacing the mutant genes in the epithelial cells lining the lungs and nasal passages. But the human body has evolved a complex system to prevent invasion of foreign DNA, and the lungs have a particularly strong defense system. So scientists need to find a way to "trick" the cells into accepting replacement genes. In a report in the June 2000 issue of Nature Biotechnology, Silvia M. Kreda, a clinical research associate of medicine at the University of North Carolina at Chapel Hill, and colleagues describe research with a particular type of airway receptor molecule that may offer a reliable pathway into cells.

Instead of producing the normal protein that forms channels in cell membranes for the passage of salt out of cells, people with CF produce a faulty protein, which causes a thick mucus that impairs function of the lungs, intestines, and other organs. If healthy genes can be transferred into the CF patient's cells, theoretically they should produce the protein product necessary to form the channels for proper electrolyte transport out of the airway cells.

Kreda and colleagues identified the P2[Y.sub.2] molecule receptor on the membrane surface of the lungs as a potential target for shuttling healthy genes into the airway passages. Their research strategy was to combine the nucleotide UTP, which binds to certain receptor molecules on the outside of the epithelial cells, with a commonly used vector known as an adenovirus. Using reporter genes to signal a successful transgenic event, Kreda's group demonstrated that UTP linked to the vector and facilitated its uptake into the cell. Additional experiments revealed that when UTP was linked to an adenovirus vector, in vitro transfer of the reporter gene into human lung cells was successful.

Kreda emphasizes that her lab is working on a model system for improving entry mechanisms in gene replacement. Before clinical trials with humans can be considered, however, vector construction needs to be refined for improved efficiency. Richard C. Boucher, the principal investigator for the study, says that although UTP is effective at binding to receptor sites on the membrane, it is rapidly degraded by the body's enzymes and is therefore not efficient for delivering the gene payload. Future in vitro experiments using the CFTR gene will measure channel activity to determine gene therapy's ability to correct the mutant gene. When the in vitro system is perfected, efficacy will be tested in animal models. A reporter gene will be transferred via the adenovirus vector into normal mice to verify gene expression. Then the CFTR gene will be introduced into knockout mice to determine whether the corrected gene is accepted into live cells in the respiratory tract.

The adenovirus, a DNA-containing virus that causes upper respiratory tract infections, is widely used as a vector in CF research because it is an established model that works well. It can pose a danger of infection to patients, however, and research is ongoing to address the safety issues of using adenovirus vectors in human studies, and to find safer vectors for gene replacement therapy. In September 2000, a panel of the American Association for the Advancement of Science called for a moratorium on gene-altering research and for the government to establish an oversight board as soon as possible.