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August 2006--As a boy, Bill Guggino loved the ocean. So when he became a graduate student and needed a research project, he thought back to a question he had pondered as a child: “How do fish live in the sea?” As a result, he began studying the chloride-secreting cells that enable fish to maintain homeostasis in a salty environment.
Three decades later, Guggino—now the chair of the Department of Physiology here—is not only an international authority on chloride transport, but also a key figure in the development of a gene therapy for cystic fibrosis.
“The protein (CFTR) that’s involved in secreting chloride in fish is the homologue to the one that’s defective in CF,” he points out. “So I’ve continued to study the basic science of this chloride channel, how it functions normally, how it traffics the membrane, and especially how it malfunctions in that disease.”
Despite a vastly improved quality of life and recent increases in the lifespan of CF patients due to gains in managing their repeated lung infections, Guggino says “a breakthrough still needs to come at the basic level, one that attacks the fundamental problem of the mutant gene and its defective protein.”
CF is a monogenetic disorder with a well-characterized etiology; mutations in the CFTR gene create flawed chloride channels that prevent the thin layer of fluid that typically lines mucosal membranes from forming.
“Cells don’t secrete salt onto the surface,” Guggino says, “and that keeps osmosis from creating the fluid layer.”
In airways, lack of fluid, in turn, thickens patients’ respiratory mucus, creating a breeding ground for repeated infections, scarring and, finally, end-stage lung disease.
Approximately 70 percent of people with CF share the same gene mutation, but the other 30 percent exhibit up to 1,400 different ones. Some of these alternate mutations, identified by geneticist Garry Cutting, a collaborator, “have provided us a rich understanding of how the protein traffics to membrane and clarified important domains involved in function,” Guggino says.
For example, Cutting found that the mutation S1455X causes a premature termination of CFTR, eliminating the last 25 amino acids. Guggino’s team then began focusing on the last amino acids of intact CFTR and identified a new protein, CFTR-associated ligand (CAL), that binds to them and regulates CFTR trafficking to the plasma membrane. The mutation, of course, disrupts this process.
Investigating a more usual mutation of the gene F508 CFTR, which results in a misfolded protein, prompted work with Pamela Zeitlin on protein repair. Zeitlin, a pediatric pulmonologist, and Guggino hope to develop strategies to prevent CFTR degradation, allowing the protein to reach the membrane. Zeitlin’s team has also been searching for an alternate chloride pathway, one temporarily used by the fetus to produce fluid in the lungs in utero. Recently, a high-throughput screen identified over 80 compounds with the potential to activate that dormant pathway. Guggino’s lab is testing those compounds on a human cell line developed by Zeitlin.
The translational impact of basic research in the field has been almost immediate: “Every two years of research extends the lifespan about six months,” Guggino says, “and that’s fantastic.”
Still, he would like to avert respiratory damage entirely by inserting a normal copy of the gene before chronic disease takes hold.
Guggino uses adeno-associated virus (AAV) as the vector in his gene therapy protocols. Its great virtue is safety. The downside is that it is also very small—“it just fits CFTR” says Guggino—and not very potent. He and colleagues are trying strategies to boost expression of the gene, including adding a promoter, tweaking capsid proteins and exploring more potent serotypes. Despite its limitations, AAV still looks like the best vector in his view—adenovirus having proved highly toxic and nonviral vectors not nearly potent enough.
“Viruses know how to bind to a receptor, avoid destruction in the cell and deposit DNA in the nucleus,” he says. “That whole process has not yet been replicated in these nonviral synthetic compounds.”
Guggino—who has not only trained a generation of clinician scientists working on CF but also practiced a highly collaborative multidisciplinary research style long before it became fashionable—says that despite the translational studies, he remains a bench scientist at heart.
“Since the mid-1980s, we’ve worked closely with the clinical departments,” he says. “But basic science is our major focus.”
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