Cystic fibrosis splicing experiments offer hope for improved treatments

December 15, 1999

CHAPEL HILL -- Although not yet ready to try their new technique in patients, University of North Carolina at Chapel Hill scientists have successfully repaired a genetic problem that accounts for a form of cystic fibrosis commonly seen in patients of European Jewish descent.

Drs. Kenneth Friedman, research fellow in pathology and laboratory medicine, and Ryszard Kole, professor of pharmacology, at the UNC-CH School of Medicine developed the method to correct a mutation involving unnecessary information inside a gene. When that gene is imperfect, cystic fibrosis results.

The two conducted the research in collaboration with Drs. Lawrence Silverman and Michael Knowles at UNC-CH and Dr. Jonathan Cohn and technician Jolanta Kole at Duke University.

"This work builds on a strategy first explored by Dr. Kole in his studies of beta-thalassemia, a genetic disease often seen in Mediterranean and some Asian countries and involving defective hemoglobin molecules that lead to red blood cell destruction in bone marrow," Friedman said. "We applied his method to a cystic fibrosis gene mutation discovered here at UNC-CH in 1991 and given the somewhat arcane name 3849+10kbC>T."

That mutation results in a milder-than-most, but still debilitating, form of the illness and many years of chronic lung problems, he said. Cystic fibrosis, the most common lethal inherited disease among whites, is a complicated and highly variable condition resulting from errors in the gene controlling salt and water balance in the lungs and other tissues.

A report on their work appears in the Dec. 17 issue of the Journal of Biological Chemistry.

"We first genetically engineered healthy cells in laboratory culture to carry a version of this mutation, and, as expected, messenger RNA production in these cells was abnormal," Friedman said. "We then explored whether the effects of this mutation could be overcome by administration of pharmacological agents collectively known as 'antisense.'"

Messenger RNA molecules carry copies of genetic instructions from the DNA "blueprints" in a cell nucleus into the cell's cytoplasm, where proteins are produced in factory-like fashion, he said. Antisense molecules are short segments of chemically altered RNA designed to stick tightly to the mutated spot in the defective messenger RNA and nowhere else.

"After we incubated the genetically engineered cells with the antisense, lo and behold the cells began to synthesize significantly more normal messenger RNA," Friedman said. "Furthermore, the ability to promote normal splicing was directly related to the dose of antisense administered. Presumably the antisense blocked the mutation and limited the opportunity for incorrect splicing."

Friedman has also demonstrated that higher levels of normal cystic fibrosis messenger RNA boost production of normal protein by the cystic fibrosis gene. He and Kole have no data yet on how treated cells affect salt and water balance in larger tissues.

"Our work, done in transfected human and animal cells in culture, is preliminary, and it's too early to apply this potential therapy to CF patients," he said. "Nevertheless, our initial results are sufficiently encouraging to warrant further study and may one day prove useful to patients.

"They suggest that instead of total gene replacement, we might be able to correct the flow of incorrect information coming from abnormal genes to make normal messenger RNA. This technique may be applicable to many other genetic illnesses as well."

About one in 25 whites carries a defective cystic fibrosis gene. When two carriers have children, the odds are that each child has a 25 percent chance of having the illness, a 25 percent chance of avoiding it entirely and a 50 percent possibility of being an unaffected carrier. Members of other races also can be born with the condition, but far more rarely.

"Research teams nationwide including the UNC Cystic Fibrosis Center have made remarkable strides in the last 10 years toward understanding this very complicated disease," Friedman said. "Ground-breaking successes achieved in cystic fibrosis may ultimately prove invaluable to researchers of other inherited diseases."

Kole is a member of the UNC Lineberger Comprehensive Cancer Center. The National Institutes of Health and the Cystic Fibrosis Foundation support the continuing UNC-CH studies.
-end-
Note: Friedman and Kole can be reached at (919) 966-0713 and 966-1143, respectively. Contact: David Williamson, 962-8596.

University of North Carolina at Chapel Hill

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