Dec. 16, 2002 Vanderbilt University Medical Center investigators are reporting success with a novel gene therapy approach. Working with cells grown in the laboratory, the group is the first to repair a defective gene and demonstrate that the resulting protein product is functional, said Dr. Alfred L. George Jr., senior author of a study published Dec. 15 in the Journal of Clinical Investigation.
Although use of the approach in patients is still years in the future, the findings are an important step in showing that a particular method of gene repair is possible, said George, director of Vanderbilt's division of Genetic Medicine.
"We have very solid evidence that we can repair messenger RNA (the copy of DNA that is used to manufacture proteins), and that the repair results in a protein that has normalized function," he said. "That's a good sign and makes us optimistic about moving forward with this type of gene therapy strategy."
Gene therapy is a phrase that describes many different modes of gene-based treatments. The most widely used strategy seeks to put normal copies of a gene into cells with a defective gene. An alternative approach targets a defective gene for repair, either of the DNA itself or of the messenger RNA copy of the gene -- the strategy favored by George's group. Repairing messenger RNA offers advantages over other types of gene therapy, George said, because it works specifically in cells that have messenger RNA copies of the gene. Cells that are not actively using the targeted gene will not contain any messenger RNA copies to be repaired.
"We think this approach may have a niche. It could be useful for any inherited disease, but it may have a special ability to correct a problem in a dominant disorder, George said.
The RNA repair method studied by George and colleagues employs molecules called ribozymes -- repair machines that can be engineered to correct a defect in a selected messenger RNA. The current work targets for repair a mutation that causes myotonia congenita, an inherited muscle disease with symptoms including muscle stiffness. Because myotonia congenita is not a debilitating disease, gene therapy may not be appropriate for some patients, George said, but the disease serves as an excellent model for testing ribozymes as potential gene therapeutics.
"We know a great deal about myotonia congenita," George said. "We know about the genetics and the physiology, and we have cell culture and animal models. We have many experimental armaments to study the disease."
In addition, George said, myotonia congenita provides a good test case for more severe inherited muscle diseases, such as muscular dystrophy and related disorders.
Myotonia congenita is caused by mutations in chloride channels -- donut-like pores that allow chloride ions to pass across the cell membrane. Because chloride channels are important participants in the contraction-relaxation cycle of skeletal muscle, defects in these proteins affect muscle relaxation and cause muscle stiffness.
Over 80 different myotonia congenita-associated chloride channel mutations have been identified, George said. His team targeted one of these for repair, a mutation that George and colleagues first identified in a Pennsylvania dog named Sparky.
Dr. Christopher Rogers, a former graduate student in George's laboratory, engineered ribozymes to correct the "Sparky" defect and then introduced the ribozymes into cells harboring the mutant chloride channels. He demonstrated that the ribozymes could indeed repair the messenger RNA for the defective channels and that the resulting repaired proteins had normal chloride channel function. The repair was not effective in all cells, George said, but in a small percentage of cells, chloride channel function was completely restored.
"We know now that the ribozyme method can work; it's effective at producing a protein with completely normal function," George said. "It would be nice if we knew it worked in the dog, and that's the next step."
The investigators will continue studies in cells to improve the efficiency of the method before they test it in a group of Sparky's descendants -- a dog model of myotonia congenita.
Other contributors to the Journal of Clinical Investigation study include Drs. Carlos G. Vanoye and Bruce A. Sullenger. The work was supported by the National Institutes of Health and the Muscular Dystrophy Association.
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