June 8, 1998 DURHAM, N.C. -- In a first step toward an effective treatment for sickle cell anemia, researchers at Duke University Medical Center have shown that they can use a new type of gene therapy to correct the defect in human blood cells.
The results of their laboratory studies, published in the June 5 issue of the journal Science, show that successful gene therapy may lie not in correcting faulty DNA, the storehouse of genetic information, but in correcting the RNA, which translates that genetic information to the protein synthesis machinery of a cell. It also provides a potential path toward ridding the sickle cell defect from people born with the debilitating disease.
The researchers plan to begin testing the therapy in sickle cell patients within a few years.
"We have shown for the first time that it is possible to correct a genetic defect in blood extracted from patients for experimentation, not just in laboratory-grown cells," said Bruce Sullenger, the paper's senior author. "In a disease like sickle cell anemia, if we could get even 10 to 20 percent correction of the defect, it could make a huge difference for patients."
Sullenger and his colleagues, Ning Lan, Dr. Richard Howrey, Dr. Seong-Wook Lee and Dr. Clayton Smith, all from Duke's Center for Genetic and Cellular Therapies, tried their new therapy on sickle cell anemia because there is no effective treatment for the underlying genetic defect and because the gene is under complex genetic controls that make it unsuitable for other types of gene replacement therapy. The research was funded in part by a grant from the National Heart, Lung and Blood Institute to Sullenger and from a Korean Academic Research Fund grant to Lee.
Sickle cell anemia is an inherited disease that is most common among people whose ancestors come from Africa and the Middle East. About one in 12 African Americans carries the sickle cell trait. The red blood cells of people with sickle cell disease contain an abnormal type of hemoglobin, the molecule that carries oxygen throughout the body. The defect is caused by a single tiny change in the protein portion of the hemoglobin molecule. This single mutation distorts the red blood cells into a sickle shape, making them fragile and easily destroyed, leading to anemia. Complications of sickle cell disease can include stroke, bone pain, kidney damage and breathing problems.
The key to Sullenger's strategy to correct the sickle cell defect is a molecule called a ribozyme, a type of RNA enzyme that can find a specific sequence of RNA code, chemically cut out a section, and splice in another section. Ribozymes are key players in editing the flow of genetic information from DNA to protein. Before the discovery of ribozymes, scientists believed only proteins could perform such cutting and splicing activities. Sullenger has devised a strategy to use this natural process to fix defective RNA -- in this case, a defective globin gene.
"The fundamental role of RNA molecules in cells is to help manage the use of genetic information," Sullenger said. "We believe the correcting RNA is the most promising strategy for correcting many types of genetic defects."
In contrast to traditional gene therapy schemes, which try to replace defective genes in the genome, Sullenger's ribozymes correct defective RNA, the message copied from DNA. Many genetic diseases, such as sickle cell anemia, are caused by defective genes that get made into defective protein. Simply adding back a normal gene to cells doesn't decrease the amount of the "bad" gene product, because the defective copy would still be present and the added gene would not be placed under the cell's precise regulatory scheme. Other scientists have shied away from tackling gene therapy for sickle cell anemia because the cell's production of beta globin is precisely regulated.
Sullenger's strategy simultaneously eliminates defective protein and creates new working copies of the globin protein. This corrected gene is also designed to make a fetal form of the globin protein. The fetal globin protein is normally made before birth and is shut down when mature globin begins being made after birth. But in some people, small amounts of fetal globin continue to be produced. In sickle cell patients who make some fetal globin, the disease symptoms are greatly reduced because fetal globin not only carries oxygen effectively, but also inhibits the mutant globin protein from sickling.
In their experiments, the Duke Medical Center scientists collected blood from sickle cell patients and from umbilical cord blood, the afterbirth of normal infants. From this blood they isolated precursor red blood cells -- the cells that produce mature red blood cells. They added ribozyme molecules that carried the corrected fetal globin genetic sequence into the cells using slippery, fatty spheres called liposomes. Once inside the cell, the ribozymes located the faulty RNA by matching up letters of the genetic code to the defective globin RNA. They then snipped off the defective piece and added in the corrected sequence.
When the scientists broke apart the cells and read the genetic sequence of the globin RNA, in each case the ribozyme had spliced in the new sequence correctly.
Next, the researchers will test the procedure in an animal model of sickle cell disease to see if the corrected globin gene can prevent development of sickling. If this procedure works, Sullenger and his colleagues would like to begin testing the ribozyme therapy in sickle cell patients within two to three years.
The most likely candidates to test the ribozyme therapy would be sickle cell patients who have developed a rare condition called alloimmunization. Patients with severe sickle cell disease often require blood transfusions to replenish their supply of healthy red blood cells. Some of these people develop antibodies to elements in transfused blood, making further transfusions dangerous to the patient. Sullenger envisions taking some of a patient's own blood, correcting the sickle cell trait, and giving it back in a transfusion. Since it is a patient's own blood, he or she would not develop antibodies against it.
In addition, promising experiments with umbilical cord blood suggest possibly this unique source of blood stem cells as a permanent treatment for sickle cell disease.
"If we could find a good method for integrating the instructions for making the ribozyme into the blood stem cells, that would be a permanent source of good, working copies of the globin gene that could at a minimum lessen the symptoms of the disease," said Sullenger. "We know that sickle cell patients who make 10 to 20 percent fetal globin do much better. If we could reach that level or better, we could perhaps eliminate the most serious manifestations of the disease."
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