Sep. 23, 2002 CHAPEL HILL -- Scientists for the first time have engineered a harmless virus to correct, rather than replace, the genetic defect causing the most common single gene disorder.
The new research presents a novel approach to gene therapy in treating the most common inherited anemias: the thalassemias. Thalassemias are genetic blood diseases that result in failure to produce sufficient hemoglobin, the oxygen-carrying protein component of blood cells. This failure is caused by defects in the genetic code responsible for the production of this protein.
Scientists have explored gene therapy for these disorders for more than 20 years; only recently has this area seen a glimmer of hope. The report from the University of North Carolina at Chapel Hill is slated to appear in the Dec. 15 issue of Blood, the journal of the American Society of Hematology, and is currently online at www.bloodjournal.org.
"This research offers a new way to treat the thalassemias, by blocking a deleterious process that causes several forms of the disease," said senior study author Dr. Ryszard Kole, professor of pharmacology, and a member of UNC Lineberger Comprehensive Cancer Center and the curriculum in genetics and molecular biology at the UNC School of Medicine.
The thalassemias are caused by a variety of different mutations in the globin gene, many of which adversely affect a process known as RNA splicing.
Three billion bases - molecules that constitute DNA - form the human genome. Only a small percentage actually code for the gene products necessary for existence. These small coding regions are like words that make sense in a long continuous string of gibberish, which must be spliced out to create a meaningful message that can then be read to make the corresponding protein. Short sequences at the border between the sense and nonsense regions, called splice sites, tell the splicing machinery where to cut, where to paste and what information can be disregarded.
Some mutations can affect splicing by creating additional splice sites in places where they should not occur. Even though the correct splice sites are still intact, the splicing machinery preferentially recognizes the incorrect splice sites, resulting in the inclusion of extra, noncoding sequences, which interfere with subsequent production of hemoglobin. Kole and colleagues found that using molecules designed to bind specific regions of RNA to block aberrant splice sites could prevent splicing machinery from using those sites.
With the aberrant sites blocked, the splicing machinery goes back to the original, correct splice sites and uses these to cut and paste the correct globin message.
"In our approach the defect in the RNA is 'masked,' thereby effectively 'repaired.' This is different from replacing the gene with a good copy, which will then produce additional RNA and hemoglobin," Kole said. "Since we only repair the existing RNA, we do not need to worry that too much of the good thing will be made. This can sometimes be harmful."
In collaboration with Dr. Tal Kafri, co-senior author of the study, assistant professor of microbiology and immunology and a member of the Gene Therapy Center, the antisense sequences were incorporated into a lentiviral vector.
"Lentiviral vectors appear to be the most attractive vehicles to carry therapeutic genes into non-dividing target cells such as the hematopoietic stem cells," Kafri said. "Gene delivery into these cells allows us to reconstitute a patient's bone marrow with vector-corrected stem cells that confer a lifelong remedy. The use of antisense technology coupled with these lentiviral vectors allows us to propel this field forward in the treatment of hematopoietic disorders."
In the UNC study, the genetically modified lentiviral vector was used to treat blood cells obtained from a thalassemic patient. The treatment partially restored correct splicing in the cells and resulted in a marked increase in correct hemoglobin protein. "The results are very encouraging because even hemoglobin levels that reach less than 50 percent of normal can have a therapeutically relevant effect," said Kole.
The antisense technology explored in this study is not limited to thalassemia. The finding that the human genome comprises far fewer genes than previously thought suggests that the complexity of human beings must arise from mechanisms of gene regulation, such as splicing. According to current estimates, at least 60 percent of the human genes may be alternatively spliced.
"We have been successful in the laboratory in shifting the aberrant splicing of a few other genes involved in genetic disorders and cancer," said Kole. "With the discovery that thousands of genes are spliced, as far as potential targets for this new form of gene therapy are concerned, the sky's the limit."
Along with Kole and Kafri, UNC co-authors include Marla M. Vacek, first author of the study and a doctoral student in the curriculum in genetics and molecular biology, and Hong Ma, a researcher in the Gene Therapy Center.
The National Heart, Lung and Blood Institute, a component of the National Institutes of Health, provided funding for the new research.
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