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Gene therapy corrects sickle cell disease in mice, Science authors report

American Association for the Advancement of Science

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A new gene therapy method corrects sickle cell disease in mice, and may suggest future therapies to treat the genetic disease in humans. The method, developed by an international research team, is described in the 14 December issue of the journal, Science.

The therapy counteracts the faulty gene that causes red blood cells to "sickle" or deform, by transferring an anti-sickling variant of the gene to bone marrow with a viral delivery system. Once there, the anti-sickling gene incorporates itself into the stem cells that give rise to red blood cells.

In two mouse models, the new gene was rapidly expressed in 99 percent of all circulating red blood cells, preventing sickling and other signs of the disease, says study author Philippe Leboulch of the Harvard Medical School and Massachusetts Institute of Technology.

Leboulch says that there are still several obstacles to overcome before the therapy can be tested in humans, but that the method is "clearly corroborating evidence that these types of vectors based on lentiviruses are highly efficient with stable expression."

Sickle cell disorders are most common in individuals of African, Mediterranean, Indian, and Middle Eastern descent, and one in every 13 African Americans carries the sickle cell trait, according to Leboulch. The disease is caused by a single nucleotide mutation in the human beta globin gene.

Why does a mutation of the beta globin gene cause disease? Red blood cells need oxygen. In healthy people, oxygen is carried to red blood cells by hemoglobin, a molecular complex. Beta globin plays a role in getting oxygen to red blood cells, too, because it contributes two protein "chains" to hemoglobin. When a person inherits the sickle cell mutation from both parents, he or she manufactures an abnormal version of hemaglobin, in which the beta globin chains develop sticky patches when they lose their oxygen.

These sticky patches cause the molecules to adhere to one another and link themselves into long fibers that stretch the red blood cell into its characteristic sickle shape. Sickled cells can get stuck in blood vessels and block blood flow, leading to anemia, stroke, and organ damage.

Despite the fact that sickle cell was the first genetic disorder for which a mutation was recognized at the molecular level, treating the disease through gene therapy has proved difficult. A potential globin gene "replacement part" is relatively large on the genetic scale of things, making it difficult to transport into the genome.

"It was difficult to transduce an anti-sickling gene to bone marrow because it is so large, and then the expression level of this gene was very low and often 'silenced' once it entered bone marrow stem cells," says Leboulch.

To overcome these obstacles, the Science authors combined recent discoveries in their own labs with advancements from other researchers to help them choose an optimal gene replacement and build a more efficient delivery vehicle.

First, the scientists designed a beta globin gene containing a residue that confers anti-sickling action in another globin called gamma globin. Then, Leboulch and colleagues outfitted a retrovirus with a flap of DNA from the HIV-1 virus that previous researchers had shown to be effective in turning retroviral carriers into better delivery systems to resting stem cells like the blood-forming stem cells in bone marrow. They also optimized the elements surrounding the new gene that control its expression in the red blood cell lineage.

Loaded with its cargo of modified beta globin gene, the improved viral vector quickly took up residence in the blood-forming stem cells of mice that had undergone irradiation to kill off their old bone marrow. Ten months after transplantation, all of the mice expressed the new gene in up to 99 percent of their red blood cells.

"Usually when a copy of a new gene lands in the genome this way, it is strongly influenced by its surroundings, and often gets silenced. But when the expression level is very high, and spread evenly through the cells, as it is in the case with lentiviral vectors, the gene can do its work," noted Leboulch.

In two different models of sickle cell disease in mice, the gene therapy caused an eightfold reduction in sickled cells in one model, and elimination of sickled cells in the second model. Other characteristics of sickle cell disease, including spleen enlargement, a urine concentration defect, and dehydration of red blood cells were also corrected in the mice.

Leboulch says that a potential dosage for human bone marrow stem cells could be kept similarly low if the therapy is targeted to enriched stem cells separated from marrow, rather than simply being transduced to crude marrow as with the mouse models.

Establishing "clean packaging lines"--large-scale production of viral vectors that can't replicate--and investigating a way to introduce the therapy without toxic irradiation to existing bone marrow would be desirable before the method can be used in clinical trials, say the researchers.


Philippe Leboulch is also at INSERM EMI in Paris, France, and Brigham and Women's Hospital, Boston, MA. The other members of the research team are R. Pawliuk, K.A. Westerman, R. Tighe, and I.M. London of the Harvard-MIT Division of Heath Sciences and Technology, in Cambridge and Boston, MA, M.E. Fabry, E.E. Bouhassira, S.A. Acharya, and R.L. Nagel at Albert Einstein College of Medicine in Bronx, NY, E. Payen, and Y. Beuzard at INSERM in Paris, France, J. Ellis at Hospital for Sick Children, Toronto, Canada, and C.J. Eaves and R.K. Humphries at The Terry Fox Laboratory and University of British Columbia, Vancouver, BC. Pawliuk, Westerman,Tighe, and Leboulch are also at Genetix Pharmaceuticals in Cambridge, MA.

This research was funded in part by NIH. INSERM, Association Française contre la Myopathie, and Genetix Pharmaceuticals.

Founded in 1880 by Thomas A. Edison, Science has been the official journal of the American Association for the Advancement of Science (AAAS) since 1900. The nonprofit AAAS is the world's largest general scientific organization.

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