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Gene therapy corrects sickle cell disease in mice, scientists report

CAMBRIDGE, Mass. -- An international team led by an MIT research affiliate has developed a method of gene therapy that corrects sickle cell disease in mice, suggesting future therapies designed to treat the genetic disease in humans. The new method is described in the Dec. 14 issue of Science.

The lead researcher, Professor Philippe Leboulch, is an assistant professor of medicine at Harvard Medical School, a research affiliate in the Harvard-MIT Division of Health Sciences and Technology, and vice president and chief scientific officer of Genetix Pharmaceuticals of Cambridge, MA.

The therapy transfers an anti-sickling variant of the faulty gene to the bone marrow, where it 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, Leboulch said.

Leboulch said 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 really efficient."

Sickle cell disorders are most common in individuals of African, Mediterranean, Indian and Middle Eastern descent; 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.

Beta globin contributes two protein "chains" to hemoglobin, the molecular complex that carries oxygen within red blood cells. When a person inherits the sickle cell mutation from both parents, he or she manufactures an abnormal version of hemoglobin, 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.

Sickle cell disease was the first genetic disorder for which a mutation was recognized at the molecular level. This was a seminal discovery published in Nature in 1957 by MIT's Vernon Ingram, the John and Dorothy Wilson Professor in the Department of Biology. However, treating the disease through gene therapy has proved especially difficult. A potential globin gene "replacement part" is relatively large on the genetic scale of things, for example, 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 the bone marrow stem cells," Leboulch said.

To overcome these obstacles, the Science authors turned to recent discoveries by them and other researchers to help 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 at turning retroviral carriers into better delivery systems to resting stem cells like the blood-forming stem cells in bone marrow. Finally, they optimized the elements surrounding the gene that control its expression in the red blood cell lineage.

An important component incorporated in the vector were the Dnase I hypersensitive sites of the beta globin locus control region, which were discovered in 1985 by MIT Professor Emeritus Irving M. London and Dorothy Tuan, a former principal research scientist at MIT.

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 and at very high levels.

"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 this case, the gene can do its work," Leboulch said.

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. 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 said a potential dosage of the vector for human bone marrow stem cells could be kept low if the therapy targeted enriched stem cells separated from marrow, rather than simply being transduced to crude marrow as with the mice.

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 are necessary before the method can be used in preclinical trials, say the researchers.

Leboulch also is affiliated with INSERM EMI in Paris and Brigham and Women's Hospital in Boston. The other members of the research team are R. Pawliuk, K.A. Westerman, R. Tighe and I.M. London of Harvard and MIT; M.E. Fabry, E.E. Bouhassira, S.A. Acharya and R.L. Nagel of the Albert Einstein College of Medicine; E. Payen, and Y. Beuzard at INSERM; J. Ellis at the Hospital for Sick Children in Toronto; and C.J. Eaves and R.K. Humphries at the Terry Fox Laboratory and the University of British Columbia in Vancouver.

This research was funded in part by the National Institutes of Health, INSERM, the Association Fran��aise Contre la Myopathie and Genetix Pharmaceuticals Inc.

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