Therapeutic Target Found for Sickle Cell Anemia

Studies in Human Cells Identify Regulator of Hemoglobin Switch

Sickle cell anemia and beta-thalassemia syndromes occupy a central place in biomedical research. Most famously, sickle cell was the first inherited disease found to be caused by a specific amino acid change. But this has not meant an easy road to relief for patients: many basic science hurdles have not budged for decades.

Graham Ramsay

Following a lead from genomewide association studies, Vijay Sankaran (left), Stuart Orkin, and colleagues have found evidence that the transcription factor BCL11A directly regulates the long-studied fetal-to-adult hemoglobin switch and thereby represents a new target for treating sickle cell anemia and beta-thalassemia.

New work from the labs of Stuart Orkin and others indicates that this may soon change. Led by Vijay Sankaran, an MD–PhD student in Orkin’s lab, the team has found evidence that the zinc finger transcription factor BCL11A downregulates expression of fetal hemoglobin (HbF). Keeping HbF expression “on” is known to reduce disease severity, so the results point to BCL11A as a new therapeutic target. The work was published in the Dec. 19 Science.

The researchers were led to BCL11A by genomewide association studies, which aim to uncover disease-associated variants in a population of unrelated individuals. “Hundreds of variants have been found to be associated with various diseases,” said Alan Michelson, associate director for basic research at the National Heart, Lung, and Blood Institute, who wrote a commentary accompanying the paper. But rarely have such findings moved “from hypothesis-generating to an actual pathway.” The current study provides hope that this new generation of human genetic studies can yield useful returns.

Sickle cell disease and beta-thalassemia arise from mutations in the beta subunits of hemoglobin. In sickle cell, misshapen red blood cells tend to clump and block blood vessels, causing severe pain crises and shortening patients’ lives. The only FDA-approved drug for sickle cell, hydroxyurea, exhibits considerable toxicity and is not universally effective. Beta-thalassemia patients generally rely on lifelong blood transfusions. Some patients have greatly reduced symptoms, however, because their bodies produce elevated levels of HbF, which is built from gamma-globin, rather than switching to the beta-containing, adult form.

“That switch had been known for years, and ever since the genes were first cloned in the early 1980s, the major focus of the field has been to try to understand how you regulate the switch,” said Orkin, who is a Howard Hughes investigator and the David Nathan professor of pediatrics at HMS and Children’s Hospital Boston; he is also the chair of pediatric oncology at Dana–Farber Cancer Institute. Understanding the fetal-to-adult switch could lead to strategies for reactivating HbF expression therapeutically.

The new study is a major advance for basic developmental biology and for therapeutic aims, said Michelson, who was Orkin’s first MD–PhD student and formerly an HMS faculty member at Brigham and Women’s Hospital. It uncovers “an entirely new pathway that nobody knew about before.”

A Wide-angle Lens
Sankaran entered Orkin’s lab with his sights set on the hemoglobin switch, but both of them were frustrated by the limitations of existing approaches. As Orkin tells it, “Vijay came in and said he wanted to work on this problem, and I said, ‘You’re out of your mind.’” But Sankaran told him he wanted to take a human genetic approach, so Orkin said, “OK, we’ll see how we do.”

Sankaran contacted Joel Hirschhorn, HMS associate professor of genetics at Children’s and the Broad Institute, about trying genomewide association studies. According to Sankaran, they were lucky to get involved just when researchers were making serious advances in designing these broad-based studies.

In other labs, several single-nucleotide polymorphisms (SNPs) had been found to be associated with variations in HbF levels in normal subjects, and in two studies published earlier this year, the Hirschhorn and Orkin labs found that these variants were also associated with clinical outcomes in sickle cell and beta-thalassemia patients. Most interesting was that variants in an intron of BCL11A were associated with more than 10 percent of the variation in HbF levels in the patients, who represented several different ethnic groups. This was an unusually “common, widespread, and powerful association,” Orkin said.

“Hundreds of variants have been found to be associated with various diseases.” But rarely have such findings moved “from hypothesis-generating to an actual pathway.”

So Sankaran, along with colleagues in the Orkin lab and the lab of Alan Cantor, an HMS assistant professor of pediatrics at Children’s, went to the bench to figure out how BCL11A might be influencing HbF levels. In primary adult erythroblasts, dialing down BCL11A using siRNA or shRNA increased gamma-globin expression and HbF, supporting their hypothesis that BCL11A might be a negative regulator of HbF. Cell morphology and microarray profiling indicated that knocking down BCL11A increased HbF not through a general perturbation of cell state but rather through a limited number of targets. In fact, chromatin immunoprecipitation (ChIP) showed that BCL11A binds at several spots in the beta-globin gene cluster. “We were a little surprised that it had such a direct effect,” Sankaran said.

The researchers found evidence that even though BCL11A acts in complex with other proteins, it is the component regulated by the fetal-to-adult switch. As a result, they believe that downregulating BCL11A in patients could boost their HbF levels and ameliorate symptoms.

Toward Therapy
The next step is to see whether these results hold true in animals. “People really want to know, will this work?” Sankaran said. “We feel pretty confident, because in some ways nature has done an in vivo experiment for us. We know that humans vary, and we know it affects clinical course.” In vivo studies would also address concerns that knocking down BCL11A might affect other cell lineages.

The researchers envision several approaches to therapy: identifying drugs to lower expression or activity of BCL11A or knocking down BCL11A in bone marrow by gene therapy and transplanting the marrow into patients.

Meanwhile, Philippe Leboulch, a visiting professor of medicine at Brigham and Women’s, is pursuing the converse gene therapy strategy: adding a “good” beta-globin gene. Perhaps, Leboulch proposed, both strategies could be used together: “Combining the two would not be a bad approach.”

In the United States, sickle cell afflicts an estimated 70,000 people, mostly African Americans. Globally, the hemoglobinopathies are some of the most common morbid genetic diseases, Orkin said, afflicting many different ethnic groups.

Unfortunately, the bulk are in underdeveloped countries, so they’re not so visible to us.” But Maude Tessier of the Children’s intellectual property office, who worked with the researchers to file a patent application, is optimistic that companies might be able to take up the task of moving toward treatments targeting BCL11A, both because of FDA incentives for addressing diseases with “orphan” status in the United States and because the need is so great internationally.

But for now, it’s back to the lab. The team does not yet understand, for example, the roles of different versions of BCL11A and thus which ones would be best to target. “The more we know about the mechanism by which BCL11A and its variants control globin expression, the better able we’re going to be to think about how that can be applied,” Orkin said.

Students may contact Stuart Orkin at for more information on this or other lab projects.

Conflict Disclosure: The authors report no conflicts of interest.

Funding Sources: The National Institutes of Health, Howard Hughes Medical Institute, Leukaemia Research Fund, and Kay Kendall Leukaemia Fund