In Our Blood: A Profile of Stuart Orkin
In Our Blood: A Profile of Stuart Orkin

In Our Blood: A Profile of Stuart Orkin

By unraveling the molecular underpinnings of inherited blood disorders, the Boston Children’s Hospital researcher has provided the basis for therapies now being tested for beta-thalassemia and sickle cell disease.

Jul 15, 2019
Anna Azvolinsky

ABOVE: THE SCIENTIST STAFF

In the late 1970s, Stuart Orkin’s research landed him on the front page of The New York Times—and in a bit of hot water with a few colleagues. Orkin, then a newly minted assistant professor of pediatrics at Harvard Medical School and Boston Children’s Hospital, was developing a way to prenatally diagnose genetic disorders using DNA from the fetus. He was working with Boston Children’s hematologist David Nathan, as well as with collaborators at Yale who had developed a clinical technique to draw blood directly from a fetus during the second trimester using a fine needle. The technique “was quite a tour de force at the time,” Orkin recalls, but it was also risky, increasing the chance of miscarriage.  

Despite the risk, the researchers wanted to use the technique to help a family from Turkey that had been referred to Nathan. The family carried a deletion of globin genes that leads to a frequently fatal form of beta-thalassemia, a genetic blood disorder. Hemoglobin, the iron-harboring protein that ferries oxygen to tissues, is expressed in red blood cells, but in beta-thalassemia, globin production in developing red blood cells is reduced, and sufferers can experience low oxygen levels, severe anemia, and developmental problems. The couple had already lost one child to the disease and wanted to know if their unborn baby also had or was a carrier of the condition. Using the technique, the team at Yale extracted fetal blood and also amniotic fluid, which included fetal cells, from the unborn child. The team then examined the synthesis of globin chains in the fetal blood to check the unborn baby for beta-thalassemia.

© Sam Ogden, Dana Farber Cancer Institute
  • David G. Nathan Distinguished Professor of Pediatrics, Harvard Medical School
  • Investigator, Howard Hughes Medical Institute
  • Jessie Stevenson Kovalenko Medal of the National
  • Academy of Sciences (2013)
  • Lifetime Impact award, Boston Children’s Hospital Global
  • Pediatric Innovation Summit (2015)
  • Clotten Prize, Clotten Foundation, Freiburg, Germany (2017)
  • George M. Kober Medal, American Association of Physicians (2018)Northwestern University’s Mechthild Esser Nemmers Prize in Medical Science (2018)

At the same time, the Yale researchers cultured and expanded the fetal cells from the amniotic fluid and shipped them to Orkin. He then extracted and cut the fetal cell DNA, separated it on a gel, and used Southern blotting to detect the presence of the mutations in globin genes. When Orkin saw DNA bands, he knew that the globin genes were intact in the fetus; had the baby carried the deletion, there would have been no bands. His DNA-based diagnosis was confirmed by the results from the fetal blood cell analysis. Orkin reported the finding in 1978 in the New England Journal of Medicine.

The study marked the first prenatal screening for any heritable disorder using genetic material from fetal cells. The New York Times picked up on the importance of the work, and, by chance, the article about Orkin’s study appeared the same day as a front-page story about the first person born from in vitro fertilization, “test-tube baby” Louise Brown.

A few of Orkin’s colleagues criticized him for the wide media attention and said he claimed to have isolated the gene responsible for beta-thalassemia rather than developing a way to prenatally diagnose the disorder using fetal DNA. Other labs, his critics claimed, were working to purify the actual globin genes to be able to better study their function. Orkin, in their minds, had merely published an image of fuzzy DNA bands corresponding to the gene. “[Other researchers] attacked us for claiming to have isolated this gene,” Orkin recalls. “There was one scientist who was quoted as saying, ‘They saw a fingerprint and we have the finger.’”

Undeterred, Orkin continued his basic science research on beta-thalassemia. Over the next five years, his lab created the first comprehensive molecular analysis of the mutations associated with the disorder. He then went on to study the molecular biology of other diseases, work that has led to the development of novel gene– and cell–based therapies for thalassemias and sickle cell disease. Those therapies are now in clinical trials.

LOVING THE LAB

Orkin was born on the upper west side of New York City. His father, Lazarus, was a urologist and surgeon, and his mother, Sylvia, worked at his father’s office and took care of Orkin and his older brother. When Orkin was in grade school, the family moved from Manhattan to Riverdale, in the Bronx. There, he had an inspiring high school chemistry teacher, an MIT graduate who fueled Orkin’s excitement for solving chemistry and physics problems. In 1963, Orkin entered MIT as a freshman.

He thought of majoring in physics but then realized that “there were 200 classmates that were going to be better physicists than me,” he says. Orkin took an introductory biology course, taught by microbiologist Salvador Luria just five years before Luria earned a Nobel Prize for his discoveries about the genetic structure of viruses and how they replicate. Orkin was introduced to the burgeoning field of molecular biology; he learned how genes are organized and expressed, and how to identify DNA sequences and decipher how certain mutations lead to disease. Orkin became one of the 15 or so biology majors at MIT at the time and spent summers working in different labs in New York City. One year it was at the Albert Einstein College of Medicine in the Bronx, studying cultured mammalian cells with Matthew Scharff. Another summer it was in Jules Hirsch’s lab at the Rockefeller University, engaging in some of the first research on the biological basis of obesity.

During his final year at MIT, Orkin did a senior thesis project in biophysics, using a serum protein from cows to study how the molecule’s three-dimensional structure dictated its biological properties. He then accepted a spot at Harvard Medical School and started the program but took a year off in the middle of the program to do laboratory research with John Littlefield, an early pioneer in somatic cell genetics, which was, at the time, a novel and expanding field.

That research, combined with his work in the clinic as a med student, helped Orkin home in on blood disorders. It was straightforward to obtain blood samples for genetic analysis, and there were many blood disorders to tackle, Orkin says. Knowing he wanted to pursue a research career, and as an alternative to military service at the time of the Vietnam War, he applied to the US Public Health Service to participate in a training program at the National Institutes of Health (NIH) for physician-scientists, and was selected. The program paid for Orkin’s last year of medical school. He then went to do two years of postdoctoral research in Philip Leder’s lab at Harvard.

Leder was among the first to use molecular biology to study mammalian cells, and it was in his lab that Orkin began to study globin gene expression. His experiments involved inducing immature red blood cells from mice to differentiate synchronously and then purifying globin RNA. Using this system, Orkin showed that, although each hemoglobin molecule contains two alpha and two beta subunits, the genes encoding the alpha and beta subunits are differentially expressed, and their messenger RNAs are not present in equal amounts in cells. The results raised the question of how the expression of these genes is controlled.

Greatest Hits

  • Discovered the vast majority of mutations that result in the inherited blood disorder beta-thalassemia, leading to better diagnosis of the disease.
    In collaboration with Louis Kunkel and other colleagues, was the first to use positional cloning to identify CYBB as the gene responsible for X-linked chronic granulomatous disease.
  • Led the first DNA-based prenatal diagnosis for a thalassemia.
  • Identified the master transcription factor, GATA1, and other transcription factors that coordinate blood cell differentiation and development.
  • Provided the first functional evidence that loss of BCL11A expression results in the reactivation of the fetal hemoglobin gene, which is normally repressed following birth. The work established that loss of BCL11A alone is sufficient to reverse sickle cell disease in mice, providing the basis for human gene therapy and genomic editing.

DIVING INTO BLOOD

In 1975, Orkin returned to Boston Children’s Hospital for a one-year residency in pediatrics, along with a clinical fellowship at Harvard Medical School, and then went on to do a hematology fellowship at the hospital, which he completed in 1978. That year, David Nathan called Orkin and told him that if he applied for and won grant money from the NIH and the nonprofit organization March of Dimes, he could start his own research lab at the hospital. “I was completely naive and agreed,” Orkin recalls. He applied for and received the funding and became “simultaneously a fellow and also a junior faculty member.” 

That paper was accepted two days after it was submitted to Nature and then published a week later. . . . That doesn’t happen anymore.

—Stuart Orkin, Harvard Medical School

Orkin became an assistant professor of pediatrics at Harvard Medical School in 1978, the same year that he, Nathan, and their collaborators published the controversial paper demonstrating that fetal DNA could be used to diagnose thalassemias prenatally. Orkin then teamed up with geneticist Haig Kazazian at Johns Hopkins University. Kazazian had a hypothesis about how to classify thalassemia patients for systematic study, and together the researchers used the early methods of gene cloning in bacteriophages to isolate mutant globin genes. To identify mutations, Orkin used Maxam–Gilbert sequencing, one of two DNA sequencing methods used at the time, to characterize the vast majority of mutations that lead to beta-thalassemia. “This [study] was important not only because it was the first instance where the comprehensive genetics of a molecular disease were worked out, but also for prenatal genetic diagnosis, which is still used all over the world,” Orkin says.

Following his thalassemia research, Orkin tackled the molecular biology of X-linked chronic granulomatous disease (CGD), a severe form of immunodeficiency. With help from Boston Children’s Hospital geneticist Louis Kunkel, Orkin and colleagues identified the gene responsible for the disease, which turned out to encode a subunit of cytochrome b, a protein necessary for phagocytic immune cells to produce superoxide and other active oxygen species that destroy microbes and tumor cells. It was 1986, and this was the first use of “positional cloning,” a method to isolate a disease-causing gene by its place in the chromosome. “That paper was accepted two days after it was submitted to Nature and then published a week later,” Orkin says. “That doesn’t happen anymore.”

By then, Orkin had decided to focus on laboratory research full time at Boston’s Children Hospital, leaving clinical practice. While he had succeeded in linking genes and mutations to specific blood disorders, he also wanted to understand what regulates the differentiation of blood cells. He and his colleagues had already identified a four-nucleotide sequence (GATA) that sits upstream of all the genes expressed in red blood cells. Presumably that’s what a putative master regulator would bind to, the researchers thought. So in 1989 they used a novel approach to express proteins encoded by red blood cell genes and found the one that bound to the GATA sequence: a zinc finger protein they named GATA1, now known to be the master transcription factor in hematopoietic cells. The work became a model of how to decipher the gene expression changes that result in the variety of blood cells that arise from hematopoietic stem cells, and how dysregulation of red blood cell progenitors can lead to leukemia. Orkin’s lab then went on to identify related GATA transcription factors, including GATA2 and GATA3.

“Almost every one of the transcription factors we identified has been involved in childhood and adult leukemias in some way,” Orkin says. “It is either mutated, overexpressed, or translocated in these blood cancers.”

gene therapy AND BEYOND

Orkin spent the next two decades focusing on the GATA transcription factors, and work on the globin genes and identifying the mutations that cause beta-thalassemia and sickle cell disease took a backseat. It wasn’t until 2005 that Orkin and his team returned to this line of research. This time the focus was on an old, unresolved question: how do red blood cells shut down production of the fetal form of hemoglobin after birth and begin to express an adult form of the protein?

Orkin’s graduate student Vijay Sankaran chose to tackle the problem. The fetal form of hemoglobin is specialized to help babies extract oxygen from their mothers’ blood, and the switch to a different, adult form happens late in gestation and after birth. The mutations that cause sickle cell disease or beta-thalassemia only occur in the adult beta globin gene—the fetal beta-like genes are normal. If researchers could figure out how to switch fetal hemoglobin production on, they thought, the fetal hemoglobin could compensate for the faulty adult hemoglobin, offering an effective, gene-based treatment for the blood disorders.

If we want to make a dent in the global burden of these diseases as a society, gene therapy is probably not the answer—it is cumbersome and too expensive. . . . We need pills.

Stuart Orkin, Harvard Medical School

Orkin was cautious about pursuing this line of research. “My first comment to Sankaran was, ‘It’s a tough problem and a lot of people have failed, but we’ll give it a go,’” he recalls. Sankaran, now a human geneticist at Harvard Medical School, was determined. He and Orkin decided to focus on a gene called BCL11A, which was implicated as a possible regulator of beta globin through population studies in which fetal hemoglobin levels were determined. In 2008, the lab showed that reducing BCL11A expression in cultures of primary developing red blood cells increased fetal hemoglobin production. The researchers then showed that eliminating BCL11A expression could reverse sickle cell disease in a mouse model. Most recently, the lab identified a short sequence in the BCL11A locus called a regulatory enhancer, the excision of which disrupts BCL11A expression and is enough to boost fetal hemoglobin significantly, indicating that BCL11A is in large part responsible for silencing fetal hemoglobin expression in adults.

Uncovering the details of how BCL11A is regulated and, in turn, how it regulates fetal hemoglobin expression has spurred a flurry of gene editing– and gene therapy–based approaches to treat both beta-thalassemia and sickle cell disease in humans. Orkin welcomes the recent developments. “We knew about these mutations 40 years ago. It’s wonderful that we can now use molecular genetics to help people,” he says. But the work isn’t done yet. “If we want to make a dent in the global burden of these diseases as a society, gene therapy is probably not the answer—it is cumbersome and too expensive,” he explains. “We need pills.”

A pill to target the BCL11A transcription factor is a tough goal, Orkin admits. There have been few successes in using drugs to act on transcription factors. “We are trying to learn everything we can about how the BCL11A locus is regulated and the protein functions so that we can develop ways to target it using small molecules,” he says. “It will require a multidisciplinary approach, and it’s challenging, but there’s no reason it’s not theoretically possible.”