Stemming Sickle Cell
Can two gene-tweaking scientists from Tamil Nadu take down a global scourge?
A few months back, Sivaprakash Ramalingam, PhD, focused his microscope on a crowded clump of human stem cells and saw a reddish glow—a chemical signal he had successfully inserted a gene into a “safe harbor” site in the cells’ DNA where it wouldn’t interrupt vital functions.
It was a crucial step in a drive by the postdoc and his advisor, Environmental Health Sciences (EHS) Professor Srinivasan Chandrasegaran, PhD, to develop a practical cure for sickle cell disease. The painful and debilitating genetic illness affects millions of people around the world, including in the U.S. and some of the poorest regions of India.
Ramalingam, 34, began his life on a small plot of land near the Bay of Bengal in southeastern India. While his father toiled in the family’s banana grove, sugar cane field and rice paddy, young Siva helped out by milking the family cow.
Today, Ramalingam works with his mentor Chandrasegaran—also a native of the southern Indian state of Tamil Nadu—on the frontiers of genetic medicine, trying to find gene-based cures for major health challenges like cystic fibrosis and HIV, as well as sickle cell disease. In their common quest, it’s hard not to see a torch passing from one generation of scientists to the next, from basic science to applied medical research and from Western institutions to young researchers from the rapidly advancing scientific institutions of the developing world.
The two public health scientists are collaborating with stem cell expert Curt I. Civin, MD, of the University of Maryland, on the sickle cell project. They are racing with labs around the world pursuing similar goals. In May, the Maryland Stem Cell Research Fund awarded Ramalingam one of 17 grants worth up to $200,000 over the next two years for his sickle cell work, as part of a program to support Maryland scientists pursuing novel approaches to stem cell therapies.
Ramalingam and Chandrasegaran say the painstaking research could take two or three more years before it is ready for testing in animals, in preparation for human trials. Asked whether they worried about the intense competition, Ramalingam admits that he sometimes loses sleep over the publication of an important paper by a rival lab.
Chandrasegaran just smiles. The 30-year veteran scientist, who pioneered the development of man-made gene-editing tools called zinc finger nucleases, takes a philosophical approach. “If you’re asking me, do you want to be first? Yes. But it’s not in our hands. If others do it, we will be happy that it was done since it will help a lot of people,” he says.
“I want to keep it low-key. Let’s take it one step at a time and do careful science.” —Srinivasan Chandrasegaran
There is no guarantee of success in this latest assault on the scourge of sickle cell. Except for mice and yeast, the DNA of most animals, including humans, is notoriously difficult to fiddle with and many efforts to repair human genes have failed.
Some early gene therapy patients died when viruses carrying engineered DNA inserted it at random locations on the genome and switched on genes that caused cancer.
Chandrasegaran says he’s leery of overselling his lab’s progress. “I want to keep it low-key,” he says. “Let’s take it one step at a time, and do careful science.”
But for Chandrasegaran, as for many other scientists, the relatively recent discovery that stem cells can be “induced” or derived from adult cells has opened exciting new avenues for medical research. “I hope that I can see it in my lifetime,” he says. “I’d like to see people cured of HIV, cured of sickle cell—any monogenic disease where you can replace the cells. It will help a lot of people, and that’s the ultimate goal.”
Sickle cell is among the most common disorders caused by a single genetic defect, and it can be devastating.
People with the disease produce crescent-shaped red blood cells that are stiff, sticky and prone to piling up or breaking apart, clogging small vessels. These misshapen cells only live about one-tenth as long as normal blood cells, and a patient’s bone marrow can’t make replacements fast enough to keep delivering sufficient oxygen to the body.
Clogged vessels often trigger attacks, called “crises,” that produce acute pain in the back, chest, arms or legs and can last for hours or days. Patients may suffer leg ulcers, small strokes, blindness, and kidney failure and be prone to lethal infections.
The disease is found in certain populations around the world but is most common in Africa, parts of the Middle East, India, Central America and the Caribbean. It affects an estimated 90,000 to 100,000 people in the U.S., including about 1 in 500 African Americans.
The mutation that causes the disease is thought to have evolved in the tropics. For those carriers of a single gene (said to have “sickle cell trait”), most do not have symptoms of sickle cell disease but do have some protection against malaria.
In the U.S., the universal screening of newborns and early, aggressive treatment of sickle cell disease with blood transfusions, antibiotics and other drugs have helped reduce infant mortality and prolong lives. But the disease can still have a devastating impact on patients: Life expectancy in the U.S. for women with the disorder is still only about 48 years. For men, it’s 42.
In recent years, doctors have cured sickle cell disease in a few hundred patients using a technique that combines stem cells from healthy donors with bone marrow transplants. But the procedure is expensive and risky, Chandrasegaran says, while matching patients with healthy donors can be very difficult.
So Ramalingam and Chandrasegaran, working with Civin, are trying a different approach. Instead of using donors, they plan to take a sickle cell patient’s own stem cells, repair the faulty gene, and turn the repaired stem cells into blood and blood-producing cells. The hope is that these healthy cells, put back in the body, will outlast and replace the diseased ones without the need for a bone marrow transplant.
The aim is to make the repair of a patient’s sickle cell gene safer, simpler and cheaper, putting the procedure within the reach of more patients. “We’d like everybody to have access to it, so we want to make it as inexpensive as possible,” says Chandrasegaran.
If the technique works, the senior researcher says it could have wide applications. The biotech company Sangamo Biosciences of California has licensed some of Chandrasegaran’s work and is using a similar strategy to knock out a gene known as CCR5 with zinc finger nucleases, eliminating a route HIV uses to invade and hijack the body’s immune system.
The Mentor Chain
When he was still in India, Ramalingam, who earned his doctorate in molecular biology from the University of Madras, studied strategies for boosting the iron content of rice through manipulating the crop’s genes.
That’s how he heard about Chandrasegaran’s groundbreaking work on zinc finger nucleases, called ZFNs, as a tool for tweaking DNA. “Chandra was the expert,” the younger scientist says. “I sent him my CV and wrote that I was interested. I was very fortunate to work with him.” He came to Hopkins in 2008 as a postdoc to work with Chandrasegaran in EHS.
One advantage of ZFNs and similar gene-editing technologies, Chandrasegaran says, is that, made carefully, they can be targeted at one and only one point in the genome, avoiding the damage that can be caused by random insertion. (Sickle cell disease is caused by an error in a single chemical “letter” in the 3.2-billion-letter-long library of human DNA.)
But making these precise tools for cutting and editing DNA isn’t always simple. Ramalingam says he probably faces another two or three years of working on this crucial phase of the effort. “The success rate is very, very low here,” he says. “So you need a lot of patience doing this research.”
The postdoc, who is married with a 10-month-old son, says he was very proud when his parents traveled the 7,000 miles from his tiny home village of Kullampalayam to visit the Baltimore lab. “They were very excited, they were very happy,” he says. “I tried to explain it to them and the basics, they understand. But the technology, they may not yet.”
While the sickle cell project could accelerate Ramalingam’s career, his senior partner in the lab is considering retiring after three decades at the School.
“Whenever I end up with problems, I discuss them with [Chandra]. He’s a great advisor.” —Sivaprakash Ramalingam
Chandrasegaran, who grew up as one of 10 children, is the son of a customs official working in what was then the French colonial city of Pondicherry on the Bay of Bengal. Accepted to an elite state-run military secondary school, Chandrasegaran rose to the rank of house captain, excelled at physics and graduated with honors. “All my friends who right now are in India? They’re generals and air marshals,” he says.
But he decided to become a scientist rather than an officer, earning a degree in chemistry from the University of Madras in India and his doctorate from Georgetown. He came to the School as a postdoc in late 1981 and joined the faculty in 1986.
At Johns Hopkins, Chandrasegaran learned molecular biology at the bench of Hamilton Smith, professor emeritus at the School of Medicine and a key scientific strategist for a private company that published a working draft of the human genome in 2001. Smith shared the 1978 Nobel in physiology or medicine with Hopkins’ Dan Nathans and a Swiss scientist, Werner Arber, for the discovery of restriction enzymes, the first chemical tools for editing DNA.
It was Smith, in fact, who suggested that Chandrasegaran pursue the synthesis for new gene-editing tools. That suggestion eventually led to Chandrasegaran’s groundbreaking work on ZFNs—technology that, Smith notes, “is now leading to discoveries of several new ways to cleave DNA in site-specific fashion without using restriction enzymes. It’s a hot new field with implications for gene therapy and genome engineering.”
“I had a great teacher,” Chandrasegaran says. “I followed him. He got me interested in molecular biology, in restriction enzymes.”
Ramalingam, in turn, says Chandrasegaran has inspired him by spending long hours in the lab and generously sharing his skills. “Whenever I end up with some problems, I discuss them with him,” he says. “He’s a great advisor to me.”