The Gene

by Siddhartha Mukherjee

  ES cells also possess an unusual third characteristic—a quirk of nature. They can be isolated from the embryo of an organism and grown in petri dishes in the lab. The cells grow continuously in culture. Tiny, translucent spheres that can cluster into nestlike whirls under the microscope, they resemble a dissolving organ more than an organism in the making. Indeed, when the cells were first derived from mouse embryos in a laboratory in Cambridge, England, in the early eighties, they generated little interest among geneticists. “Nobody seems to be interested in my cells,” the embryologist Martin Evans complained.

  But the real power of an ES cell lies, yet again, in a transition: like DNA, like genes, and like viruses, it is the intrinsic duality of its existence that makes this cell such a potent biological tool. Embryonic stem cells behave like other experimentally amenable cells in tissue culture. They can be grown in petri dishes; they can be frozen in vials and thawed back to life. The cells can be propagated in liquid broth for generations, and genes can be inserted into their genomes or excised from their genomes with relative ease.

  Yet, put the same cell into the right environment in the right context, and life literally leaps out of it. Mixed with cells from an early embryo and implanted into a mouse womb, the cells divide and form layers. They differentiate into all sorts of cells: blood, brain, muscle, liver—and even sperm and egg cells. These cells, in turn, organize themselves into organs and then become incorporated, miraculously, into a multilayered, multicellular organism—an actual mouse. Every experimental manipulation performed in the petri dish is thus carried forward into this mouse. The genetic modification of a cell in a dish “becomes” the genetic modification of an organism in a womb. It is a transition between lab and life.

  The experimental ease permitted by embryonic stem cells also surmounted a second, and more intractable, problem. When viruses are used to deliver genes into cells, it is virtually impossible to control where the gene is inserted into the genome. At 3 billion base pairs of DNA, the human genome is about fifty thousand or a hundred thousand times the size of most viral genomes. A viral gene drops into the genome like a candy wrapper thrown from an airplane into the Atlantic: there is no way to predict where it might land. Virtually all viruses capable of gene integration, such as HIV or SV40, generally latch their genes randomly onto some spot in the human genome. For gene therapy, this random integration is an infernal nuisance. The viral genes might fall into a silent crevasse of the genome, never to be expressed. The genes might fall into an area of the chromosome that is actively silenced by the cell without much effort. Or worse, the integration might disrupt an essential gene or activate a cancer-causing gene, resulting in potential disasters.

  With ES cells, however, scientists learned to make genetic changes not randomly, but in targeted positions in the genome, including within the genes themselves. You could choose to change the insulin gene and—through some rather basic but ingenious experimental manipulations—ensure that only the insulin gene was changed in the cells. And because the gene-modified ES cells could, in principle, generate all the cell types in a full mouse, you could be sure that a mouse with precisely that changed insulin gene would be born. Indeed, if the gene-modified ES cells eventually produced sperm and egg cells in the adult mice, then the gene would be transmitted from mouse to mouse across generations, thus achieving vertical hereditary transmission.

  This technology had far-reaching implications. In the natural world, the only means to achieve a directional or intentional change in a gene is through random mutation and natural selection. If you expose an animal to X-rays, say, a genetic alteration might become permanently embedded in the genome—but there is no method to focus the attention of an X-ray to one particular gene. Natural selection must choose the mutation that confers the best fitness to the organism and thereby allows that mutation to become increasingly common in the gene pool. But in this scheme, neither mutation nor evolution has any intentionality or directionality. In nature, the engine that drives genetic alteration has no one in its driver’s seat. The “watchmaker” of evolution, as Richard Dawkins reminds us, is inherently blind.

  Using ES cells, however, a scientist could intentionally manipulate virtually any chosen gene and incorporate that genetic change permanently into the genome of an animal. It was mutation and selection in the same step—evolution fast-forwarded in a laboratory dish. The technology was so transformative that a new word had to be coined to describe these organisms: they were called transgenic animals—from “across genes.” By the early 1990s, hundreds of strains of transgenic mice had been created in laboratories around the world to decipher the functions of genes. One mouse was made with a jellyfish gene inserted into its genome that allowed it to glow in the dark under blue lamps. Other mice, carrying variants of the growth hormone gene, grew twice the size of their normal counterparts. There were mice endowed with genetic alterations that forced them to develop Alzheimer’s disease, epilepsy, or premature aging. Mice with activated cancer genes exploded with tumors, allowing biologists to use these mice as models for human malignancies. In 2014, researchers created a mouse carrying a mutation in a gene that controls the communication between neurons in the brain. These mice have substantially increased memory and superior cognitive function. They are the savants of the rodent world: they acquire memories faster, retain them longer, and learn new tasks nearly twice as fast as normal mice.

  The experiments brimmed over with complex ethical implications. Could this technique be used in primates? In humans? Who would regulate the creation of animals with transgenes? What genes would be, or could be, introduced? What were the limits on transgenes?

  Fortunately, technical barriers intervened before the ethical mayhem had a chance to become unmoored. Much of the original work on ES cells—including the production of transgenic organisms—had been performed using mouse cells. In the early 1990s, when several human embryonic stem cells were derived from early human embryos, scientists ran into an unexpected barrier. Unlike the mouse ES cells, which had proved so amenable to experimental manipulations, human ES cells did not behave themselves in culture. “It may be the field’s dirty little secret: human ES cells do not have the same capabilities as mouse ES cells,” the biologist Rudolf Jaenisch said. “You can’t clone them. You can’t use them for gene targeting. . . . They are very different from mouse embryonic stem cells, which can do everything.”

  At least temporarily, the genie of transgenesis seemed contained.

  Transgenic modification of human embryos was out of the question for a while—but what if gene therapists could settle for a less radical goal? Could viruses be used to deliver genes into human nonreproductive cells—i.e., to neurons, blood, or muscle cells? The problem of random integration into the genome would remain—and most crucially, no vertical transmission of genes from one organism to the next would occur. But if the virally delivered genes could be put into the right kind of cells, they might still achieve their therapeutic purpose. Even that goal would represent a leap into the future of human medicine. It would be gene-therapy lite.

  In 1988, a two-year-old girl named Ashanti DeSilva, or Ashi, for short, in North Olmsted, Ohio, began to develop peculiar symptoms. Children have dozens of transient ailments in infancy, as any parent knows, but Ashi’s illnesses and symptoms were markedly abnormal: bizarre pneumonias and infections that seemed to persist, wounds that would not heal, and a white blood cell count that hovered consistently below normal. Much of Ashi’s childhood was spent in and out of the hospital: at age two, a run-of-the-mill viral infection spun out of control, causing life-threatening internal bleeding and a prolonged hospitalization.

  For a while, her doctors were mystified by her symptoms, attributing her periodic illnesses loosely to an underdeveloped immune system that would eventually mature. But when the symptoms refused to abate as Ashi turned three, she underwent a barrage of tests. Her immunodeficiency was attributed to her genes—to rare, spontaneous mutations in both copies of a ge
ne called ADA on chromosome twenty. By then, Ashi had already suffered several near-death experiences. The physical toll on her body had been immense—but the emotional anguish that she had experienced was more pronounced: one morning, the four-year-old awoke and said, “Mommy, you shouldn’t have had a child like me.”

  The ADA gene—short for “adenosine deaminase”—encodes an enzyme that converts adenosine, a natural chemical produced by the body, into a harmless product called inosine. In the absence of the ADA gene, the detoxification reaction fails to occur, and the body gets clogged with toxic by-products of adenosine metabolism. The cells that are most acutely poisoned are infection-fighting T cells—and in their absence, the immune system collapses rapidly. The illness is fleetingly rare—only one in 150,000 children is born with ADA deficiency—but it is even rarer still, because virtually all children die of the disease. ADA deficiency is part of a larger group of notorious illnesses called severe combined immunodeficiency, or SCID. The most famous SCID patient, a boy named David Vetter, had spent all twelve years of his life in a plastic chamber in a Texas hospital. The Bubble Boy, as David was called by the media, died in 1984, still imprisoned in his sterile plastic bubble, after a desperate attempt at a bone marrow transplant.

  David Vetter’s death gave pause to doctors who had hoped to use bone marrow transplants to treat ADA deficiency. The only other medicine, being tested in early clinical trials in the mideighties, was called PEG-ADA—the purified enzyme derived from cows and wrapped in an oily chemical sheath to make it long-lived in the blood (the normal ADA protein is too short-lived to be effective). But even PEG-ADA barely reversed the immunodeficiency. It had to be injected into the blood every month or so, to replace the enzyme degraded by the body. More ominously, PEG-ADA carried the risk of inducing antibodies against itself—depleting the levels of the enzyme even more acutely and precipitating a full catastrophe, making the solution infinitely worse than the original problem.

  Could gene therapy correct ADA deficiency? After all, only one gene needed to be corrected, and the gene had already been identified and isolated. A vehicle, or vector, designed to deliver genes into human cells had also been identified. In Boston, Richard Mulligan, a virologist and geneticist, had designed a particular strain of retrovirus—a cousin of HIV’s—that could potentially shuttle any gene into any human cell with relative safety. Retroviruses can be designed to infect many kinds of cells; their distinct capability is their ability to insert their own genome into the cell’s genome, thereby permanently affixing their genetic material to that of a cell. By tweaking the technology, Mulligan had created partially crippled viruses that would infect cells and integrate into their genomes, but would not propagate the infection from cell to cell. Virus went in, but no virus came out. The gene fell into the genome but never popped out again.

  In 1986, at the National Institutes of Health in Bethesda, a team of gene therapists, led by William French Anderson and Michael Blaese,I decided to use variants of Mulligan’s vectors to deliver the ADA gene into children with ADA deficiency.II Anderson obtained the ADA gene from another lab and inserted it into the retroviral gene-delivery vector. In the early 1980s, Anderson and Blaese had run some preliminary trials hoping to use retroviral vectors to deliver the human ADA gene into blood-forming stem cells of mice and then monkeys. Once these stem cells had been infected by the virus carrying the ADA gene, Anderson hoped, they would form all the cellular elements of blood—including, crucially, T cells into which the now-functioning ADA gene had been delivered.

  The results were far from promising: the level of gene delivery was abysmal. Of the five monkeys treated, only one—Monkey Roberts—had cells in the blood that showed long-term production of the human ADA protein from the virus-delivered gene. But Anderson was unfazed. “Nobody knows what may happen when new genes enter the body of a human being,” he argued. “It’s a total black box, despite what anyone tells you. . . . Test-tube and animal research can only tell us so much. Eventually you have to try it in a person.”

  On April 24, 1987, Anderson and Blaese applied to the NIH for permission to launch their gene-therapy protocol. They proposed to extract bone marrow stem cells from the children with ADA deficiency, infect the cells with the virus in the lab, and transplant the modified cells back into the patients. Since the stem cells generate all the elements of blood—including B and T cells—the ADA gene would find its way into the T cells, where it was most required.

  The proposal was sent to the Recombinant DNA Advisory Committee, or RAC, a consortium set up within the NIH in the wake of the Berg recommendations of the Asilomar meeting. Known for its tough oversight, the advisory committee was the gatekeeper for all experiments that involved recombinant DNA (the committee was so notoriously obstreperous that researchers called getting its approval being “taken through the Rack”). Perhaps predictably, the RAC rejected the protocol outright, citing the poor animal data, the barely detectable level of gene delivery into stem cells, and the lack of a detailed experimental rationale, while noting that gene transfer into a human body had never been attempted before.

  Anderson and Blaese returned to the lab to revamp their protocol. Begrudgingly, they admitted that the RAC’s decision was correct. The barely detectable infection rate of bone marrow stem cells by the gene-carrying virus was clearly a problem, and the animal data was far from exhilarating. But if stem cells could not be used, how could gene therapy hope to succeed? Stem cells are the only cells in the body that can renew themselves and therefore provide a long-term solution to a gene deficiency. Without a source of self-renewing or long-lived cells, you might insert genes into the human body, but the cells carrying the genes would eventually die and vanish. There would be genes, but no therapy.

  That winter, mulling over the problem, Blaese found a potential solution. What if, rather than delivering the genes into the blood-forming stem cells, they merely took T cells from the blood of ADA patients and put the virus into the cells? It would not be as radical or permanent an experiment as putting viruses into stem cells, but it would be far less toxic and much easier to achieve clinically. The T cells could be harvested from peripheral blood, not bone marrow, and the cells might live just long enough to make the ADA protein and correct the deficiency. Although the T cells would inevitably fade from the blood, the procedure could be repeated again and again. It would not qualify as definitive gene therapy, but it would still be a proof of principle—gene-therapy double-lite.

  Anderson was reluctant; if he was to launch the first trial of human gene therapy, he wanted a definitive trial and a chance to stake a permanent claim on medical history. He resisted at first, but eventually, conceding Blaese’s logic, relented. In 1990, Anderson and Blaese approached the committee again. Again, there was vicious dissent: the T cell protocol had even less supportive data than the original. Anderson and Blaese submitted modifications, and modifications to the modifications. Months passed without a decision. In the summer of 1990, after a prolonged series of debates, the committee agreed to let them proceed with the trial. “Doctors have been waiting for this day for a thousand years,” the RAC’s chairman, Gerard McGarrity, said. Most others in the committee were not as sanguine about the chances of success.

  Anderson and Blaese searched hospitals around the country to find children with ADA deficiency for their trial. They came upon a small trove in Ohio: all of two patients with the genetic defect. One was a tall, dark-haired girl named Cynthia Cutshall. The second, Ashanti DeSilva, was a four-year-old daughter of a chemist and a nurse, both from Sri Lanka.

  In September 1990, on an overcast morning in Bethesda, Van and Raja DeSilva, Ashi’s parents, brought their daughter to the NIH. Ashi was now four years old—a shy, hesitant girl with a fringe of shiny hair, cut pageboy-style, and an apprehensive face that could suddenly be brightened by a smile. This was her first meeting with Anderson and Blaese. When they approached her, she looked away. Anderson brought her down to the hospital’s gift shop and asked her
to pick out a soft toy for herself. She chose a bunny.

  Back at the Clinical Center, Anderson inserted a catheter into one of Ashi’s veins, collected samples of her blood, and rushed it up to his lab. Over the next four days, 200 million retroviruses, in an enormous cloudy soup, were mixed with 200 million T cells drawn from Ashi’s blood. Once infected, the cells grew in petri dishes, forming lush outcroppings of new cells, and even newer cells. They doubled by day and doubled by night in a silent, humid incubator in Building 10 of the Clinical Center—just a few hundred feet from the lab where Marshall Nirenberg, almost exactly twenty-five years before, had solved the genetic code.

  Ashi DeSilva’s gene-modified T cells were ready on September 14, 1990. That morning, Anderson ran out of his home at dawn, skipping breakfast, near nauseous with anticipation, and dashed up the steps to the lab on the third floor. The DeSilva family was already waiting for him; Ashi was standing by her mother, her elbows planted firmly on her seated mother’s lap as if waiting for a dental exam. The morning was spent running more tests. The clinic was silent, except for the occasional footfall of research nurses running in and out. As Ashi sat on her bed in a loose yellow gown, a needle was put into one of her veins. She winced slightly but recovered: her veins had been cannulated dozens of times before.

  At 12:52 p.m., a vinyl bag carrying the murky swirl of nearly 1 billion T cells infected by the ADA-gene-carrying retrovirus was brought up to the floor. Ashi looked apprehensively at the bag as the nurses hooked it to her vein. Twenty-eight minutes later, the bag had run dry, its last dregs emptied into Ashi. She played with a yellow sponge ball on her bed. Her vital signs were normal. Ashi’s father was sent downstairs with a pile of quarters to buy candy from the vending machine on the ground floor. Anderson looked visibly relieved. “A cosmic moment has come and gone, with scarcely a sign of its magnitude,” one observer wrote. It was celebrated, in style, with a bag of multicolored M&M’s.