The Gene

by Siddhartha Mukherjee

  The protocol for this test was simple: ten men with a severe variant of the disease were injected with a single dose of a virus carrying a gene for factor IX. The presence of the virus-encoded protein was monitored in the blood for several months. Notably, this trial tested not just safety, but efficacy: the ten virus-injected patients were monitored for bleeding episodes, and for their use of additional factor IX by injection. Although the injection of the virus-borne gene increased the factor IX concentration to just 5 percent of the normal value, the effect on bleeding episodes was startling. The patients experienced a 90 percent reduction in bleeding incidents, and an equally dramatic reduction in their use of injected factor IX. The effect persisted over three years.

  The potent therapeutic effect of a mere 5 percent replacement of a missing protein is a beacon for the aspirations of gene therapists. It reminds us of the power of degeneracy in human biology: if only 5 percent of a clotting factor is sufficient to restore virtually all clotting function in human blood, then 95 percent of the protein must be superfluous—a buffer, or reservoir, possibly maintained in the human body as a backup in the event of a truly catastrophic bleed. If the same principle holds for other genetic diseases caused by single genes—for cystic fibrosis, say—then gene therapy might be vastly more tractable than had previously been imagined. Even the inefficient delivery of a therapeutic gene to a small subset of cells might be sufficient to treat an otherwise fatal disease.

  But what about that perennial fantasy of human genetics, the alteration of genes in reproductive cells to create permanently amended human genomes—“germ-line gene therapy”? What about the creation of the “post-humans” or “trans-humans”—i.e., human embryos with permanently modified genomes? By the early 1990s, the challenge of permanent human genome engineering had been reduced to three scientific hurdles. Each of these had once seemed like an impossible scientific challenge, but each is on the verge of being solved. The most remarkable fact about human genomic engineering today is not how far out of reach it is, but how perilously, tantalizingly near.

  The first challenge was the establishment of a reliable human embryonic stem cell. ES cells are stem cells derived from the inner pith of early embryos. They live in transit between cells and organisms: they can be grown and manipulated like a cell line in the lab, but are also capable of forming all the tissue layers of a living embryo. The alteration of the genome of an ES cell is thus a convenient stepping-stone to the permanent alteration of an organism’s genome: if the genome of an ES cell can be changed intentionally, then that genetic change can potentially be introduced into an embryo, into all organs formed within the embryo, and thus into an organism. The genetic modification of ES cells is the rather narrow pass through which every fantasy of germ-line genomic engineering has to travel.

  In the late 1990s, James Thomson, an embryologist in Wisconsin, began to experiment with human embryos to derive stem cells from them. Although mouse ES cells had been known since the late 1970s, dozens of attempts to find human analogues had failed. Thomson traced these failures to two factors: bad seed and bad soil. The starting material for the establishment of human stem cells was often of poor quality, and the conditions for their growth were suboptimal. As a graduate student in the 1980s, Thomson had studied mouse ES cells intensively. Like a hothouse gardener capable of coaxing exotic plants to live and propagate outside their natural environments, Thomson had gradually learned the many eccentricities of ES cells. They were temperamental, volatile, and fussy. He knew of their propensity to fold up and die at the slightest provocation. He learned about their requirement for “nurse” cells to coddle them, their peculiar insistence on clumping together, and the translucent, refractive, hypnotic glow of the cells that transfixed him each time he saw them under a microscope.

  In 1991, having moved to the Wisconsin Regional Primate Center, Thomson began to derive ES cells from monkeys. He plucked a six-day-old embryo from a pregnant rhesus monkey, then let the embryo grow in a petri dish. Six days later, he peeled away the outer layer of the embryo, as if skinning a cellular fruit, and extracted single cells from the pith of the inner cell mass. As with mouse cells, he learned to culture these cells in nests of nurse cells that could supply crucial growth factors; without these nurse cells, the ES cells died. In 1996, convinced that he could try his technique on humans, he asked the regulatory boards at the University of Wisconsin to allow him to create human ES cells.

  But mouse and monkey embryos had been easy to find. Where might a scientist find freshly fertilized human embryos? Thomson stumbled on an obvious resource: IVF clinics. By the late 1990s, in vitro fertilization had become a common treatment for various forms of human infertility. To perform IVF, eggs are harvested from a woman after ovulation. A typical harvest yields multiple eggs—sometimes up to ten or twelve—and these eggs are fertilized by a man’s sperm in a petri dish. The embryos are then grown briefly in an incubator before being implanted back into the uterus.

  But not all IVF embryos are implanted. Implanting more than three embryos is unusual and unsafe, and the spare embryos are typically discarded (or rarely, implanted into other women’s bodies, who carry such embryos as “surrogate” mothers). In 1996, having obtained permission from the University of Wisconsin, Thomson obtained thirty-six embryos from IVF clinics. Fourteen of them grew into glistening cellular spheres in the incubator. Using the technique that he had perfected on monkeys—the peeling away of the outer layers, the gentle coaxing of cell growth on “feeders” and nurse cells—Thomson isolated a few human embryonic stem cells. Implanted into mice, these cells were capable of generating all three layers of the human embryo—the primordial sources of all tissues, such as skin, bones, muscles, nerves, intestines, and blood.

  The stem cells that Thomson had derived from IVF-discarded embryos recapitulated many features of human embryogenesis, but they still had a major limitation: although they were capable of making virtually all human tissues, they would not efficiently generate some tissues, such as sperm and egg cells. A genetic change introduced into these ES cells could thus be transmitted into all cells of the embryo—except to the most important ones: the cells capable of transmitting the gene to the next generation. In 1998, soon after Thomson’s paper had been published in Science, groups of scientists around the world, including researchers from the United States, China, Japan, India, and Israel, began to derive dozens of embryonic stem cell lines from fetal embryonic tissues in the hopes of discovering a human ES cell capable of germ-line transmission of genes.

  But then, with little warning, the field was frozen shut. In 2001, three years after Thomson’s paper, President George W. Bush sharply restricted all federal ES cell research to the seventy-four cell lines that had already been created. No new lines could be derived, even from IVF-discarded embryonic tissues. Laboratories working on ES cells faced stringent oversight and cuts in funding. In 2006 and 2007, Bush repeatedly vetoed federal funding for the establishment of new cell lines. Advocates of stem cell research, including patients with degenerative diseases and neurological impairments, thronged the streets of Washington, threatening to sue federal agencies responsible for the ban. Bush countered these requests by holding press conferences flanked by children produced by the implantation of “discarded” IVF embryos that had been brought to life via surrogate mothers.

  The ban on federal funding for new ES cells froze the ambitions of human genomic engineers, at least temporarily. But it could not stop the advance of the second step required to create permanent heritable changes in the human genome: a reliable, efficient method to introduce intentional changes in the genomes of ES cells that were already in existence.

  At first, this too seemed like an insurmountable technological challenge. Virtually every technique to alter the human genome was crude and inefficient. Scientists could expose stem cells to radiation to mutate genes—but these mutations were sprinkled randomly throughout the genome, defying any attempt to directionally influence the mutation.
Viruses carrying known genetic changes could insert their genes into the genome, but the site of insertion was typically random, and the inserted gene was often silenced. In the 1980s, another method to introduce a directional change in the genome—flooding cells with pieces of foreign DNA carrying a mutated gene—was invented. The foreign DNA was inserted directly into a cell’s genetic material, or its message was copied into the genome. But although the process worked, it was notoriously inefficient and prone to errors. Reliable, efficient, intentional change—the deliberate alteration of specific genes in a specified manner—seemed impossible.

  In the spring of 2011, a researcher named Jennifer Doudna was approached by a bacteriologist, Emmanuelle Charpentier, about a conundrum that seemed, at first, to have little relevance to human genes or genomic engineering. Charpentier and Doudna were both attending a microbiology conference in Puerto Rico. As they walked through the alleyways of Old San Juan, past the fuschia-and-ochre houses with arched doorways and painted façades, Charpentier told Doudna of her interest in bacterial immune systems—the mechanisms by which bacteria defend themselves against viruses. The war between viruses and bacteria has gone on for so long, and with such ferocity, that like ancient, conjoined enemies, each has become defined by the other: their mutual animosity has been imprinted in their genes. Viruses have evolved genetic mechanisms to invade and kill bacteria. And bacteria have counter-evolved genes to fight back. “A viral infection [is a] ticking time bomb,” Doudna knew. “A bacterium has only a few minutes to diffuse the bomb—before it gets destroyed itself.”

  In the mid-2000s, a pair of French scientists named Philippe Horvath and Rodolphe Barrangou stumbled on one such mechanism of bacterial self-defense. Horvath and Barrangou, both employees of the Danish food company Danisco, were working on cheese-producing and yogurt-making bacteria. Some of these bacterial species, they found, had evolved a system to deliver coordinated slashes in the genomes of invading viruses to paralyze them. The system—a molecular switchblade of sorts—recognized serial-offender viruses by their DNA sequence. The cuts were not delivered at random places, but at specific sites in viral DNA.

  The bacterial defense system was soon found to involve at least two critical components. The first piece was the “seeker”—an RNA encoded in the bacterial genome that matched and recognized the DNA of the viruses. The principle for the recognition, yet again, was binding: the RNA “seeker” was able to find and recognize the DNA of an invading virus because it was a mirror image of that DNA—the yin to its yang. It was like carrying a permanent image of your enemy in your pocket—or, in the bacteria’s case, an inverted photograph, etched indelibly into its genome.

  The second element of the defense system was the “hitman.” Once the viral DNA had been recognized and matched as foreign (by its reverse-image), a bacterial protein named Cas9 was deployed to deliver the lethal gash to the viral gene. The “seeker” and the “hitman” worked in concert: the Cas9 protein delivered its cuts to the genome only after the sequence had been matched by the recognition element. It was a classic combination of collaborators—spotter and executor, drone and rocket, Bonnie and Clyde.

  Doudna, who had been immersed in the biology of RNA for most of her adult life, was intrigued by the system. At first, she thought of it as a curiosity—“the most obscure thing I ever worked on,” as she later put it. But working with Charpentier, she began to meticulously break it down to its constituent components.

  In 2012, Doudna and Charpentier realized that the system was “programmable.” Bacteria, of course, only carry the images of viral genes so that they can seek and destroy viruses; they have no reason to recognize or cut other genomes. But Doudna and Charpentier learned enough about the self-defense system to trick it: by substituting a decoy recognition element, they could force the system to make intentional cuts in other genes and genomes. Switch the “seeker,” they found, and a different gene might be sought and cut.

  There is a phrase buried in that last sentence that should send a restless ping of fantasy through the mind of any human geneticist. An “intentional cut” in a gene is the potential source of a mutation. Most mutations occur randomly in the genome; you cannot command an X-ray beam or direct a cosmic ray to selectively change only the cystic fibrosis gene or the Tay-Sachs gene. But in Doudna and Charpentier’s case, the mutation was not delivered randomly: the cut could be programmed to occur exactly at the site recognized by the self-defense system. By changing the recognition element, Doudna and Charpentier could redirect it to attack a selected gene, thereby mutating the gene at will.I

  The system could be manipulated even further. When a gene is cut open, two ends of DNA are revealed, like severed string, and the ends are trimmed back. The cutting and trimming is meant to repair the broken gene—and the gene then tries to recover the lost information by seeking an intact copy. Matter has to conserve energy; the genome is designed to conserve information. Typically, a cut-open gene tries to recover the lost information from the other copy of the gene in the cell. But if a cell is flooded with foreign DNA, then the gene witlessly copies the information from this decoy DNA, rather than from its backup copy. The information written into the decoy DNA fragment is thus copied permanently into the genome—akin to erasing a word in a sentence and then forcibly writing a substitute in its place. A defined, predetermined genetic change can thus be written into a genome: the sequence ATGGGCCCG in a gene can be altered to ACCGCCGGG (or any desired sequence). A mutant cystic fibrosis gene can be corrected to the wild-type version; a gene to confer viral resistance can be introduced into an organism; the mutant BRCA1 gene can be reverted to wild type; the mutated Huntington’s gene, with its mirthless, singsong repeat, might be disrupted and deleted. The technique has been termed genome editing, or genomic surgery.

  Doudna and Charpentier published their data on the microbial defense system, called CRISPR/Cas9, in Science magazine in 2012. The paper immediately ignited the imagination of biologists. In the three years since the publication of that landmark study, the use of this technique has exploded. The method still has some fundamental constraints: at times, the cuts are delivered to the wrong genes. Occasionally, the repair is not efficient, making it difficult to “rewrite” information into particular sites in the genome. But it works more easily, more powerfully, and more efficiently than virtually any other genome-altering method to date. Only a handful of such instances of scientific serendipity have occurred in the history of biology. An arcane microbial defense, devised by microbes, discovered by yogurt engineers, and reprogrammed by RNA biologists, has created a trapdoor to the transformative technology that geneticists had sought so longingly for decades: a method to achieve directed, efficient, and sequence-specific modification of the human genome. Richard Mulligan, the pioneer of gene therapy, had once fantasized about “clean, chaste gene therapy.” This system makes clean, chaste gene therapy feasible.

  One final step is necessary to achieve the intentional permanent modification of the genome in human organisms. Genetic changes that have been created in human ES cells have to become incorporated into human embryos. The direct transformation of a human ES cell into a viable human embryo is inconceivable, for both technical and ethical reasons. Even though human ES cells can generate all types of human tissues in a laboratory setting, it is impossible to envision implanting a human ES cell directly into a woman’s womb in the hopes that the cell will autonomously organize itself into a viable human embryo. When human ES cells have been transplanted into animals, the most that the cells can achieve is a loose organization of the vital tissue layers of the human embryo—far from the anatomical and physiological coordination achieved by a fertilized egg during human embryogenesis.

  One potential alternative is to attempt the genetic modification of an embryo in toto after it has achieved its basic anatomical form—i.e., a few days or weeks after conception. This strategy is also intractable: once organized, the human embryo becomes fundamentally obdurate to gene modificatio
n. Technical hurdles aside, the ethical qualms about such an experiment would vastly outweigh any other considerations: attempting genome modification in a living human embryo obviously raises a gamut of questions that reverberate far beyond biology and genetics. In most nations, such an experiment lies beyond the conceivable boundaries of permissibility.

  But there’s a third strategy that may be the most approachable. Suppose a genetic change is introduced into human ES cells using standard gene-modification technologies. And now imagine that the gene-modified ES cells can be converted into reproductive cells—sperm and eggs. If ES cells are truly pluripotent stem cells, then they should be able to give rise to human sperm and eggs (a real human embryo, after all, generates its own germ cells—sperm or egg).

  Now consider a thought experiment: if a human embryo can be created by IVF using such gene-modified sperm or eggs, then the resultant embryo will necessarily carry these genetic changes in all its cells—including its sperm and egg cells. The preliminary steps of this process can be tested without changing or manipulating an actual human embryo—and can thus safely skirt the moral boundaries of human embryo manipulation.II Most critically, the process mimics the well-established protocols of IVF: a sperm and an egg are fertilized in vitro, and an early embryo is implanted into a woman’s body—a procedure that raises few qualms. It is a shortcut to germ-line gene therapy, a back door to transhumanism: the introduction of a gene into the human germ line is facilitated by the conversion of ES cells into germ cells.

  This final challenge was largely on its way to being solved exactly as Doudna was perfecting her system to alter genomes. In the winter of 2014, a team of embryologists in Cambridge, England, and at the Weizmann Institute in Israel developed a system to make primordial germ cells—the precursors of sperm and eggs—out of human embryonic stem cells. Earlier experiments, using earlier versions of human ES cells, had failed to create such germ cells. In 2013, Israeli researchers modified these earlier studies to isolate new batches of ES cells that might be more capable of forming germ cells. A year later, collaborating with scientists at Cambridge, the team found that if they cultured these human ES cells under specific conditions, and shepherded their differentiation using specific coaxing agents, the cells would form clusters of sperm and egg precursors.