Editing Humanity: The CRISPR Revolution and the New Era of Genome Editing

CHAPTER 8 GENOME EDITING B.C.

“If I had a ruble for every time I’ve heard about the promise of gene editing, I’d be an oligarch!” declares Fyodor Urnov. “What hypothetical promise? It’s been in the clinic for nearly a decade!”¹ Urnov should know: for more than a decade, he was one of the molecular musketeers at a biotech company called Sangamo that took the lead in developing genome editing and brought it to the clinic, developing a therapy for HIV. It wasn’t an unequivocal success by any means, but it opened the door for CRISPR and an avalanche of new therapies, some of which might turn into cures.

Now back in academia working with Doudna at the Innovative Genomics Institute, Urnov is all in on CRISPR, allied with the biggest name in the field. “I’m happy Jennifer Lopez is doing a TV show [on CRISPR], but what the other Jennifer is doing is a lot more interesting,” he joked the first time I heard him give a lecture in 2018. He speaks fast and enunciates crisply in a vestigial Russian accent mellowed by more than two decades on the West Coast. If there is a guru in the world of genome editing, Urnov is the man. But before we consider what CRISPR means for humankind now and in the future, I first need to tell a bit more of the back story regarding CRISPR.

Genome editing did not burst onto the scene fully formed like Athena, with what Urnov termed the “immortal” Charpentier-Doudna CRISPR discovery in 2012. In fact, the year before, the journal Nature Methods declared gene editing its “Method of the Year” based on the promise of two forerunners of CRISPR—zinc finger nucleases (ZFNs) and TALENs.² Although expensive and difficult to deploy, the technologies entered the clinic years ahead of CRISPR—ZFNs developed commercially by Sangamo, and TALENs championed by Paris-based Cellectis.

If Doudna and Charpentier’s teamwork in 2012 is the pillar for genome editing in the modern era, the New Testament if you will, then Urnov brands the era leading up to that moment as “Genome editing B.C.”—before CRISPR.

Few Nobel laureates have a more remarkable personal story than Mario Capecchi. Creativity and success in science requires “the abrasive juxtaposition of unique sets of life experiences that are too complex to pre-orchestrate.”³ Remarkably, Capecchi survived outrageous odds during World War II to become the first scientist to conduct a form of gene editing in mammalian cells. Capecchi was born in Verona in October 1937 as fascism flared across Italy. His father, an officer in the Italian air force, had an affair with a beautiful poet who lectured at the Sorbonne in Paris. After Capecchi’s birth, “my mother wisely chose not to marry him.”⁴ As a bohemian, she staunchly opposed fascism and took her baby to the Italian Alps. But in 1941, Capecchi recalls the Gestapo arriving in Tyrol and arresting his mother, who was incarcerated in Dachau, Germany.

For a year, Capecchi lived with a neighboring family, living on homemade bread; he remembers jumping naked in barrels of freshly picked grapes. But when the money Capecchi’s mother had provided ran out, he was left to fend for himself. Only four years old, Capecchi headed south, “sometimes living in the streets, sometimes joining gangs of other homeless children, sometimes living in orphanages and most of the time being hungry.” Many memories of that period “are brutal beyond description.” After the liberation of Dachau in 1945, Capecchi’s mother returned to Italy to search for her son. Miraculously she found him in a hospital in Reggio Emilia, where he was being treated for malnourishment. In Rome, Capecchi had his first bath in six years. Later, the Capecchis sailed to America. “I was expecting to see roads paved with gold,” he wrote. “I found much more: an opportunity.” Capecchi settled with his Uncle Edward, a physicist, just outside Philadelphia, He reveled in wrestling and still has the physique to prove it.

After graduating from Antioch College, Capecchi interviewed at Harvard with Jim Watson. When he asked Watson where he should conduct his PhD, Watson snorted: “You’d be crazy to go anywhere else.” Capecchi joined the effort to defragment the genetic code. Capecchi admired Watson’s bravado and stark honesty, as well as a sense of justice. “He taught us not to bother with small questions, for such pursuits were likely to produce small answers,” he said. A few years later, Capecchi set up his own group at the University of Utah. By microinjecting DNA into the nucleus of living cells, he developed a method to swap a gene for a near-identical copy. In the early 1980s, an NIH panel rejected Capecchi’s proposal, but he’d overcome tougher odds than that.

Capecchi’s groundbreaking work, along with Oliver Smithies, a British geneticist then at the University of Wisconsin, and Martin Evans, provided researchers with a means to “knock out” a mouse gene using homologous recombination. By inactivating a gene in embryonic stem cells and then injecting those modified cells to create a chimeric embryo, scientists could do in small, furry mammals what they’d been able to do routinely in yeast and bacteria for decades. The technique was demanding, inefficient, and took months to perform, but the ability to create an animal model lacking a key gene was a godsend for geneticists and developmental biologists. Like a genetics gold rush, the journals were flooded with papers reporting what happened when one mouse gene after another was muted, many providing critical models of human genetic diseases. And it earned Capecchi, Evans, and Smithies the Nobel Prize in Physiology or Medicine in 2007.

Although a major advance for biology, the problem with targeted, or homologous recombination was its very low efficiency in mammalian cells—only about 0.01 percent. But Maria Jasin, a molecular biologist at Memorial Sloan Kettering Cancer Center in New York, knew that rates were much higher in yeast. In 1994, her group tested the idea that introducing a break in both strands of DNA could trigger the cell to repair the break. Using a restriction enzyme called I-SceI, she found higher rates of recombination when she cut the DNA. Moreover, those breaks could be repaired with an exogenous piece of DNA. Jasin detected two different forms of DNA repair: homologous recombination and non-homologous end-joining (NHEJ). The former produced a clean repair, the latter a series of short deletions flanking the target site. Jasin’s demonstration that a double-strand break was “editogenic” was a major landmark, arguably the first gene-editing experiment, even though its significance was not truly appreciated for a decade. In the Old Testament of Genome Editing, Urnov calls this the “gospel according to Jasin.”⁵

“I am a textbook example of ‘right place, right time.’ My entire life!”⁶ I’ve managed to corral Urnov for an hour in a hotel lobby in Florence, Italy, to reflect on the Old Testament of genome editing.

Urnov was born in the former Soviet Union during the calm of the 1970s. Socialism more or less worked. Moscow was a cultural Mecca. Friends and families argued about the meaning of life at the kitchen table. His father was a literary professor, his mother a linguist. Both were published biographers. His grandfather had edited the works of Charles Dickens. Urnov grew up on the works of Lewis Carroll, Dickens and Twain, while becoming an obsessive Beatles fan. Although not a scientific household, Urnov’s father was friends with the family of the great Russian molecular biologist Vladimir Engelhardt, who lent him a copy of The Double Helix. Watson’s book had a profound effect on the fourteen-year-old Urnov, especially the “incomparable taste of having discovered a secret.” After one reading, any other career plans were canceled. He was hooked on DNA.

Urnov enrolled at Moscow State University in 1985, studying biology the year that Mikhail Gorbachev came to power. Glasnost was instituted in Urnov’s freshman year, perestroika in his sophomore year. The Chernobyl disaster in 1986 also had a major effect on him. By the time he graduated, the Soviet Union had essentially fallen apart. Going west would no longer mean being smuggled in a suitcase or emigrating via Israel. With his parents’ support, Urnov enrolled at Brown University. His PhD advisor, Sue Gerby, patiently domesticated the ex-Soviet into her lab, where he spent six years studying how genes turned on and off. At a conference, a “starstruck” Urnov asked Alan Wolffe if he could join his lab as a postdoc. Wolffe was dynamic, charismatic, and the youngest institute director ever at the NIH. He said yes.

Urnov was catapulted into the premier league of genetics research. Wolffe was an expert in the emerging field of epigenetics, the study of chemical modifications to DNA that regulate gene activity. Joining Wolffe’s lab reminded Urnov of the Red Queen’s race in Through the Looking-Glass, as Alice says, when we run this fast, we generally get somewhere else. Everybody in the lab was top-notch, working around the clock. He had to up his game and be ready when Wolffe ambled up to his bench and asked: “Ah, Dr. Urnov, what have you discovered?”

Alfred Hershey, one of the founders of molecular biology, once described “Hershey Heaven” as coming to the lab, running an experiment, and having it work every day. Urnov says he was “truly in Hershey Heaven.” But then in 2000, in a surprising move, Wolffe accepted an offer to head research at a young biotech company in California. At age forty, Wolffe was ready for a new challenge. Urnov readily agreed to go west. It was the first time he heard the name Sangamo.

The home office of Edward Lanphier, the retired founding CEO of Sangamo BioSciences, is a carriage house in Marin County, about ten miles north of San Francisco. I stop to admire the mementoes of a successful biotech career. A framed front page of the San Francisco Examiner from 1981 has the headline: GENENTECH JOLTS WALL STREET. A bookshelf houses the obligatory vanity license plate (“SANGAMO”) that Lanphier belatedly detached from his car. A framed photo shows Lanphier and his daughter in New York outside the Nasdaq stock exchange when Sangamo went public, the neon sign flashing his name.

I don’t have to look far for the origin of the company’s name: a Sangamo Electric meter has been converted into the base of a table lamp. In the 1890s, Lanphier’s great-grandfather, a Yale-educated electrical engineer, Robert Lanphier, cofounded a company in Sangamon County, Illinois. Lanphier designed and patented the watt-hour meter, with its familiar rotating wheel.⁷ Sangamo Electric became a public company before being acquired by Schlumberger in 1975. Lanphier asked his father if he could borrow the name and the logo for his own engineering start-up in the mid-’90s.

Lanphier got his start in the pharmaceutical industry in the early 1980s at Eli Lilly, which had just licensed recombinant human insulin from Genentech. In 1992, he joined Somatix, a “first-generation” gene therapy company. Three years later, Lanphier launched Sangamo, and soon became enamored with the potential of a class of gene regulators for gene therapy.

Zinc finger proteins (ZFPs) are an abundant^I class of gene activators that were discovered a decade earlier by Nobel laureate Aaron Klug, a Lithuanian Jew who emigrated to England for his PhD and worked with Rosalind Franklin shortly before her death in 1958. These transcription factors had an unusual structure—a series of digit-like projections that make direct contact with the DNA. Each digit consisted of some thirty amino acids, anchored at the knuckle by a zinc atom binding to a quartet of amino acids. “Zinc structural domain” didn’t have much of a ring to it, so Klug coined the term “zinc finger” for each module. Further work showed each finger recognizes a specific three-base sequence of DNA, like a blind person reading braille. Thus, a DNA-binding protein containing three “zinc fingers” can recognize a specific nine-base stretch of DNA.

Sangamo’s initial goal was to use zinc finger proteins to switch certain genes on (or off). Lanphier approached the leaders in the field, including Carl Pabo at MIT. In London, he sat outside the office of Klug, who was president of the Royal Society, like a schoolboy waiting for the headmaster, before sealing a partnership over a three-hour lunch. Klug became a key advisor to Sangamo after selling his own company, Gendaq, to Lanphier in 2001. Sangamo also brought some biotech muscle onto the board, including Bill Rutter and Herb Boyer, cofounders of Chiron and Genentech respectively.

In 2000, Lanphier decided to ride the wave of irrational exuberance in the markets and go public, raising $150 million at the peak of the biotech bubble. To launch this new chapter, he lured Wolffe to become head of research. “It was an enormous coup,” Lanphier recalls. “Alan was just a frickin’ rock star” with an encyclopedic knowledge. “One of the most brilliant men I’ve ever known. Every brilliant, twentysomething alpha-male postdoc wanted to work for Alan.”⁸ Joining Urnov on the expedition was another Russian expat, Dmitry Guschin. Together with zinc finger designer Ed Rebar (from Pabo’s lab), Michael Holmes (from Bob Tijan’s lab at UCSF), Jeffrey Miller, and Andrew Jamieson, Sangamo assembled a boiler room of fearless young talent. Their mission was to take Sangamo, Lanphier said, “from the concept of a steam engine to an internal combustion engine to a freakin’ Ferrari.” The most critical hire was an English postdoc named Philip Gregory. “Amongst a group of unbelievably talented people, Philip organically rose to be the first amongst equals,” Lanphier said.

Wolffe’s expertise was crucial in creating artificial transcription factors—proteins that bind to specific DNA motifs to switch genes on and off—that can drive cells to particular developmental fates. Pabo had shown that by mixing and matching individual zinc finger units, researchers could design a new hybrid transcription factor to recognize a specific DNA sequence. By the time Urnov arrived, Sangamo had designer zinc finger proteins to show this approach was feasible, setting the stage to test their efficacy in human cells.

Then in May 2001, tragedy struck. While attending a conference in Rio de Janeiro, Wolffe was struck by a bus and killed while out running one morning. “Alan was the hub of the spokes… and then he’s gone.” Pabo, the chair of Sangamo’s scientific advisory board, acted as the research team leader until Gregory assumed the role two years later.

Just as Sangamo was preparing to enter the clinic, using a novel ZFP to treat nerve damage in diabetes patients by switching on the VEGF gene, news arrived of another setback. In the summer of 2002, just months after Alain Fischer and colleagues had published exciting gene therapy trial results,⁹ reports emerged that one of his patients had developed leukemia. The virus carrying the therapeutic gene had inserted itself into the patient’s genome, causing cancer. Following the 1999 Jesse Gelsinger tragedy, gene therapy trials were put on hold or abandoned.

By this time, however, a glimmer of light for zinc fingers had emerged, offering the possibility of not merely replacing a disease gene with a healthy copy, or switching on a silent gene, but actually editing and fixing the mistake at the DNA level. A team at Johns Hopkins had devised a means to modify zinc fingers to target them to genes of interest.

In 1978, Hamilton “Ham” Smith, a 6’5” biochemist at Johns Hopkins University, received the call almost every scientist dreams about: he had won the Nobel Prize. Smith, never comfortable in social situations, survived the scrum of press photographers and well-wishers, and a mild case of Imposter Syndrome. Even his mother was surprised: when she heard the news on the car radio, she turned to her husband and said: “I didn’t know there was another Hamilton Smith at Hopkins.”¹⁰

Smith’s Nobel was awarded for his serendipitous discovery of restriction endonucleases, a large family of bacterial enzymes that recognize and cut specific DNA sequences or motifs. Genetic engineers turned these enzymes into the catalysts of the recombinant DNA revolution. “Everything about modern biology, from the idea of determining a DNA sequence to the idea of recombinant DNA to DNA fingerprinting, it all starts with restriction enzymes,” said geneticist David Botstein.¹¹ By the mid-1990s, thousands of restriction enzymes had been catalogued, shipped commercially around the world in polystyrene buckets of dry ice. But their usefulness as a scalpel for precision gene editing was limited. These enzymes cut DNA at very short recognition sites, typically only four to six base pairs. While those motifs might only occur a few times in the tiny genome of a virus, they crop up thousands of times scattered across the human genome.

Smith often discussed the idea of engineering artificial enzymes that could be more selective in cutting DNA with his students, including in 1986 a visiting chemist named Srinivasan “Chandra” Chandrasegaran. Years later, Chandra set out to engineer a chimeric restriction enzyme, a new kind of nuclease. Flicking through the enzyme catalogue offered by New England Biolabs, Chandra and his colleague Jeremy Berg settled on the amusingly named FokI from Flavobacterium okeanokoites. Like a Star Wars TIE fighter seeking the thermal exhaust vent, FokI scans the DNA in search of a specific landmark—a sequence of five bases, GGATG. But once it settles on the DNA, the actual cutting is carried out by a different domain of the enzyme about ten bases downstream. As the two domains were separate, Chandra reckoned he could alter the target parameters by tethering a different DNA recognition domain to the cutting site.

Chandra published his “hybrid restriction enzyme” breakthrough in 1996.¹² His team fused the DNA-cutting domain of FokI with zinc-finger domains that supplied the specificity. “In theory,” Chandra wrote, “one can design a zinc finger for each of the sixty-four possible triplet codons, and, using a combination of these fingers, one could design a protein for sequence-specific recognition of any segment of DNA.” These zinc-finger nucleases (ZFNs) could be programmed to latch onto any DNA sequence that would serve all manner of applications. Interestingly, Chandra’s choice has stood the test of time. “Like the fact that a [soccer] match lasts ninety minutes or the QWERTY keyboard starts with the letter Q, it is widely accepted,” says Urnov. “People haven’t seen the need to evolve beyond that.”

Chandra was in no doubt that his chimeric nucleases—“a new type of molecular scissors”—could transform gene therapy: in 1999 he said his goal was to excise a gene mutation and replace it neatly with its normal counterpart. Ethical issues aside, he wrote, “gene therapy will be routinely used in clinical practice, signifying a paradigm shift in the treatment of human disease.”¹³

Chandra teamed up with Dana Carroll, a biochemist at the University of Utah, who wanted to customize a ZFN to engineer a mutation in a classic animal model such as the fruit fly. If done right, the Drosophila cells would turn from brown to yellow. Carroll’s colleague saw the yellow bristles down the microscope.¹⁴ “If I were you, I’d be pretty excited,” he told his boss. By 2002,¹⁵ Carroll’s group had demonstrated the ability to engineer DNA in living organisms, the first use of ZFNs not merely to modulate the expression of certain genes, but actually to change their DNA sequence. Carroll’s development of ZFNs coupled with the editogenic insights from Jasin and colleagues laid the foundation for genome editing in humans.¹⁶

From 1997, Sangamo’s headquarters was in a building in Point Richmond, shared with Pixar, the animation studio behind Toy Story and A Bug’s Life later acquired by Disney. When Pixar moved to a larger headquarters in Emeryville, Sangamo expanded into the space. For three years, Urnov and Holmes shared an office that was formerly Pixar’s screening room. Urnov says their partnership was akin to Lennon and McCartney, before conceding that might be a bit of a stretch. Assisting the Sangamo team was Matthew Porteus, a physician-scientist at Stanford who had trained with David Baltimore. He’d also been inspired by Carroll’s ZFN papers and wanted to get them to work in human cells. Porteus developed an assay using the green fluorescent protein that could report successful gene targeting using ZFNs.¹⁷

Sangamo’s young musketeers were on a mission and there was no time for failure. “Nothing creates a sense of urgency like being on Nasdaq,” says Urnov. Over the next few years, Sangamo figured out how to turn good ZFNs into effective gene editors. There were multiple disease targets—sickle cell disease, hemophilia, and severe combined immunodeficiency (SCID). (Urnov and Holmes even dabbled with editing the CCR5 gene.) With the French gene therapy setback in everyone’s minds, Sangamo began looked to repair the genetic glitch in those SCID patients—a mutation in the gene for the interleukin-2 gamma receptor (IL2Rζ).

One day, Urnov was reading the results skipping off a lab instrument called a phosphoimager. It looked like “we’d achieved a one-in-five efficiency of gene editing. Efficiency like this happens spontaneously in about one in a million cells.” Urnov shared the results with Holmes. “If this is real, we’ve just entered into a new era!” Holmes concurred, already planning the next experiments to pressure test the result while an exhilarated Urnov paced around the room. “Extraordinary claims demand extraordinary evidence. We both knew nobody would believe us!” Urnov recalls. They kept the results to themselves, while secretly running every control experiment that they could think of. Sangamo’s expertise was starting to pay off. “We’d finally built the fast engine in the car with the superb tires, a super-aerodynamic frame, and a super-flat racing track.”

Urnov and Holmes had glimpsed gene correction—the ZFN created a DNA break, replaced by a piece of genetic information. They looped in Gregory, Rebar, and Miller to make a circle of five, and came up with a definitive experiment to rule out the possibility of an artefact caused by sample contamination. Urnov selected a cell that was homozygous for the gene being edited. If there was contamination, there would always be two forms of the gene. But if the only signal was the sequence being introduced, that would indicate that the native gene had been replaced—edited—by the external sequence. Over one long weekend, Urnov tested a group of edited cells. The first few were normal, unchanged, boring. The next was a heterozygote—one normal variant, one altered. This continued until—“Ode to Joy!”—he found a cell in which both copies of the gene had been changed. Urnov dashed off an email to Holmes: “A HOMOZYGOTE!!!” That was soon followed by “ANOTHER homozygote!!!”

There’s a Russian proverb that says: “If you grab the rope, don’t complain that the cart is too heavy.” It was time to open the curtain and put the finishing touches on the all-important scientific report. Urnov ran the most important experiment of his life—a wondrously low-tech experiment devised by Ed Southern in the 1970s, in which DNA fragments are ingeniously sucked out of a gel and transferred onto a nylon membrane with the absorbent assistance of a 4” stack of paper towels (the Southern blot). Holmes showed they could reverse the edited change, while the experiments in cancer cells were repeated in clinically relevant white blood cells.

Before submitting the paper to Nature, one of Sangamo’s key advisors, Sir Aaron Klug, proposed Urnov and colleagues use the term “high-efficiency gene correction” rather than modification. (The manuscript copy bearing Klug’s handwritten comments remains one of Urnov’s most prized possessions.) After two rounds of review, Nature published the report that rewrote the gene therapy playbook in April 2005.¹⁸ Sangamo had demonstrated the feasibility of correcting human genetic mutations. Moreover, the method avoided the problem of insertional mutagenesis that had marred the French gene therapy trial. “The ‘hit and run’ mechanism of ZFN action uncouples the therapeutically beneficial changes made to the genome from any need to integrate exogenous DNA, while still generating a permanently modified cell,” Urnov wrote.

When the Nature editors asked for ideas for a cover headline, Urnov suggested “genome editing.” (His father had just become the editor in chief of a Russian journal of literary criticism.) Five years after the completion of the first draft of the human genome, scientists had demonstrated the feasibility of rewriting the language of life to fix a genetic disease.

WIRED magazine’s Sam Jaffe reported on the landmark “nano-surgery” technique with a headline that hopefully earned the copy editor a bonus: “Giving Genetic Disease the Finger.”¹⁹ Jaffe quoted David Baltimore: “This doesn’t just deliver a foreign gene into the cell. It actually deletes the miscoded portion and fixes the problem.” The potential to target any gene in the genome was plain to see. Chandra’s review of the paper for Nature Biotechnology was entitled: “Magic scissors for genome surgery.”²⁰

The next step was to move toward treating SCID patients, which required performing gene editing in stem cells. But to the team’s despair, all they found were small DNA sequence insertions and deletions, gene knockouts not precision repair. “This was, putting it mildly, not the droid we were looking for,” says Urnov. The impasse was broken in style by the company’s new chief medical officer, Dale Ando.

“I know exactly what to do,” Ando said. “And I know what gene, and what disease. We’re not going to do bubble boy disease. We’re going to do HIV.”

“Um, okay,” Urnov said

“We’re going to do CCR5 in T cells”

“Okay.”

“And we’re going to collaborate with Carl June.”

“Who’s that?”

Ando started laughing. Not a bad way to make an impression on your first day in the job.

Few areas of medical research were more urgent or competitive in the mid-1990s than HIV, which was first described as an acquired immune deficiency syndrome (AIDS) in a handful of patients in 1981. As the epidemic spread, scientists in the Bay Area observed that some people possessed a natural immunity to the virus. Meanwhile, several groups identified the protein receptor footholds—CD4 and a co-receptor called CXCR4—that enable HIV to gain entry into white blood cells.

In June 1996, five separate reports incriminating another membrane protein, CCR5 (C-C chemokine receptor-5), as a second co-receptor were rushed into print by the top three journals, all within a week of each other. If HIV was a blimp that is snagged by the Empire State Building (CD4), then CCR5 was the cable car ferrying passengers—the HIV genetic material—to the ground. Like tabloid newspapers, premier science journals can get competitive trying to be the first to publish a research breakthrough. Alas, one of those CCR5 reports²¹ in Cell was pushed into production so hastily that several pages ended up being printed upside down.

One of the senior authors of that report was Marc Parmentier, a Belgian physician-scientist, who had a hunch that abnormalities in CCR5 might explain the slow disease progression in some people exposed to HIV. Parmentier’s team took samples from three such individuals and found a glaring thirty-two-base gap (Δ32 or “delta 32”) in the middle of the CCR5 gene.²² The size of this deletion left little doubt that the function of the truncated protein was compromised. After testing hundreds of samples and volunteers, he found that the Δ32 variant was surprisingly common in Europeans—a carrier frequency (meaning one copy) of about 10 percent—but not a single HIV patient carried two copies of the Δ32 variant. A colleague showed that white blood cells with the Δ32 gene were resistant to HIV infection. By the time Parmentier submitted the report to Nature in July,²³ another group had found the same results on a larger cohort of patients.

In the 1980s, Stephen O’Brien, a lab chief at the NIH, embarked on a search for genetic factors that influence HIV susceptibility and progression. O’Brien and geneticist Michael Dean began systematically screening candidate genes in their HIV population. After twelve years, the NIH team had examined more than one hundred candidate mutations in thousands of HIV patients without success. But the glut of CCR5 papers revealed one of the best candidates in years.²⁴ On July 4, while O’Brien was at the cinema watching the premiere of Independence Day, his team was furiously sequencing samples. They too uncovered the Δ32 variant, but didn’t observe any Δ32 homozygotes in more than 1,300 HIV patients. About 1–2 percent of the American population is a Δ32 homozygote, but HIV patients almost never are. Without a portal into the white blood cell, HIV can land but it can’t infect.^II

The geographic distribution of CCR5 Δ32 is interesting: it is most common in northern Europeans at a frequency of 5–15 percent. But as you travel farther south and east, the frequency drops—Δ32 is almost nonexistent in Africans and Asians. This pattern suggests that it must have been positively selected for a reason that has nothing to do with HIV, which didn’t cross over to humans until the early 20th century. O’Brien felt the only reasonable explanation was “a mysterious, but breathtaking, fatal infectious disease outbreak which, like AIDS, exerted a huge mortality, and from which CCR5 Δ32 carriers were resistant.” The prime candidate is the Black Death, which ravaged Europe throughout the Middle Ages. Perhaps the Δ32 variant arose in Scandinavia in response to an earlier plague.

One year before the wave of CCR5 discoveries, a Seattle man named Timothy Ray Brown was diagnosed with HIV while studying in Berlin. He staved off the disease using the antiretroviral drug cocktail, but in 2006, after attending a wedding in New York, fell ill upon his return home. His doctor diagnosed anemia, but a painful biopsy revealed that Brown had acute myelogenous leukemia. The only treatment was a bone marrow transplant: fortunately, a search for potential donors turned up more than 250 matches. Brown’s hematologist had the idea to select a donor who had the defective CCR5 gene. On the list of prospects, donor number sixty-one possessed the Δ32 variant.

Brown didn’t want to be a guinea pig,²⁵ but he signed up for a transplant. Three months after the operation in February 2007, the virus was undetectable in Brown’s blood and he ceased taking his HIV medications. After his leukemia returned, Brown had a second operation in February 2008. Doctors eventually declared him HIV-free with a normal T cell count.²⁶ “My name is Timothy Ray Brown and I am the first person in the world to be cured of HIV,” the Berlin Patient proudly wrote.

Brown’s experience gave Sangamo a lot to chew on. Mutation repair was still the Holy Grail for many diseases but inactivating key genes could have important medical benefits in certain situations, including the one Ando was advocating. The goal was to attempt “to recreate this HIV-protective genotype in the cells of HIV-positive individuals, in the hopes of essentially creating a compartment of the immune system that is protected from HIV infection,” Urnov recalled.²⁷

With Holmes leading the HIV program, Sangamo finally entered the clinic in 2009, in collaboration (as Ando had advocated) with Carl June, a leading gene therapy physician at the University of Pennsylvania. Five years later, Sangamo reported results on the first dozen HIV patients who had been treated with their own CCR5-edited T cells.²⁸ The results were mixed: the therapy was safe and there was some evidence of an antiviral effect, enabling some subjects to remain off standard antiretroviral therapy. In 2015, Sangamo received approval from the U.S. Food and Drug Administration (FDA) to extend the concept from T cells to stem cells, with the goal of protecting other cellular compartments of the immune system from harboring HIV. To date, Sangamo has treated more than one hundred HIV patients.

While Sangamo is known as the zinc finger specialist, a French company has taken the TALEN gene-editing technology to the clinic. The CEO of Cellectis, André Choulika, thought CRISPR was “super cool” when he first heard about it, but decided to stick with TALENs, mostly for immunotherapies. “We found them to be more accurate, precise, and powerful, and we thought they would be safer for patients,” he says.²⁹

Lanphier retired from Sangamo in June 2016 after twenty-one years, partly for health reasons but also because he felt it was time “to bring in the real pros.” On CNBC’s Mad Money, Jim Cramer asked Lanphier about CRISPR and Sangamo’s faith in the ZFN platform. “The key to human therapeutics is specificity—the ability to target exactly the gene you want and only that gene,” Lanphier replied. “That’s where zinc finger nucleases have a complete monopoly.”³⁰ Years later, I asked Lanphier if he still felt the same way. “CRISPR is bacterial. It’s nonspecific. It’s immunogenic,” he said. “It’s a great research tool. It’s going to give a lot of visibility to genome editing. And when people actually want to use it therapeutically, that’s where they’ll end up talking to us.” It must have been a wrench to remove that vanity license plate. “Nobody can do it the way Sangamo does it, on this scale, with the kind of precision,” he said.

Before retiring, Lanphier launched therapeutic programs in blood disorders, including sickle-cell disease, beta thalassemia, and hemophilia. Ed Rebar came up with a clever strategy to switch on genes in the liver. Albumin, the most abundant protein in human blood, is produced by a gene that is extremely active in the liver. Rebar reckoned: what was to stop Sangamo from smuggling in a gene like a Trojan horse and taking advantage of this powerful albumin gene promoter? The method, dubbed in vivo protein replacement, or “invisible mending”, involved snipping the albumin gene in the first intron, plugging in a transgene into this “safe harbor,” and using the constitutive power of the albumin promoter to fire up the gene of interest. That fueled programs to target rare inherited disorders of genes normally expressed in the liver such as mucopolysaccharidosis (MPS) types I and II (also known as Hurler and Hunter syndromes, respectively) and hemophilia B.

On November 13, 2017, forty-four-year-old Brian Madeux climbed onto a bed in Room 1037—the Infusion Room—of the UCSF Benioff Children’s Hospital in Oakland. Dressed in a gray sweatshirt and khaki shorts, Madeux nervously watched a nurse hook up an IV. He was no stranger to hospitals, having endured more than two dozen surgeries for hernias, bunions, spinal, eye, and ear problems resulting from Hunter syndrome. Surrounded by doctors, nurses, and a film crew, he was about to become the first patient to receive a direct infusion of a gene-editing drug. “It’s kind of humbling,” he told the Associated Press.³¹

Madeux’s infusion took place on the centenary of the first description of his disease. Charles Hunter, a Scottish physician who had emigrated to Winnipeg, Canada, published a case report of two brothers, ages ten and eight, with a syndrome of physical abnormalities that would later bear his name.³² The brothers had several common features—undersized, large head, short neck, broad chest, easily winded. We now know the disease is caused by a deficiency of an enzyme called iduronate-2-sulphatase. Hunter syndrome patients are unable to break down two particular carbohydrates, which consequently accumulate in various tissues. Enzyme replacement treatments involve weekly infusions that can cost more than $100,000 per year. Madeux’s doctors hoped his treatment would stem the progression of his disease and serve as an inspiration for other patients. For the first few days, he felt weak and dizzy, later he suffered a partially collapsed lung (probably unrelated to the therapy). Encouragingly, his liver appeared to be functioning normally. More patients were enrolled, some receiving higher doses. But initial results were equivocal.

Lanphier’s successor, Sandy Macrae, is a Scottish physician who trained in the 1990s as a molecular biologist with the great Sydney Brenner. “My wife said it would never be of any use to me, and then this job came up,” Macrae jokes. After revising the name of the company to Sangamo Therapeutics, he began inking deals for different disease targets with big pharma partners. Sangamo wasn’t going to discard decades of expertise on ZFNs, but it is no longer just a zinc finger company. “If I was back doing my postdoc, I’d be using CRISPR,” Macrae admitted.

Rarely does a biotech CEO acknowledge mistakes or failures, but Macrae has done both. Success in clinical genome editing comes down to three things: editing, delivery, and biology. The Hunter syndrome story showed that the albumin promoter strategy works beautifully in cells and animal models, and appears safe in human patients. Any complications in the trials were due to the AAV vector that was used. The trial was a “remarkably unremarkable event,” Macrae said.³³ But the boost in enzyme levels only proved significant in a patient who received the highest dose. He then developed a side effect called transaminitis, which shut down production of the enzyme. “We succeeded in the editing, but it wasn’t good enough for the biology,” Macrae said. A new effort in the clinic with improved vectors is underway.

As for HIV, results could have been better. “We didn’t understand enough about the biology. It was not the dramatic cure we hoped for,” Macrae said. “We’re not an HIV company.” More promising data, albeit so far only in mice, suggests that a zinc finger approach can distinguish and shut down the faulty expanded version of the Huntington’s disease gene from the normal counterpart.³⁴ But providing these medicines at an affordable cost to patients will be a challenge for the entire industry. Macrae says a typical Sangamo gene-editing drug costs about $300 million to move from idea to clinical trials to FDA approval.

Sometimes the pioneers are not the ones who reap the rewards. But veteran Ed Rebar, who briefly headed the Sangamo R&D team before joining Sana Biotechnology in 2020, remains a staunch believer in the power of zinc. “For therapeutic applications, ZFNs can do everything we need them to do,” he told a crowd of genome engineers.³⁵ “Precision, any base, high levels of specificity.” CRISPR is a great tool for basic research and has enjoyed widespread adoption. “But therapy is a different type of application.”

Rebar wasn’t exactly preaching to the choir. The answer to most genome editing applications in the clinic is to be found in the New Testament of CRISPR-based therapies. But ultimately, patients and their families won’t care which technology is used if it answers their prayers and delivers a cure.

I. There are about seven hundred genes—3 percent of the total—encoding zinc finger proteins in the human genome.

II. Subsequent studies revealed that some Δ32 homozygotes are infected by a different strain of HIV, which gains entry via the CXCR4 co-receptor.