“Given her continued neutralizing antibody response to rabies virus in the cerebrospinal fluid and blood, and our inability to isolate the virus or detect viral nucleic acid in saliva, the patient was considered cleared of transmissible rabies and removed from isolation on the 31st day.” A month later, she was released from the hospital.*
Jeanna eventually returned to school and had no difficulties with learning or memory, although she still suffered some weakness in her left hand and foot, and walked with a lurching gait. Today, eight years after being bitten by a rabid bat, Jeanna Giese appears in more than a dozen videos on YouTube, in which she looks and sounds healthy and pleasant. She even has her own website: jeannagiese.com.
Her experience is a case study in the powers and limitations of the human immune system. She was one of only two survivors out of twenty-five attempts at using the first Milwaukee (or Wisconsin) protocol, as Willoughby’s pioneering procedure is now known, and two out of ten in the revised version. Plainly there is ample room for improvement. If synthetic genomics were used to enhance our immune response, we would possess a deliberately engineered superimmunity to a vast array of diseases. This would represent a fundamental advance over the immune systems that we were natively endowed with, and which were born during the Paleocene.
Today’s cutting-edge practice as applied to Jeanna Giese might one day seem incremental and brutal as we look back on the remarkable alternatives described in this chapter. All the more reason to embrace them.
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* In June 2011, an eight-year-old California girl became the third person in the United States known to have recovered from rabies infection through the use of the Wisconsin protocol by a team of physicians, nurses, and therapists at UC Davis Children’s Hospital.
CHAPTER 6
-30,000 YR, PLEISTOCENE PARK
Engineering Extinct Genomes
When people think about extinct species such as the dodo, the passenger pigeon, or Tyrannosaurus Rex, what often comes to mind is the old adage, “Extinction is forever.” Only it isn’t. As we have seen, one member of an extinct species has already been brought back to life, albeit briefly, and it wasn’t even through genomic engineering.
The Pyrenean ibex is a type of mountain goat also known as the bucardo. When they were still in existence, bucardos were one of Europe’s most striking wild animals, with handsome faces, distinctive curving horns, and short, thick wool. They roamed all over the Pyrenees mountain range along the border between Spain and France. But during the late nineteenth century they were massively hunted, and by 1900 fewer than one hundred of the animals were left. The Spanish Ministry of Environment confined a small remaining population of forty bucardos to Ordesa National Park, a spectacular mountainous region in Huesca province.
In 1993 only ten individuals were left, and by 1999 there was only one, a twelve-year-old female named Celia. In the spring of that year, two biologists at the Center for Agro-Nutrition Research and Technology in Aragon, Jose Folch and Alberto Fernández-Árias, captured Celia, took a tissue scraping from her ear for the purpose of preserving the bucardo cell line, and put a radio-tracking collar around her neck. In the laboratory, the researchers multiplied the cells and then stored them away for safe-keeping in liquid nitrogen. Less than a year later, on January 6, 2000, Celia died.
According to the conventional wisdom, that should have been the end of the matter; one more species gone forever. But Folch and Fernández-Árias had a plan for bringing the animal back through a process known as nuclear transfer cloning. Nuclear transfer cloning was the same technology that gave us the first cloned mammal, Dolly the sheep, in 1996. In an ironic twist of fate, Dolly had not been cloned from the cells of a living sheep. Rather, she had been produced from a frozen udder cell of a six-year-old ewe that had died three years prior to Dolly’s birth. Dolly had been literally raised from the dead. But if a live sheep can be cloned from a dead one, then why not a mountain goat? It made no difference to the frozen cells that they happened to be the last of the line: cells were cells, and so long as they contained intact DNA and the other normal structures within the cell nucleus, then they ought to be acceptable candidates for cloning.
Nuclear transfer cloning was still in its infancy, with many more failures than successes. Conceptually, the process was simple enough: take the nucleus from a cell of the animal to be cloned, transfer it to an embryonic cell from which the nucleus had been removed (an “enucleated” cell), and then implant that newly re-nucleated embryonic cell into the uterus of a surrogate mother. In theory, supposing that neither the recipient cell’s cytoplasm or other organelles, nor the transferred nucleus itself, were damaged in the process, the procedure ought to work. In practice, it mostly didn’t in the first few experimental trials. Apparently the process of disrupting cells in this gross manner often injured them beyond repair.
The first animal to be cloned by nuclear transfer was a northern leopard frog, produced at a research institute in Philadelphia in 1951 by experimenters Robert Briggs and Thomas J. King. They moved the relatively large DNA-bearing nuclei into the large frog eggs with a simple hollow glass pipette. During their first few attempts, the implanted embryonic cells withered and died. But the researchers persisted, and over the course of 197 attempts, Briggs and King were able to turn out twenty-seven tiny tadpole clones.
Forty-five years later, anyone might think that the nuclear transplantation batting averages would have improved. In fact, the reverse was true. When Ian Wilmut tried to clone the first sheep from an adult mammalian cell, he started out with an initial pool of 277 mammary gland (udder) cells taken from a six-year-old Finn Dorset ewe. Those 277 cells yielded up only twenty-nine embryos. Those twenty-nine embryos were transferred into the uteruses of Scottish blackface ewes—the recipient surrogate mothers. This resulted in only thirteen pregnancies. And out of the thirteen, only one cloned little lamb was born, Dolly.
So when Jose Folch, Alberto Fernández-Árias, and an international team of experts tried to clone the extinct Pyrenean ibex, they had no illusions about their prospects for success. Indeed, they were well prepared for the series of failures that they experienced. For one thing, Celia had been the last of the species, which meant that the surrogate mothers would be of a different species than Celia. That constituted no insuperable barrier, however, because interspecies nuclear transfer cloning was already a going concern. In 2001, for example, a common domestic cow was successfully used as a surrogate mother for the cloning of a wild ox. Domestic dogs have been used as surrogate mothers for gray wolf clones (although it took 372 embryos to get three live wolves), and so on.
On a date that has never been reported in the scientific literature or in news accounts, and that we here disclose for the first time, the experimenters began their first species regeneration attempts, their so-called Experiment One, in the fall of 2002.* First they had to turn Celia’s somatic (skin) cells into embryos, a delicate biological black-arts process. To do it, they needed a substantial collection of viable goat egg cells. They obtained them by placing thirty domestic goats into a state of superovulation, using hormones to stimulate the ovaries to produce mature egg cells.
Working under a microscope and using micromanipulators, the experimenters removed the nucleus from each egg cell and replaced it with one of Celia’s somatic cells. Next came the step that transformed the two cellular parts into a single working entity: the researchers applied two short pulses of electrical current to each cell, a process known as electrofusion. They then incubated the resulting fused cells, creating fifty-four reconstructed embryos. These were now essentially Celia’s egg cells, as crafted through biotechnology.
Finally, according to Folch, “we transferred the cloned bucardo embryos to thirteen female recipients [who were either Spanish ibex goats or mixed hybrids], having two pregnancies that terminated spontaneously before day 75 of pregnancy.”
All that toil and trouble resulted in no live births. So ended Experiment One.
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In the winter of 2003, the researchers tried again with Experiment Two. This time they transferred 154 cloned embryonic bucardo cells into the wombs of 44 recipient goats. This yielded five pregnancies, one of which continued normally until term.
And then, according to the scientific publication describing the experiment, “at day 162 postfusion, we performed a caesarean section . . . One bucardo female weighing 2.6 kg [5.7 pounds] was obtained alive without external morphological abnormalities. The newborn displayed a normal cardiac rhythm as well as other vital signs at delivery (i.e., open eyes, mouth opening, legs and tongue movements) . . . To our knowledge, this is the first animal born from an extinct subspecies.”
It was Wednesday, July 30, 2003, a turning point in the history of biology. For on that date, all at once, extinction was no longer forever.
The Pleistocene epoch lasted from about 2.5 million years ago to about 10,000 years ago. That is getting awfully close to the present. The modern continents were about where they are today. But if ours is the age of global warming, the Pleistocene was the age of massive glaciation and global cooling. Glaciers covered as much as 30 percent of the earth’s total land area, and in North America the ice sheet at one point extended as far south as what is now Chicago.
The Pleistocene witnessed the rise of the charismatic megafauna, animal species that included the woolly mammoth, Neanderthal man, and Homo sapiens. It also saw the extinction of many of them, including the mammoth and Neanderthal, although the reasons for the extinctions are unclear. While the advancing glacial ice depopulated the affected regions of plant and animal life, it did not necessarily destroy them: in many cases the animals retreated southward and continued to thrive. The wooly mammoth seems to have evolved in very cold habitats.
One explanation offered for the extinctions is that modern humans hunted many of the species into extinction. And they may have been at least partly responsible for the extinction of the Neanderthals, our genetically closest-related hominid species, as well as for the demise of the woolly mammoth. If this is true, the question arises whether we have an obligation to bring these creatures back, not as circus sideshow attractions but as part of a focused scientific attempt to increase genetic diversity by reintroducing their extinct genomes into the global gene pool.
The mammoth almost cries out for resurrection. Some specimens unearthed from permafrost are so lifelike that they appear to be merely sleeping, not dead, much less extinct. Consider, for example, the baby mammoth Dima, discovered in a northern Siberian gold mine in 1977.
Resurrecting such a beast looks almost easy, but when it was actually attempted, it proved to be anything but. In 1980 Viktor Mikhelson of the Leningrad Institute of Cytology tried to reconstruct a mammoth embryo from cells recovered from Dima, but gave up after several months of failure. In the case of Neanderthal man, moreover, we have only fossils, not cells, making a resurrection attempt even more challenging.
Neanderthal man was first discovered in August 1856, when miners working in a limestone quarry in the Neander Valley, near Düsseldorf, Germany, came upon a pile of bones. The workers thought they were the remains of a bear. In reality they were a partial skeleton of an ancient, lost species of humans.
Figure 6.1 Dima
Neanderthal man has since become a cultural icon, a fabled creature with a trademark name on the order of Godzilla or King Kong. But the Neanderthals were real, and for all the negative associations called up by their name, they were for a time the pinnacle of the animal kingdom.
They were bigger and stronger than modern humans, with larger skulls. And while “Neanderthal” has long been considered synonymous with “dumb brute,” these people in fact manifested ample signs of reasonably high intelligence. They used stone and wood tools, as well as axes and spears. They applied body ornamentation, made fires, built relatively complex shelters, hunted and skinned animals, and ate meat. Neanderthals also buried their dead, sometimes together with flowers, a practice that suggests that they possessed some sort of primitive ideology or belief system.
Paleontologists have attributed these practices to Neanderthals on the basis of artifacts recovered from sites containing Neanderthal skeletal remains. Further knowledge of these people has come from an analysis of the Neanderthal genome. Scientists had long thought that the reconstruction of ancient DNA sequences was unlikely if not impossible, something that occurred only in the wilder reaches of Michael Crichton–style science fiction. The argument against it was based on the great age of the samples: any DNA recovered from ancient fossils would probably be too fragmented or corrupt to be readable. But in the 1980s Swedish paleontologist Svante Pääbo demolished that view once and for all.
Even as a child, Pääbo was fascinated by archeology and ancient civilizations, and at thirteen (in 1968) he persuaded his mother, who was a chemist (his father would win a Nobel Prize in Physiology or Medicine in 1982) to take him to Egypt. Later, while enrolled in a PhD program at the University of Uppsala, he obtained skin and bone samples from twenty-three mummies and hoped to extract DNA from them. To prevent contamination from human DNA, he did his work in an exceptionally clean laboratory, with a ventilation system sanitized by ultraviolet light. The lab was run in accordance with hot-zone-style biosafety procedures, with workers clad in sterile gloves, masks, and boots.
Pääbo ended up extracting and analyzing short stretches of DNA from the 2,400-year-old mummy of an infant boy. It was the first time in history that anyone had done such a thing, and his 1985 paper reporting his findings—his very first scientific paper, published while he was still a grad student—ran as a cover story in Nature: “Molecular Cloning of Ancient Egyptian Mummy DNA.”
Pääbo then turned his attention to Neanderthal man. In 1997 he obtained from the Rhineland Museum in Bonn a half-inch (1 cm) bone sample of a 42,000-year-old Neanderthal fossil. He ground up the sample, dissolved it in a chemical solution, and extracted recognizable mitochondrial DNA fragments. Later, he and colleagues tested more than seventy Neanderthal tooth and bone samples and obtained useful DNA from six of them.
In May 2010 Pääbo and an international team published “A Draft Sequence of the Neanderthal Genome” in Science. Four of the gene sequences discovered by members of that team have brought us somewhat closer to a more accurate picture of Neanderthal man. Fragments of the MC1R gene suggest that the Neanderthals were likely to have light rather than dark skin. Another discovery was more portentous: the presence of parts of the FOXP2 gene, which is involved in speech and language. This means that if we ever clone a Neanderthal into existence, we might actually be able to converse with him or her.
But why resurrect a Neanderthal? Or for that matter, any other animal?
The most obvious reason for resurrecting extinct species is to attenuate, even partially, the wave of mass extinction that is currently taking place and is a hallmark of the Holocene—our own epoch. If the continuing loss of countless species is a tragedy, then the introduction of effective countermeasures, and the increase in species diversity that will accompany them, can only be viewed as a benefit.
If we can rescue one species from permanent extinction, then we can rescue others as well. Zoos are already becoming agents of species conservancy, keeping germ lines intact in living examples. In addition to living zoos there are also “frozen zoos,” repositories of DNA as well as frozen, viable cell cultures, semen, embryos, oocytes, and ova, as well as blood and tissue specimens of extinct, rare, or endangered species. The Frozen Zoo at the San Diego Zoo, for example, houses samples from more than 8,400 individuals representing more than 800 species or subspecies. There is a smaller collection of genetic samples from rare and endangered species in cryo-preservation at the American Museum of Natural History in New York.
Worldwide, there are about a dozen frozen zoos. The genetic material in storage there could be used in the same type of nuclear transfer cloning experiments that produced Dolly and the bucardo clone. Frozen zoos exist to amplify the gene pool,
increase genetic diversity, and rescue populations of endangered species; successful efforts to revive species that are already extinct will have the same effect. Extinct species by definition have been removed from the gene pool. Cloning them back into existence will bring their lost genetic material back into circulation.
But how can cloning, which produces only carbon copies, be used to increase genetic diversity? If you clone from frozen tissues of dead animals, cloning them back into existence reintroduces their lost genetic material into the global population.
Some organizations are doing this systematically, for example, the Audubon Center for Research on Endangered Species (ACRES) near New Orleans. Established in 1997 with $20 million in public and private financing, its long-term goal is to “unlock the secrets that could make extinction extinct.” Its immediate goal is to use nuclear transfer cloning to increase the gene pool, genetic diversity, and populations of endangered species.
ACRES researchers have been enormously successful, cloning several examples of a species of endangered African wildcat. But then they did something new: they bred together some of their clones, which then gave birth naturally. This was first done in the summer of 2005; the cloned wildcats produced two litters totaling eight wildcat kittens, all of them through natural birth. ACRES researchers thus have established that cloned animals can mate with each other and produce natural offspring. This creates additional specimens of the endangered animals, specimens that contain entirely new combinations of genetic material. Using these methods, ACRES has been able to increase the population of endangered Mississippi sandhill cranes by more than 20 percent in two years.
The director of the center, Betsy Dresser, makes several good points with respect to the benefits of cloning endangered species:
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