Team that resurrected dire wolf has Pitt, Penn State connections

Some of the scientific groundwork that paved the way for a rebirth of the long-extinct dire wolf took place in Pennsylvania.

With much fanfare, Texas company Colossal Biosciences announced April 7 that its scientists had returned the dire wolf, a species extinct for more than 10,000 years, “to its rightful place in the ecosystem.”

They did so by way of Romulus and Remus, two genetically modified pups born using a gray wolf surrogate that exhibits several key traits of the long-extinct Ice Age species.

Colossal Biosciences’ Chief Science Officer Beth Shapiro, 49, was an assistant professor at Penn State University. And Vice President for Artificial Womb Technology Trevor Snyder, 50, spent nearly a decade at the University of Pittsburgh, first earning his doctorate in bioengineering and then working as a research assistant.

Colossal’s announcement set off a spirited debate among scientists and media pundits about what exactly constitutes “de-extinction,” which is defined by the International Union for the Conservation of Nature as resurrecting the key traits that embody a particular species.

“When we say we’ve succeeded in de-extinction, we’re following the IUCN’s Species Survival Commission guidelines and have resurrected some of these key phenotypes, acknowledging that it’s not possible to bring something back that’s identical,” Shapiro, a native of Allentown in Lehigh County, said in an interview with TribLive.

Using DNA extracted from two dire wolf fossils, Colossal’s team made 20 targeted gene edits to the DNA sequence of a gray wolf, which shares a common ancestor with the dire wolf. It produced pups with a lighter coat and larger size than gray wolves.

Does that make it a dire wolf?

“Those 20 gene edits bring back some of what we think are the key traits that embody the dire wolf,” Shapiro said. “But there’s about 30 different species concepts that are circulating in the biological community, and by some of them you’d say it is a dire wolf; by others, you wouldn’t.”

For Colossal, it’s a dire wolf. And semantic and philosophical debates aside, Shapiro said she is excited about the potential applications for several of the techniques used in creating Romulus, Remus and a third pup that was not featured prominently in the Colossal announcement.

Is Colossal looking to reintroduce dire wolves to the wild? No. Shapiro said the pups are being raised at a large, undisclosed wildlife area in the United States.

But scientists believe gene editing has implications for many animals that are threatened or nearing extinction.

Ancient DNA

After an academic path that saw her study as a Rhodes Scholar at England’s Oxford University, Shapiro landed a 2007 assistant professorship at Penn State. During her time at University Park, she was part of the development of an ancient DNA lab and was awarded the MacArthur Fellowship, sometimes referred to as the “genius grant,” focused on work that helped lay the foundation for Colossal’s dire wolf announcement.

Shapiro was able to use genetic information gleaned from ancient plants and animals to learn how evolution happens over time and terrain. In the mid- to late 2000s, technical advances in genomic sequencing technology allowed researchers to reach into those specimens of ancient DNA and learn crucial information about how ancient animals responded to large-scale changes in their habitat and environment.

“We’d go out into the world, dig up bones of things that used to be alive and develop protocols to extract the tiny bits of DNA preserved in those bones,” she said. “Then we map them in a way that makes it possible to learn about how populations grow and shrink in response to climate change or how they were impacted by people turning up in their habitats.”

Shapiro’s early work focused on bison and horse bones from the Arctic because the frigid temperatures better preserved the specimens she studied.

“The ancient DNA work led to the development of tools we eventually used to sequence the dire wolf genome, assemble it, analyze it and figure out what makes a dire wolf a dire wolf,” she said.

Colossal’s team learned all it could from studying the recovered pieces of the dire wolf’s DNA sequence, focusing on how a particular sequence correlated to the way it is expressed in the animal’s physical features.

“That is one of the hardest problems in biology right now,” Shapiro said. “We wanted to choose as few edits as possible because every additional edit you make runs the risk of an unintended consequence in the host.”

The team developed computational tools to try and predict the results of gene edits they hoped to make in gray wolf DNA, Shapiro said.

“A good example is pigmentation,” she said. “When we sequenced the genomes for both fossil specimens, we saw that both had variants in three pigmentation genes. But our analysis said those same genes in gray wolves can sometimes lead to deafness and blindness. They aren’t the same variants, but they’re part of the same genes.

“That was an unacceptable risk for us, so we decided we weren’t going to make those specific changes from the dire wolf genome. We made different changes, variations that already exist in dogs and gray wolves. That way, we can de-extinct this phenotype in a safe way. And that’s a development that I think really has some applications in conservation.”

Plugging gaps

While splashy headlines like “The Dire Wolf Is Back. Or Is It?” certainly grab attention, reintroducing a long-extinct animal to the North American ecosystem is not the goal.

All of the focus on Romulus and Remus obscured the fact that some of the same technology Colossal used to create them also was used to clone four red wolves, a species that still exists but is critically endangered in the U.S. There are fewer than 20 red wolves left in existence.

“They’re genetically bottlenecked, which means the ones left are all descended from just a few individuals,” said Snyder, who earned his bioengineering degree and worked as a research assistant at the University of Pittsburgh from 1998 to 2007. “The methods Colossal has developed can be used to restore some healthy genetic diversity to this and other critically endangered species.”

Shapiro told TribLive that Colossal is collaborating with Australian scientists to try to save an endangered species called the northern quoll, a small carnivorous marsupial.

Toads are a prominent part of the quoll’s diet, and the introduction and proliferation of the invasive — and poisonous — cane toad in Australia has led to dire consequences for the quoll population.

“Northern quolls are adorable, but they’ll probably be extinct in the next 10 years because they keep eating cane toads and dying,” Shapiro said. “We’ve created cell lines of quolls that have just one edit to their genome that allows them to eat a cane toad and survive. We’ve been able to learn from comparative genomics and genome editing to be able to do something that can save this species from going extinct.”

Shapiro said staff at Revive & Restore, a California company that is also exploring the genetic rescue of endangered and extinct species, is leading a similar effort with black-footed ferrets.

They are endangered — and at one point were thought to be functionally extinct — because many contract a disease called sylvatic plague when they consume prairie dogs, a favorite prey. But their domestic cousins are genetically immune to the disease.

“The plan is to figure out what gives domestic ferrets that genetic immunity, and then put that into the black-footed ferret,” Shapiro said. “What you would ideally end up with is something that’s almost 100% black-footed ferret, with just one or two edits that allow them to survive in the habitat that has introduced the disease that’s killing them.”

What’s next?

During Snyder’s time at the University of Pittsburgh, he worked on developing medical technology in the form of life-supporting devices such as heart pumps, artificial lungs, dialysis machines and bio-artificial organs.

Today, he’s hoping to bring some of that artificial assistance to wild conservation.

“At Colossal, we’re applying those approaches to develop artificial womb technology,” said Snyder, a native of Oklahoma. “Current cloning technology, which we used for the dire wolves, requires the use of surrogates, such as dogs or wolves. Implanting clone embryos carries some risk for the surrogate.”

Artificial wombs, on the other hand, could alleviate the need for surrogates. Snyder said Colossal’s goal is to determine if the technology can be applied to a variety of larger animals.

“If we can demonstrate that it’s safe in larger animals, it may be applicable to humans and could perhaps be used to rescue prematurely born infants,” Snyder said.

It could also be used to de-extinct an animal such as the Western black rhinoceros, for which there are no surrogates available. One of the subspecies of black rhinos, it was declared extinct in 2011 by the International Union for Conservation of Nature. Several other subspecies already have gone extinct, and the species overall is listed as critically endangered in its native Africa.

Public funding

Colossal is a privately funded company. But Snyder and Shapiro said their work wouldn’t be possible without the groundwork laid by decades of publicly funded science, which is among the many areas targeted for federal spending cuts by the Trump administration.

“This is a classic standing-on-the-shoulders-of-giants moment,” Shapiro said. “None of what we’ve accomplished would be possible without the publicly funded science that led to the development of (DNA and gene editing technology) and all of the research infrastructure that exists to allow us to sequence genomes and compare them to one another.”

Publicly funded science was a big part of the reason the dire wolf was Colossal’s first project, Shapiro said.

“It was such a great example for a first species because there’s so much science that’s been developed by the National Science Foundation, the National Institutes of Health and other foundations around the country and the world,” she said. “It was something where we could come up with the idea, start working and, 18 months later, there are puppies born.”

Does this mean that extinction is a thing of the past? Not likely, Shapiro said.

“We have to stand on the back of all the traditional approaches to conservation that are already out there,” she said. “Conservation is a field where we’ve been largely doing the same thing for the past 100 years. And it’s working. But in a lot of cases, it’s not working fast enough.”