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This is a story about two phone calls and two accomplished researchers being equally surprised by the news coming from the other.
The first call came in 2011 when Randall Prather — a pig geneticist and Curators professor in reproductive physiology in the Division of Animal Sciences in the University of Missouri’s College of Agriculture, Food and Natural Resources — placed a cold call to Bob Rowland, a pig virus expert who serves as a virologist and professor of diagnostic medicine and pathobiology at Kansas State University’s College of Veterinary Medicine.
Up until that point, the two had never spoken to each other, although they both were aware of each other’s work in their respective fields. Prather, who has two degrees from KSU as coincidence would have it, had a proposition for Rowland: Take a sampling of genetically modified pigs from Prather’s laboratory that have had the cluster of differentiation (CD) no. 169 sialoadhesin molecule knocked out of their genetic code and challenge them with Porcine Reproductive and Respiratory Syndrome (PRRS) virus and see if they get sick, using Rowland’s specialized facility located 245 miles west of the MU campus.
To Rowland, it sounded like the best opportunity to finally slay a dragon of a virus that has accounted for approximately $10 billion in losses for the pork industry in the last 20 years in the U.S. alone.
“It just blew me out of my chair,” Rowland said. “I didn’t know his previous affiliation with K-State, but when he called me and told me what he wanted to do, it was just incredible. What he was working with was just a real game changer in terms of infectious disease work.”
“He already has the infrastructure to do exactly what I wanted done with these pigs, so I called him up and he said ‘Sure, I’d love to,’” Prather said.
Based on the previous research, the CD 169 molecule was thought to have been the entry mediator to the white blood cells in the lungs, where the disease would cause such maladies as an inability to reproduce, small size and a high mortality rate. It was not, but it lead to the second phone call four years later this past March.
This time Prather picked up his phone and heard an excitable Rowland, who was on vacation relaxing on a beach in Florida before talking to the technicians at his lab about the ear tag numbers of three pigs. It turned out that the trio were all pigs that had the CD 163 molecule knocked out of their genetic code at Prather’s lab during a research project that was funded through British-based biotechnology company Genus plc. They exhibited no signs or evidence or being infected with PRRS after being challenged with the virus. The study is being published in Nature Biotechnology this month.
The second time worked.
“Pleasant disbelief. ‘Did it really work?,’” recalled Prather of his initial reaction. “I thought it would work, but it was so clean. What did I expect? I expected the pigs to still get kind of sick.”
After hanging up the phone in his office in the eastern sector of the Animal Sciences Research Center, Prather went a few feet down to the office of Kristin Whitworth, a research scientist on his lab team who has worked with him for 20 years.
“We didn’t say a whole lot. We just kind of stared at each other,” Whitworth said. “He said ‘Bob was sitting on a beach, but he’s not sitting on the beach anymore.’”
The waiting game
When Prather first heard about the PRRS virus in 1989 — the year he started at MU as the newest addition of the reproductive biology cluster of the Food for the 21st Century (F21C) program — it was called Swine Mystery Disease.
Rowland first started doing research on PRRS in 1994 while on staff at South Dakota State University, where his colleagues included David Benfield, who graduated from MU in 1979 with a doctorate degree in virology/microbiology. Benfield, who now works at The Ohio State University’s College of Veterinary Medicine, is credited as being a co-discoverer of the virus. During his career, Rowland has worked on 10 major viruses that concern pigs, but “PRRS has been my bread and butter.”
In 2001, Prather’s team made the discovery that it was possible to make “knockout pigs” (as they are called by inactivating a certain gene) through the xenotransplantation, i.e. transplanting a pig organ to a human. At that point, his team had submitted a disclosure that suggested further exploration of CD 169 and CD 163 molecules, but it would take more than a decade to secure the proper funding — and as it turned out, the right technology, to come around.
For the CD 169 project, Prather’s lab used a time-intensive process known as homologous recombination. It was the only method in the animal genetics research circle until the revolutionary CRISPR/Cas9 system came out. Jennifer Doudna at the University of California-Berkeley is credited with first coming up with the system in 2012.
The system works by allowing researchers to more easily target genomes and perform precise gene editing by taking a ribonucleic acid (RNA) to target the piece of deoxyribonucleic (DNA) and then having a Cas9 protein bind the two helix structures and then precisely clip where the RNA guide bends. Simply put, researchers just have to pick a sequence of DNA that they would like to edit and inject the CRISPR/Cas9 into a zygote cell through a micropipette.
Prather’s team first used the system in July 2013 after Kevin Wells, an associate professor in the Division of Animal Sciences who works closely with Prather’s team as a genetic engineer, first told him about Doudna’s paper in November 2012. “Up until that point, every system that had been described required multiple proteins, so it would take many genes to make use of this,” Wells said. “That paper demonstrated for the first time that it was a single gene, which seemed to me to be something that we could work with.”
The proceeding CD 163 project was initially going to be carried out entirely using the older method, until — as Wells put it — “we realized that we really speed things up if these CRISPRs worked the way we thought they did.”
In a short period of time, the new system cut down significantly on time in the lab and overall costs. The lengthy periods of building a target vector and crossing fingers that the desired genetic editing would actually go through were over.
“That has just changed the way we do business, because it makes trying to do a knockout not ‘can we do it’ but ‘how quick can we do it,’” Prather said. “All of a sudden you don’t have to worry about the months to years to build that targeting construct. You have a few hours designing and then a day building it and then you’re ready to inject it into zygotes and see if it works.”
“It was a game changer for us because it worked so efficiently and it worked the first time we used it,” said Whitworth, who was in charge of designing the CRISPR systems for the CD 163 research.
From hoof to trotter
As a researcher who has become synonymous with swine on the MU campus, there are few bacon jokes or puns that Prather has not heard. As he will tell you, though, it was a career path that he did not initially envision while growing up in Manawa, Wisconsin, a small town that sits west of Green Bay.
When Prather’s family moved to Garnett, Kansas, when he was in high school, Prather had plans of one day working on the family’s cow-calf production farm after earning his undergraduate degree in animal science from K-State, the alma mater of both of his parents. “‘The farm isn’t ready for me. I need to do something else,’ so I thought ‘In a couple of years, I’ll get my master’s degree and the farm will look better.’ It didn’t, so I decided to stay in school,” Prather said.
Before he finished his undergraduate degree, his advisor, Bob Hines, who taught a course in swine sciences implored him to take it as part of his academic requirements. “I said, ‘I’m not working with pigs and I’m not taking your class.’ He said, ‘You’re not going to graduate,’ so I took his class.”
After completing his graduate degree in animal since at KSU in 1984, Prather enrolled in the doctoral program in endocrinology and reproductive physiology at the University of Wisconsin, where he had planned to do research on cattle and begin to assimilate the professional interests of his father, Elvin, who had spent a majority of his career as a doctor of veterinary medicine with a dairy practice. When Prather arrived in Madison, he found the cattle research space to be overcrowded. The ability to work with pigs was far easier — and became even more so when the lone person who was working on porcine research left for a job at Ohio State.
“I said ‘Forget this cattle stuff. I’m going to go work with pigs because I can get material to work with.’ It’s one of the best decisions of my life. That’s why I work with pigs: because someone else left and there was a vacuum and I filled it. It doesn’t have anything to do with me loving pigs. It was me taking advantage of the opportunity that was there.”
Prather came to the realization that pigs could be great models when it came to tackling human health issues, noting, among other qualities, that they have very similar cardiovascular and immune systems as compared to a human’s. “They’ve just got a whole list of things,” Prather said.
Prather’s discoveries within porcine research began to grow, leading up to the “Wall of Pork and Beef” and later the establishment of the National Swine Resource and Research Center in 2003 (where he serves as a co-investigator with Wells). To date, Prather and his team have made more than 1,000 cloned pigs representing more than 40 different genetic modifications. Prather’s milestones continue to expand, even if the size of his office does not.
“You see this pile on the shelf behind me? I don’t have any more wall space,” Prather said as he pointed to a stack of nine pictures on top of a shelf.
More work to do
In addition to their ability to fight off PRRS, the group of CD 163 knockout pigs also have other attractive characteristics. Just ask Melissa Samuel. As a senior research specialist who was worked on Prather’s team for 15 years, Samuel has seen her fair share of pigs as the person who oversees the care for the pigs at the ASRC.
“This group of pigs is my favorite ones because they’re so easy to work with,” she said. “When you work with pigs for 15 years, you have your favorite pigs and your not-so-favorite ones and certain lines are friendlier than other ones, but this group is my favorite group because they work so well and they definitely have their personalities, too.”
When pigs such as these are available to the pork producers has yet to be determined. If development stage is successful, the commercial partner will seek any necessary approvals and registration from governments before a wider market release. The University of Missouri has signed an exclusive global licensing deal for potential future commercialization of virus resistant pigs with Genus plc.
What is known is that with the partnership of Prather and Rowland and the CRISPR Cas9 method, this is an exciting time for all of those involved. Virus experts such as Rowland can now turn their focus away from vaccines and look to genetic editing to solve the other diseases that plaque the food industry.
Both Prather, who has teamed up with 52 different investigators from 37 institutions during his career, and Rowland look forward to many other joint projects down the road. “Really our power in collaboration is the fact that we can accelerate discovery,” Rowland said. “He makes the pigs. He gives them to me. I challenge the pigs and we move on. I guess it’s a good definition of the word synergy because together we’re doing so much more than we could ever do separately.”
Rowland added that although a collaboration is often referred to as an activity, he said that “it is really a study of human character.”
“All of the good collaborations that I have are with people of tremendous character. Anybody who can be patient with me has to have a lot of character,” Rowland said with a chuckle.
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