March 15, 2016

 

 Editing Animals

Gene editing applications expanding with advancing technology 

Posted March 2, 2016

Rapid developments in gene editing and genetic understanding have brought humanity to a new dawn of discovery and transformation in medicine, according to Dr. K.C. Kent Lloyd. 

He is part of an international effort to create and phenotype about 20,000 mouse strains, each with a different gene turned off, to identify how similar genes in humans may contribute to disease. The information is expected to help improve diagnostic testing, identify subtle disease signs, and develop focused treatments—including genome editing—in human and veterinary medicine.

“We’ve still got a dairy cow with all the good dairy genetics. We’ve just gone in and tweaked a little snippet of DNA at the gene that makes horns and made it so it’s the variant for Angus, which doesn’t grow horns.”
Alison L. Van Eenennaam, PhD, geneticist and cooperative extension specialist,
University of California-Davis

Gene editing in agriculture already has produced pigs and cattle resistant to particular diseases and dairy cows lacking horns, and current research promises to produce chickens resistant to heat, beef herds in which only the more profitable male calves are born, and chicken flocks in which only females needed for egg production are hatched.

Alison L. Van Eenennaam, PhD, a geneticist and cooperative extension specialist also at UC-Davis, is working with the Minnesota-based company Recombinetics on, among other things, a project that has produced some of the Holstein dairy cattle that lack horns by editing one allele to match another found in Angus cattle.

“We’ve still got a dairy cow with all the good dairy genetics,” she said. “We’ve just gone in and tweaked a little snippet of DNA at the gene that makes horns and made it so it’s the variant for Angus, which doesn’t grow horns.”

At a December 2015 International Summit on Human Gene Editing co-hosted by the National Academies of Sciences, Engineering, and Medicine; Chinese Academy of Sciences; and U.K.-based Royal Society, presenters described potential uses of gene editing in humans to reduce pregnancy loss, early childhood death, abnormalities, and disabilities; avoid transmission of genetic diseases such as cystic fibrosis and Tay-Sachs disease; and control HIV in adults. They also described the risks of off-target cleavage in gene editing, the complexity of genetics, the risks of introducing permanent changes to the human gene pool, and the potential that people could use the technology to not only enhance disease resistance but also amplify sensory perceptions or select for favored eye colors. 



Alison L. Van Eenennaam, PhD, center, and graduate student Lindsay Upperman hold Holstein cattle born with a gene edited so that they grow without horns, as is typical of Angus cattle. (Courtesy of Karin Higgins/UC-Davis)

Humans are becoming masters in the art of manipulating genes, despite limited understanding of gene function and interaction, Klaus Rajewsky, MD, head of the immune regulation and cancer research team at the Max Delbrück Center for Molecular Medicine in Berlin, said during the summit. And one recent technology, CRISPR-Cas9, is so overwhelmingly efficient and specific that it is changing the outlook of future gene editing.

Democratizing technology 

CRISPR-Cas (clustered regularly interspaced short palindromic repeats–CRISPR-associated proteins) genome editing adapts an immunologic defense system that is used by bacteria and archaea against predatory phages and plasmids. The defense system uses guide RNA with complementary sequences to target DNA, finding and cutting foreign genes matching those previously encountered, a mechanism that can be used for site-specific cuts across animal and plant sequences.

One recent article indicates companies interested in using the technology for gene therapies have raised more than $600 million in venture capital and public markets since 2013.

“The pace of this activity is remarkable given that the first granted patent for the use of CRISPR technology in eukaryotic cells was issued April 15, 2014,” it states.

CRISPR, along with other gene editing technologies, is seen as a potential source of diagnostic tests, treatments, vaccinations, and alterations in fields as varied as clinical medicine and yogurt production.

Dr. Van Eenennaam, who uses CRISPR and TALEN (transcription activator–like effector nucleases) for editing, said CRISPR is easier to use and more efficient than older technologies, and its low cost has democratized the field.

She describes the gene editing as akin to “precision breeding,” hastening selection rather than eliminating it.

The project to produce hornless Holsteins is funded through a grant from the Department of Agriculture’s National Institute of Food and Agriculture, which indicates that removing horns in conventional Holsteins has inspired criticism. Traditional breeding would require decades to produce cattle without horns but with equivalent milk production.

Researchers with the animal genetics company Genus used CRISPR-Cas9 to disable a cell receptor and produce pigs resistant to the deadly porcine reproductive and respiratory syndrome, announcing the results in correspondence published online Dec. 7, 2015, in Nature Biotechnology. The protein disabled in the modification, a scavenger receptor, is connected with removal of excess hemoglobin from blood, and a company officer has indicated further refinements could retain protein function while preventing the PRRS virus from binding to cells. Editing also could expand which species are available as animal models.

Angelika Schnieke, chair of livestock biotechnology at the Technische Universität München, Germany, said in a presentation during a December 2015 workshop on scientific and ethical considerations in editing animal genomes that pigs can be useful models for studying cancer, cardiovascular disease, and obesity, replicating human disease, physiology, and pharmacokinetics more closely than do rodents. They also are better models for imaging technology, stent placement, and surgery, she said. 

They see genetic engineering of animals as something that requires a very compelling argument in order to support it.”

Paul B. Thompson, PhD, a professor of philosophy and the W.K. Kellogg Chair in Agricultural, Food, and Community Ethics,
Michigan State University

Peter Hohenstein, PhD, a research group leader at the Roslin Institute at the University of Edinburgh in Edinburgh, Scotland, and whose laboratory has incorporated genome editing with TALEN and CRISPR-Cas9 for research projects, said in the workshop that the total number of animals needed may remain the same even as fewer are needed for individual projects. If creating animal research models becomes easier and less costly, he said, funders will expect more scientific output.

Dr. Lloyd said knockout mice produced in his laboratory already are providing insights into human diseases. Knocking out one mouse gene known as FAM151B, for example, reduced an ocular cell layer, resulting in retinal atrophy. He thinks the findings could help identify genetic causes of blindness in humans.

Knocking out a different gene produced the first mouse model of the human disease Bardet-Biedl syndrome 5, resulting in mice with both obesity and metabolic disorders, he said.

Compelling argument and risk assessment

Paul B. Thompson, PhD, a professor of philosophy and the W.K. Kellogg Chair in Agricultural, Food, and Community Ethics at Michigan State University, said genetic alteration of animals, particularly those raised for food, raises moral issues for the public.

“They see genetic engineering of animals as something that requires a very compelling argument in order to support it,” he said.  

But he thinks that most people lack well-formed opinions on genetic engineering and are susceptible to suggestions in survey questions. People’s opinions also change with shifts from general topics to specific uses, he said.

Modifying mice for biomedical research, for example, tends to be viewed as morally problematic but necessary, he said. Social science research has provided only a murky picture of public opinion on genetic modification of foods so far, but he thinks modifications intended to combat disease are more likely to be accepted than those solely to increase food production or efficiency thereof.



Left: This chimeric mouse was produced by injecting embryonic stem cells of one mouse with an agouti coat into the blastocyst of another mouse with a black coat. Some offspring from this mouse will express a mutation present in the stem cells and be useful for further study. Right: A surgical embryo transfer recipient of zygotes was modified, using CRISPR-Cas9 genome editing as part of the Knockout Mouse Project. (Photos courtesy of Dr. K.C. Kent Lloyd/UC-Davis)​

“I think that it would still generate a significant amount of disquiet and opposition, and the disquiet partly just reflects this sense that people—they would need to be convinced that this was not just some way to continue with what they would otherwise regard as an unacceptable production system,” he said.

Most corn and soybeans grown in the U.S. are genetically modified, and other modified crops include cotton, sugar beets, apples, and potatoes. The FDA also approved the first medical product of gene splicing technology, a recombinant DNA human insulin produced in bacteria, in 1983.

Michael Hansen, PhD, senior scientist for Consumers Union, said people may accept gene editing techniques as they become more familiar with the technology, in particular for modifications that improve animal welfare. But people also want disclosure when it is used as well as testing to reduce any risks associated with off-target modifications. An absence of labels distinguishing products of genetically modified animals could lead to a view producers are hiding something, he said.

Though not specific to gene editing, the Jockey Club’s rules for entry into the American Stud Book, a registry for Thoroughbreds, prevent registration of foals produced through any type of genetic manipulation. A spokeswoman for the American Kennel Club expressed doubt dogs produced in part through gene editing would be accepted for club registration and competition, but definitive answers were unavailable at press time. 


A Holstein with a gene edited to prevent horn growth (Photo by Hannah Smith Walker/Cornell Alliance for Science)

A recent “somewhat tongue-in-cheek essay” lists scenarios in which gene editing could be used for “de-extinction” of carrier pigeons, creation of whimsical animals unicornlike or dragonlike in appearance, and realization of science fiction stories of genetic manipulation for art. The authors warn that gene editing applications beyond humans could fall into legal and regulatory cracks.

Federal agencies are re-examining their controls on biotechnology products under a White House directive issued in July 2015, and the Food and Drug Administration in particular is considering how to regulate gene editing technologies.

Megan Bensette, an FDA spokeswoman, said the agency regulates any articles intended to alter an animal’s structure or function. As for “deletion” genomic editing, she said the FDA is still deliberating on its regulatory role.

We have a wonderful opportunity, actually, in veterinary medicine to lead the entire field. And what I mean is not just in veterinary medicine but lead medicine in general.”

Dr. K.C. Kent Lloyd, a veterinary scientist and director of the Mouse Biology Program,
University of California-Davis

Malini Wileman, PhD, a biologist with the FDA Center for Veterinary Medicine, further explained during the workshop on animal editing that the agency’s analysis will include considering the likelihood that introduced or altered genetic sequences could recombine with endogenous sequences or endemic viruses, the disease risks of recombination events, the methods of tracking and holding modified animals, and the durability of claims made about alterations.  

The FDA has regulated recombinant DNA constructs as drugs, notably approving in November 2015 AquaBounty Technologies’ Atlantic salmon that grow to market weight in half the time of conventional Atlantic salmon. Dave Conley, director of corporate communications for AquaBounty, declined to discuss public attitudes toward AquaBounty’s salmon and other genetically engineered foods but said in a message that the company’s salmon “is an environmentally responsible seafood choice.”

Opportunity and responsibility

While agriculture industries are taking advantage of genome editing technology, Dr. Lloyd has seen little in clinical veterinary medicine. 

“We have a wonderful opportunity, actually, in veterinary medicine to lead the entire field,” he said. “And what I mean is not just in veterinary medicine but lead medicine in general.”

Somatic cell–based treatments—involving extracting, altering, and returning cells—are in their early stages, but they have the potential to combat disease by restoring or blocking gene function, Dr. Lloyd said. Human medicine is, appropriately, proceeding with caution in therapeutic somatic cell editing and largely has limited germ line editing on the basis of ethical and moral concerns, he said.

 “We have an opportunity to proceed cautiously, judiciously, and to demonstrate the value of therapeutic editing for treating diseases in animals and in our clinical patients,” he said. 

Dr. Tom Burkgren, executive director of the American Association of Swine Veterinarians, said disease resistance is among the most logical early applications for gene editing in agriculture, not only to improve health and production but also to reduce the need for antimicrobials and vaccines. Young pigs, for example, are vulnerable before their immune systems develop and antibodies respond to vaccines.

Asked about the possibility that reducing pig diseases could reduce the number of pigs needed to meet market demands, Dr. Burkgren laughed and said to be careful what you ask for. Reducing morbidity and mortality likely would result in demand for fewer pigs and a decline in prices, but he expects veterinarians would adjust.

He also said Mother Nature tends to laugh at human plans, creating new diseases or applying old ones in new ways.

Dr. Thompson thinks veterinarians need to be prepared for a “fairly serious public conversation” on genetic modification, including within the community of veterinarians. And those who are helping develop modification technologies need to invest time and money in engaging with the public.

Dr. Lloyd said he knew of no better example of a chance to advance medicine for humans and animals.

“It’s veterinary medicine’s responsibility—the profession’s responsibility—to take advantage of that opportunity now,” he said.