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Genome Editing Reaching Farm Animals, Though Regulatory Uncertainty Looms


NEW YORK (GenomeWeb) – Academic and industry investigators are pressing forward with genome-editing efforts to tackle intractable farm animal health and welfare traits, despite uncertainty about the regulatory hurdles that products from these animals might face before entering the food chain.

From pigs that can resist deadly viruses to dairy cattle that are genetically de-horned, genome-edited animals have been turning up with increasing frequency in the scientific literature, spurred on by investment from companies such as Genus, Recombinetics, and eGenesis. But those in the field are still waiting to see how the US Food and Drug Administration (FDA) and regulatory agencies in other countries will deal with such animals.

Last January, the FDA proposed draft guidance suggesting modifications intentionally introduced into animal genomes should be regulated in a manner comparable to new drugs, meaning developers would have to show efficacy, animal and human safety, and safety for the environment. It remains to be seen whether those regulations will be adopted. And similar regulatory vacuums exist for genome-edited animals in many parts of the world.

"Having no regulations means [the products] kind of get sucked up in all of the uncertainty associated with different administrations and it becomes unworkable because there is no path to market," said Alison Van Eenennaam, an animal genomics and biotechnology cooperative extension specialist at the University of California at Davis, who is currently doing research on genome-edited cattle.

For her part, Van Eenennaam suggested that regulation of genome-edited animals should occur on a case-by-case basis — dependent on the type of genetic change(s) made and anticipated effects in the animal — rather than being triggered by the use of editing technology itself.

"It should be based on the risks associated with the product," she said. "If you haven't changed the risk profile of the product, there's nothing to regulate — if the purpose of regulation is safety, which it should be."

In the meantime, editing technologies are making it possible to more easily edit not just one gene, but many. George Church and colleagues from eGenesis, Harvard Medical School, and other centers in the US, China, and Denmark demonstrated in Science last summer that they could simultaneously inactivate large numbers of porcine endogenous retroviruses with CRISPR-Cas9 technology.

During a plenary presentation at the Plant and Animal Genome conference in San Diego last month, Bruce Whitelaw, animal biotechnology chair at the University of Edinburgh's Roslin Institute, walked attendees through the evolving technologies used for genome-editing — from restriction endonuclease enzymes to zinc finger nuclease or transcription activator-like effector nucleases (TALEN) technologies and, more recently, CRISPR-Cas9-based editing.

"I think the jury is out on which [gene-editing technology] will become dominant," Whitelaw said in an interview.

"It's probably going to be the licensing deals and the ownership of the [intellectual property] that will determine that," he added. "But from an experimental perspective — for all the research labs working on animals, plants, bacteria, viruses, you name it — CRISPR is just so easy to do."

In his own lab at the Roslin Institute, genome-editing projects in cattle, poultry, fish, and swine are already underway, though a handful of pig projects are currently furthest along.

For example, Whitelaw and his team are searching for ways to allay the consequences of African swine fever virus (ASFV) infections, which leads to deadly hemorrhagic fevers and rapid death in pigs from most parts of the world.

There is no effective vaccine or treatment for the disease, he explained, so infected herds are quarantined and destroyed. But there are a few pigs that are naturally immune to ASFV: although ASFV is endemic to Africa, and naturally infects warthogs or bush pigs, it does not produce perceivable symptoms in the African pigs, prompting investigators to search for host-specific genetic differences protecting the animals in Africa.

In a study published in the Journal of Virology in 2011, Whitelaw and colleagues from the UK, Australia, and the US used targeted, candidate gene sequencing in domestic pigs and warthogs to narrow in on variants in the NF-kappa-B transcription factor component-coding gene RELA. Because that transcription factor is involved in the pro-inflammatory "cytokine storm" that kills ASFV-susceptible pigs, they suspected that the warthog version of RELA may offer protection against this pathogenesis.

But even if the warthog version of RELA does prompt ASFV resistance, breeding warthogs with domestic pigs is not a realistic way to get the ASFV-resistance alleles into domestic pig populations. And that's where genome editing comes in.

For a proof-of-principle paper published in Scientific Reports in early 2016, Whitelaw led a team of investigators from the Roslin Institute, Sangamo Biosciences, and the animal genetics company Genus that used zinc finger nuclease-based editing and homology-directed repair to swap the warthog version of the RELA gene into domestic pig embryos, leading to three live-born piglets carrying the edited RELA gene.

"We're using editing to bring that allele into the domestic pigs on the hypothesis that this will affect how severe virus infection is," Whitelaw said. "We don't know the answer yet," he added, noting that the team plans to challenge RELA-edited pigs with ASFV later this year to determine whether they remain susceptible or have achieved resistance to the virus.

Whitelaw's team and other groups at the Roslin Institute have established strategic partnerships with Genus and other commercial sector firms or government funding agencies to advance such research.

In addition to funding the ASFV study, for example, Genus's Pig Improvement Company (PIC) branch is supporting Whitelaw's team and a team at the University of Missouri led by Randall Prather as they independently use CRISPR-Cas9-based gene editing to chase resistance to an even more widespread and economically important pig adversary: the porcine reproductive and respiratory syndrome virus (PRRSV).

In a correspondence appearing online in Nature Biotechnology in 2015, Prather and co-authors from the University of Missouri, Kansas State University, and Genus described PRRSV-resistant pigs produced using CRISPR-Cas9 to knock out CD163, a gene believed to code for a PRRSV receptor on pig cells.

The Roslin researchers did their own tweaks to CD163 to boost PRRSV resistance for a study published in PLOS Pathogens in early 2017. Rather than knocking out the gene completely, they used CRISPR-Cas9 to remove exon 7 of CD163, producing pigs with immune cells that were no longer susceptible to PRRSV infections.

Whitelaw noted that Genus has applied for a New Animal Drug Application (NADA) from the FDA for its CD163-edited, PRRSV-resistant pigs and is also taking the product to regulatory agencies in other countries.

The PRRS virus costs pig producers in the US more than $600 million each year, according to a 2013 economic assessment in the Journal of Swine Health and Production. Global annual losses linked to PRSS virus run far higher, not to mention the massive culls producers must carry out when PRRSV infections are identified.

The teams are not alone in its animal welfare-related editing pursuits. A Minnesota-based biotechnology company called Recombinetics is applying gene editing to everything from biomedical research to livestock improvement.

On the its website, the firm touts efforts to develop male pigs that are "naturally castrated" using genetics — a project being done in collaboration the swine genetics supplier to avoid boar-taint in pork meat without manually castrating male piglets.

In collaboration with researchers at the University of Minnesota and Texas A&M University, researchers at Recombinetics have also developed hornless, or "polled," dairy cattle, using TALEN-based editing to introgress a polled gene from Angus beef cattle into dairy cattle from the Holstein breed — work they reported in a correspondence to Nature Biotechnology in 2016.

"Physical de-horning of cattle, which is done to protect animals and producers from accidental injury, is not only costly but is also painful for the animals and has come under scrutiny owing to public concerns about farm animal welfare," they wrote.

Although some Holstein cattle are naturally polled, the gene often turns up in low genetic merit Holsteins — those with less-favorable genetic traits, from a dairy point of view — or in more meritorious bulls with pricy semen, Van Eenennaam explained.

Her lab is currently following offspring from one of the genome-edited polled Holstein bulls (which was crossed with horned Hereford cows), to ensure that the edited allele segregates as anticipated and continues producing the hornless phenotype.

Although the animals are still young, their meat and milk will eventually be evaluated and compared with products from several control cattle. Still, Van Eenennaam said, "it's a bit unclear for what hypothesis we are designing experiments to test."

"We eat polled animals all the time," she said. "Realistically, what's the risk of this particular gene editing application, when we're not introducing a novel protein?"

To look for potential off-target gene-editing effects, her team is collaborating with researchers in Australia to sequence the genomes of calves born from the edited bull. The Recombinetics-led team that reported on the first two edited, polled Holsteins sequenced those animals as well.

For his part, Whitelaw noted that off-target rates of existing editing technologies are low and getting lower, falling far short of the de novo mutation and recombination rates that naturally occur during sexual reproduction.

While presenting at PAG, Whitelaw predicted that genome-edited pigs might be on the market within five years. He pushed that estimate back during a more recent interview, suggesting that it will be at least five — but not more than 10 — years before CD163-edited pigs reach our plates, provided there is consumer approval and regulatory frameworks that allow the technology to go forward.

In their search for regulatory clarity, those in the genome-editing field point to regulatory holdups faced by some genetically-modified plants and animal products in the past.

For example, after seeking approval from the FDA for more than two decades, AquaBounty Technologies' genetically engineered, fast-growing Aquadvantage Atlantic salmon was deemed safe for human consumption and environmentally low-risk by the American regulatory agency in 2015.

Even so, the transgenic fish — which contains a growth hormone-regulating gene from another salmon species, the Chinook salmon, under the control of an ocean pout promoter — has not yet reached the US market. A 2016 ban prevents the Aquadvantage salmon import or sale until the FDA comes up with clearer labeling guidelines for the fish.

In contrast, the Canadian Food Inspection Agency approved the Aquadvantage salmon for sale in Canada in 2016. AquaBounty Technologies made headlines in mid-2017 when it announced that some 4.5 metric tonnes (five US tons) of Aquadvantage salmon had already been sold to Canadians.

The advent of genome-edited farm animals has raised similar, but distinct issues for regulatory agencies. Unlike transgenic animals, animals produced with genome editing do not necessarily include recombinant DNA or DNA from another species.

In the case of the Recombinetics' polled Holsteins, for example, investigators took advantage of diversity that already exists in the species, moving a "Celtic" polled allele from cattle breeds already consumed by humans into dairy cattle from a different breed.

Recombinetics has not submitted a New Animal Drug Application for the genome-edited polled cattle, Van Eenennaam noted, since it is unclear whether that will ultimately be necessary for that type of edit.

"The FDA wants us to open a New Animal Drug Application in order to tell us whether the polled allele is a new animal drug," she said. "If the only way you can find out if you're a drug is to say you're a drug, that puts you in a Catch-22, because if you're not a drug, you don't have to open a New Animal Drug Application."

Further, Van Eenennaam argued that if NADAs are required for all genome-edited animals, it may become too costly for investigators in academia, the public sector, or small companies to develop products that reach the market, leaving the technology in the hands of large companies with deep pockets.