To protect chickens from bird flu, researchers try to CRISPR in immunity

LONDON — In this case, it was neither the chicken nor the egg that came first. Rather, it started with some specially engineered cells in a lab.

The chicken cells had had their genomes edited, not to correct a faulty mutation, but in an effort to blunt the bird flu virus. The chickens that were bred from those cells were designed to be resistant to the infection.

The experiment was a sign of the times. A highly pathogenic strain of that flu has swept to nearly every corner of the globe in recent years and left an unprecedented wave of death in its wake, decimating both wild and farmed birds and tipping into mammals as well. While farmers trying to protect poultry have turned to isolating farmed birds from wild birds — no more ranging free for those chickens — and, increasingly, to vaccination, scientists in the U.K. on Tuesday offered up the idea of stitching immunity to influenza into chickens’ DNA. In a way, the research is an extension of humanity’s long history of enhancing certain traits in crops and farm animals, though with a decidedly 21st-century twist.

In the paper, published in Nature Communications, the researchers described using the genome-editor CRISPR to alter a protein in chicken cells that flu hijacks to make copies of itself. If the virus can’t take over that protein, the idea goes, it can’t establish an infection.

The results were promising: Almost all the chickens with the edited protein rebuffed a level of virus that the research team said approximated the flu exposure a chicken would encounter from a sick coop-mate or an infected wild bird drawn to a fowl farm to pilfer some feed. The edited chickens showed no health or developmental issues and laid eggs in typical numbers.

But the researchers set themselves a higher bar, and tested an extremely high dose of flu that caused a number of breakthrough infections in the CRISPR’d chickens. Out of concern that any virus that managed to circumvent the CRISPR’d immunity could harbor potentially dangerous mutations, the scientists said they wanted to demonstrate complete infection protection in the birds — even against such high levels of flu — before attempting to roll them out.

To reach that threshold, the researchers are now pursuing further experiments that involve editing three genes, not just one.

“We felt that it would be the responsible thing to be more rigorous to stress test this and ask, are these chickens truly resistant?” said Imperial College London virologist Wendy Barclay, one of the senior authors on the paper. “What if they were to somehow encounter a much, much higher dose?”

With additional research needed, the paper is more proof of concept than immediate proposal, but the authors were already thinking about whether people would consider frying up some tenders or scrambling some eggs from an edited bird. As it is, genetically modified crops and animals continue to face resistance among some groups.

“It’s not just enough to develop the technology,” said Alewo Idoko-Akoh, the first author of the paper and a research associate at the University of Bristol, who used to work as a veterinarian in his native Nigeria. “It’s got to be done in such a way that it’s culturally sensitive and also acceptable.”

No chicken has been found to be naturally immune to the H5N1 avian flu strain that’s been spreading across the world; it’s not as if chicken farms could selectively breed for such a trait. Instead, scientists at the University of Edinburgh — where Idoko-Akoh formerly worked — got the idea of CRISPR’ing it in. For their target, they selected the ANP32 family of genes that encode proteins that Barclay and her group have shown are essential for flu to make copies of itself inside cells.

“It occurred to us that if you could disrupt that interaction in some way, if you could prevent the protein from being used, perhaps by this gene editing, then the virus would not be able to replicate,” Barclay said.

Specifically, the researchers used CRISPR to make tiny tweaks to the ANP32A gene. They performed the edits in the lab in cells that go on to form eggs and sperm, and then injected those cells into chicken embryos developing in eggs. The embryos took up the edited cells, went on to hatch, and then those chickens were later mated with each other. All their offspring, in turn, inherited the edited ANP32A gene.

The researchers then put their CRISPR’d chickens up against regular birds as they exposed them to flu. (For the experiments, the researchers didn’t use H5N1, but rather a less pathogenic bird flu called H9N2.)

In their first test, they challenged the birds with a level of virus — 1,000 infectious units — that they estimated replicated what the birds would encounter during a farm outbreak. While all of the control birds were infected, nine out of 10 of the edited birds were able to block infection. The tenth shed comparatively little virus for a short period of time, the researchers reported.

In the next round, the researchers tried an extremely high flu dose — 1 million infectious units. All the control birds were infected, but this time, half of the edited chickens were also infected, though they had lower viral levels and were less likely to transmit the virus on than the control birds.

The researchers said it wasn’t clear exactly how the virus was able to infect the edited birds, but sequencing showed the viruses from the breakthrough infections had picked up some mutations. The fear is that if a gene-edited approach to blunting bird flu can’t prevent infections entirely, it could pressure the virus to evolve in ways that could make it a better spreader than it already is, particularly among mammals. Experts have worried about H5N1 spreading among people and igniting a pandemic since the virus was discovered 25 years ago, and though it would take multiple changes to the virus’s genome to do that, scientists want to make sure they’re not boosting it even one rung up that ladder.

“If this was a disease that only infected chickens, yes, then the resistance we created would be better than what we would get with a vaccine,” said Mike McGrew, a senior author on the paper and a group leader at the University of Edinburgh’s Roslin Institute. “But because this is a zoonotic disease, and can be spread potentially to humans, we really need to aim for complete resistance. Not resilience, resistance, so the virus cannot grow in the chickens.”

In an effort to generate sterilizing immunity — that is, blocking infections entirely — the researchers are now experimenting with editing a total of three genes in the ANP32 family. Their lab tests have shown the combined approach generates stronger protection, though they haven’t yet made chickens with the edits for further testing — as well as to make sure the edits don’t have any health impacts on the chickens.

Benjamin Schusser, a professor of reproductive biotechnology at the Technical University of Munich, who was not involved with the new research, said he agreed with the paper’s authors that they needed to demonstrate sterilizing immunity before the chickens could ever be used. Having viruses that pick up mutations as they cause breakthrough infections “gets dangerous, for animals and for humans.”

“This is a great piece of work showing there might be ways to do it, but it requires much more research to do it,” Schusser said.

The researchers, who have submitted a patent application on the work, framed the gene-editing strategy as an alternative to vaccination. While some governments have grown receptive to vaccination campaigns as the toll of bird flu mounts, some have resisted out of concerns that immunizing birds could render them asymptomatic, but not protected from infection. The fear is that the virus would spread without notice, or that an infected bird could unknowingly be imported by another country. France, for example, recently started vaccinating millions of ducks, and the United States responded by restricting imports of the birds.

Vaccines face other limitations. Flu viruses evolve quickly, so they could mutate in ways that allow them to outrun vaccine-generated protection.

If the CRISPR approach were proven out, experts say integrating the protective edits into the world’s billions of chickens would be feasible, though it could take some time. A small number of breeding companies supply much of the world’s Gallus gallus domesticus, so it would involve introducing the edits to the pedigree birds at the top of the companies’ “breeding pyramids,” and then waiting as the trait passed down through future generations.

The U.K. team is not alone in exploring the use of CRISPR in farm animals to protect them from diseases or adapt them to changing climates. Already, Czech researchers in 2020 described CRISPR’ing chickens to make them resistant to avian leukosis virus.

“I definitely see it as an extension of breeding tools,” Irina Polejaeva, a professor of developmental biology at Utah State University, who was not involved in the new research, said about gene editing. “Spontaneous natural mutations responsible for specific traits were identified over the previous centuries and amplified by selective breeding, leading to a generation of specialized livestock and poultry breeds. Gene editing is a new high-precision tool that can introduce specific beneficial traits.”

Lawmakers and regulators around the world are starting to think more about how to handle these technologies. In the U.S., regulators have cleared CRISPR’d cows with shorter hair to help them withstand higher temperatures; there are also genetically engineered salmon and pigs on the market. The U.K. passed legislation earlier this year that opens the door to gene-edited crops and livestock. Japan has authorized the sale of CRISPR’d fish as food.

“I think the world is changing,” said Helen Sang of the University of Edinburgh, another author on the new paper. “Countries are bringing in new legislation to bring in regulations that will look at the safety of gene-edited animals for food, and within a few years, there will be the regulations in place. And the biology will follow along behind.”