In a step toward making pig organs safe for human transplant, scientists at Egenesis Bio Inc. have used multiplexed CRISPR editing to remove porcine endogenous retroviruses (PERVs) from pigs.

The team's findings, which were published in the Aug. 11, 2017, issue of Science, have multiple implications.

"We envision our animals will provide a lot of information to the gene editing and gene therapy field, by revealing whether there are long-term effects" of multiplexed genome editing, senior author Luhan Yang told BioWorld.

Yang is a co-founder and the chief scientific officer of Egenesis, a company aiming to develop pigs whose organs can be used for cross-species transplantation, also known as xenotransplantation.

Such xenotransplantation would be one way to address the shortage of organs for transplant. The waiting list for organ transplants is currently about 120,000 individuals long, which is twice as long as it was in 1999. And every year, 8,000 people die while waiting for a transplant.

The first form of xenotransplantation – using animal blood to transfuse humans was first attempted in the 17th century, though as surgeon David Cooper put it in a 2012 review paper, "perhaps not surprisingly, the results were mixed. As a result, xenotransfusion was banned in France for a number of years."

In the intervening four centuries, despite intermittent attempts to transplant organs from chimpanzee testicles to baboon livers, the technique has not progressed to the point of being a viable option to address the organ shortage.

There are two major barriers to xenotransplantation. One is that the immune system is excellent at recognizing exposure to other species. Even mouse monoclonal antibodies lead to immune issues.

The other is that species, including pigs, come with genome elements called endogenous retroviruses (ERVs). In the context of xenotransplantation, those elements pose two separate challenges.

They can move around within the genome, and in xenotransplantation, there is the possibility that they could jump from the transplanted organ into human cells – and, in doing so, disrupt the cells they integrate into, similar to what was seen with some early gene therapy vectors.

Egenesis' strategy is to remove the PERVs via gene editing. Such removal is a technical challenge, as pigs have dozens of PERV copies in their genomes.

Recent advances in CRISPR gene editing have enabled the simultaneous editing of multiple sites in the genome, and Yang and her team found that they could indeed successfully remove all PERV copies from primary pig cells.

However, they were initially not successful in keeping such cells alive to the point where they could use them to generate embryos.

"Primary cells are very sensitive to multiple cuts – they [get] stressed out and die," Yang said. To enable survival of the cells, the team had to treat them with a chemical cocktail of pro-survival factors.

Using that cocktail enabled the team to generate PERV-free pig embryos and from those, piglets, the oldest of which are now 4 months old.

The team showed that around half the cells had some damage to their genome as a result of the procedure, which could stem from off-target effects, the chemical cocktail, or genome rearrangements that occurred in the course of on-target editing.

Yang said that such genome damage is not a problem for the team, as they can select undamaged cells to use for the creation of embryos.

Experiments to see whether removing the PERVs will ultimately affect the health of the pigs are ongoing.

"We don't know what the functionality of those elements is in the genome," Yang said, although the fact that piglets generated from edited cells have not shown any obvious negative effects up to 4 months of age is encouraging.

Still, the team is continuing to monitor the health of the pigs, looking for abnormalities in blood and histology as well as signs that the animals may be tumor-prone over the long haul.

"It's still early days," she said.

In the long run, Egenesis is working on developing a method that can address both cross-species immunocompatibility and PERVs.

"We want to create a world where there is no organ shortage," Yang said. "We understand it's challenging, but that's also the sense of mission."