Scientists who dream of transplanting organs from pigs into people have long faced a nagging question: What about PERVs? Remnants of ancient viral infections, genes from porcine endogenous retroviruses—known by their unfortunate acronym—are scattered throughout the pig genome, and could infect a person who one day receives a pig’s heart, lung, or kidney as a replacement or temporary organ. Now, a U.S. company aiming to grow transplant-friendly pig organs reports that it has crossed these viruses off the list of concerns. Using CRISPR gene editing, researchers from the company and several academic labs created dozens of apparently healthy pigs with no trace of PERV genes.
“If this is correct, it’s a great achievement,” says virologist Joachim Denner of the Robert Koch Institute in Berlin who has studied the mysterious PERV sequences. Scientists still don’t know whether the viral particles they produce can infect humans at all, he notes, much less whether they would cause disease if they did. And even with PERVs off the table, pigs will require other modifications so that their organs won’t be rejected by the human immune system or cause other harms. Still, Denner says, “If it is possible to knock [PERVs] out, you should do it.”
A shortage of human organs creates long waitlists for vital transplants; in the United States, about 22 people needing various organs die every day while they wait. Pig organs, meanwhile, can grow to a conveniently human size. The concern about PERVs has been hard to dismiss, however, especially because studies have shown that the viruses can infect human cells in a dish.
Scientists have already implanted cells from pig pancreases into people with diabetes and found no evidence of subsequent PERV infection. But those are small numbers of cells within a protective capsule, Denner notes. Large porcine organs, which will likely require immune-suppressing drugs to keep the patients’ bodies from attacking them, might pose a greater risk of PERV transmission. He and his colleagues recently attempted to chop PERV genes out of pig cells with an editing technology called zinc finger nucleases, but the many imprecise DNA cuts proved toxic to cells.
Then CRISPR came along. Two early developers of that gene-editing technology, Harvard University geneticists George Church and Luhan Yang, suspected that CRISPR’s highly efficient duo of guide RNA and a DNA-slicing enzyme could make precise, genome-wide changes to pig cells. In 2015, they co-founded the company eGenesis to focus on engineering transplantable organs, and Yang became the company’s chief scientific officer. The same year, they showed that CRISPR could knock out PERV genes at all 62 sites in the pig genome—the most widespread CRISPR editing feat to date.
But for that project, the researchers had used a line of “immortal” pig kidney cells, chosen for their ability to survive and divide indefinitely in a dish. To make PERV-free pigs, they needed to start with genetically normal cells straight from a living pig. In the new work, done with a team of Danish and Chinese collaborators, the eGenesis team applied the CRISPR system to cells derived from the connective tissue of fetal pigs. Those cells proved more fragile when subjected to CRISPR’s hack job: Once edited, they failed to grow normally, possibly because the damage to their DNA prompted them to stop dividing or self-destruct, Yang says. But by exposing the cells to a chemical cocktail that encouraged growth and tamped down on a key growth-suppressing gene, the team bumped up the portion of flourishing PERV-free cells in a dish to 100%.
To produce piglets, the researchers then used a standard cloning technique: They inserted the DNA-containing nuclei of these edited cells into egg cells taken from the ovaries of pigs at a Chinese slaughterhouse. They allowed each egg to develop into an embryo and implanted it in the uterus of a surrogate mother.
“Before our study, there was huge scientific uncertainty about whether the pig [produced after this editing] is viable,” Yang says. But in a Science paper published online today, her team reports that the technique produced live, apparently healthy pigs with about the same success rate as the cloning method without genetic modification—about one pig per 100 implanted embryos. And when they tested tissues from the 37 piglets born so far, all appeared to be PERV-free.
The technical feat has, ironically, inspired some dread among those enthusiastic about xenotransplantation, the transfer of nonhuman animal organs into people. With the actual risk of PERVs uncertain, some worry that the extra editing will needlessly add complexity to the already-difficult organ development process, especially if the regulators at the U.S. Food and Drug Administration (FDA) insist on PERV-free pigs for future human experiments. “If this is required, it will add to the time before pigs can be used for transplants in patients in desperate need,” says transplant immunologist David Cooper of the University of Alabama at Birmingham. “And it will add to the cost of providing pigs for the initial clinical trials.”
“At this moment, I don’t think we are very worried about PERV,” adds Muhammad Mohiuddin, a cardiac transplant surgeon at the University of Maryland School of Medicine in Baltimore. His group is collaborating with the company United Therapeutics to develop implantable pig hearts. (He is gearing up to swap them into the chests of baboons, after showing last year that the organs could survive for years beating in baboons’ abdomens.) Will his team add PERV modifications to its agenda? “If FDA mandates us, ‘To move forward you need to get rid of this PERV since George Church has shown you how to do it,’” he says, “then of course, yes.”
PERVs aren’t the only thing standing in the way of transplant-ready pig organs. Researchers will need to knock out pig genes that provoke the human immune system, and insert others that will prevent toxic interactions with human blood. eGenesis is working on such modifications, too. Compared to the PERV feat, Yang says, those compatibility issues are “the second challenge, and probably more challenging.”
Source & Credits: Sciencemag