Recently developed methods for editing genes could make pig organs safe for human transplant.
Pig organs have not been used for transplant partly because they carry viruses that could infect people. Harvard researchers report October 11 in Science that they have used a powerful gene-editing tool to simultaneously disable 62 of the viruses.
Pig cells contain multiple copies of embedded viruses called porcine endogenous retroviruses, or PERVs. Such viruses copy and paste themselves into pig DNA. If the retroviruses infected a person during or after a transplant, they could disrupt important human genes, leading to cancer or other diseases.
CRISPR/Cas9, as the novel gene-editing method is known, has been used to edit DNA in monkeys and in laboratory animals (SN: 3/8/14, p. 7). Cas9 is a DNA-cutting enzyme. It works in association with pieces of RNA that chemically match with a target on DNA to guide the enzyme to specific cutting spots. When Cas9 cuts DNA, the cell tries to repair the breach either by pasting the cut ends together again or by copying unbroken DNA from a twin gene on another chromosome. Tying together broken ends can result in “mistakes” that disable the gene, the goal of the research.
Usually, researchers target single genes. But doing that would disable only one PERV. “We wanted to get rid of all of them,” says George Church, a geneticist at Harvard Medical School. Church and geneticist Luhan Yang in Church’s lab headed the project. They theorized that since the retroviruses are identical, all of them could be eliminated in one fell swoop with the same guide RNAs. “We didn’t know it was possible, and we certainly didn’t know it was going to be easy,” Church says.
Yang and colleagues designed two guide RNAs that target the pol gene of the retroviruses for cutting. That gene encodes a polymerase enzyme necessary for the retrovirus to replicate itself.
At first, the experiment didn’t work at all, Yang says. The problem may have been that the researchers put in too much of the cutting enzyme, dicing up the DNA and leading cells to switch on a suicide program.
Yang and colleagues designed a more tunable system. They inserted DNA editing genes, engineered to turn on only when researchers added the antibiotic doxycycline, into pig cells. In that experiment, a maximum of 37 percent of retrovirus pol genes showed signs of editing 17 days after the team gave the signal for editing to begin.
But the efficiency of editing wasn’t uniform. The team sorted out cells that had been edited with what looked like 37 percent efficiency. When the team examined the cells individually, they found that the cells fell into one of two camps. Either about 10 percent of the virus genes were edited, or 97 to 100 percent were. Cells in which all or nearly all of the pol genes were revised carried only 16 to 20 types of edits — mostly cases in which little bits of DNA had been snipped out. That pattern indicates that as Cas9 sliced genes, the cells often repaired the damage by copying a previously edited pol gene.
“It’s very cool,” says Jennifer Doudna, a molecular biologist at the University of California, Berkeley and one of the researchers who first developed the CRISPR/Cas9 system as a gene-editing tool. Such a chain reaction “just underscores the efficiency of the system.”
With the Harvard group’s discovery, researchers now know that bulk editing is possible. Making pig organs safe for transplant is just one of the possible medical applications of the technology. Some of those applications, such as fixing mutations in human germline cells (eggs and sperm) or in embryos have been very controversial (SN: 5/30/15, p. 16).
While developing pig organs for human transplant seems to be a good use of the technology, Doudna says scientists need to give serious consideration to how and whether to apply CRISPR/Cas9 to every problem it could help solve. Human germline editing is one case that deserves particular thought and debate. “We as scientists should take a step back and say, ‘should we really go there,’” she said October 9 in Baltimore at the annual meeting of the American Society of Human Genetics.
In the new experiments, Yang and colleagues also measured how often pig cells transmit PERVs to human cells. Normally, about 1,000 pig retroviruses are found in a group of 1,000 human cells. (The researchers can’t tell how many viruses invade each human cell; they are measuring in bulk). Edited pig cells passed along 1,000 times fewer retroviruses — probably zero viruses, Yang says — than did unedited cells. The researchers suspect that the low level of PERVs they detected in human cells grown with edited pig cells are actually human viruses that are very similar to the pig viruses.
That doesn’t mean pigs will be an immediate source of transplant organs. There are still other genes in pigs that could cause organs to be rejected if transplanted into humans. It’s only a matter of time until those genes are altered to be more people-friendly, too, the researchers predict. “If we can do 62, we can do 20 more,” Church says. And the researchers need to apply the technique to produce whole animals that lack retroviruses and other troublesome genes.
About 2 million people worldwide are waiting for transplants, Church says. He and Yang cofounded a company called eGenesis to engineer pigs to produce organs suitable for human transplants. The researchers aren’t worried whether the public will want genetically engineered pig organs. “Human life is at stake,” Yang says. “I would think the public would be very accepting if it is proven safe and effective.”