WASHINGTON, Aug. 10th. Today scientists in Massachusetts announced that by using the CRISPR–Cas9 gene-editing system they were able to inactivate all 25 viruses in the pig genome, yielding seemingly healthy piglets and moving research one step closer to a future of xenotransplantation. The breakthrough fundamentally addressed the risk of cross-species viral transmission in xenotransplantation. In producing the first batch of live pigs free of dangerous viruses, it set the stage for transplanting life-saving organs from pigs into humans.
Luhan Yang, co-founder and chief science officer of eGenesis, the Cambridge, Mass.–based start-up that led the research described, in the U.S. journal Science, how they used the powerful gene-editing tool CRISPR to modify pig cells and produce embryos free of the so-called porcine endogenous retroviruses (PERVs) in order to create the desired piglets.
“We are pushing the envelope of technology day by day. I think that such innovation is required to tackle as challenging a problem as xenotransplantation.”
Worldwide, human organs for transplant are in short supply. Researchers and clinicians have long hoped that the challenge could be alleviated through the availability of suitable animal organs for transplant, a concept known as xenotransplantation.
Pigs in particular have been especially promising candidates due to their similar size and physiology to humans. But one of the largest safety concerns has been the fact that most mammals including pigs contain repetitive, latent retrovirus fragments in their genomes — present in all their living cells — that are harmless to their native hosts but can cause disease in other species.
The presence of PERVs in pigs brought more than a billion dollars’worth of pharmaceutical industry investments in developing xenotransplant methods to a standstill by the early 2000s, according to the study.
Then, Yang and colleagues demonstrated in a 2015 Science paper a method to inactivate all 62 copies of PERVs in porcine cells and eliminated PERV transmission to human cells.
In the new study, the researchers developed a strategy to enable efficient and precise gene editing to deactivate all 25 genomic sites related to PERVs in pig fibroblast cells using the CRISPR technology.
In conjunction with a method to inhibit cell death during gene editing, they successfully produced viable PERV-free porcine embryos via somatic cell nuclear transfer, the same method that created Dolly, the world’s first cloned sheep, in 1996.
The team then implanted the PERV-free embryos into surrogate sows and demonstrated the absence of PERV re-infection, initially in fetuses and finally in recently born piglets.
“At least for those who are four months old, we did not observe difference in physiology between the modified piglets and normal ones,” Yang said.
“We will continue to use this platform to engineer the pig genome, on the basis of PERV-free pigs, to enhance pig-to-human immunological compatibility for the clinical xenotransplantation of porcine organs as early as possible,” she said.
The next stage of the research, Church and Yang say, will be to essentially “humanize” the pigs—modifying them enough that their organs can function in the human body. This involves immunological changes as well as making the tissues compatible and fixing blood-clotting issues. They have already begun such work and are writing it up for submission to a peer-reviewed journal, Church adds.
Researchers expect to start by transplanting kidneys, where the human waiting list is the longest, followed by other organs like the heart and liver; pancreatic islet cells to combat type 1 diabetes; skin; and corneas.
Studying the eGenesis-edited pigs will also give researchers the opportunity to see whether editing a significant number of genes with CRISPR causes any long-term problems in mammals. Pigs are the biggest animals that have undergone CRISPR, he says, and he wants to see what happens when they are allowed to “grow to a ripe old age” of over 20. There has been some speculation that CRISPR might lead to cancers, but that has not been adequately tested, he says.
Whether or not pig retroviruses would truly pose a risk of causing disease in humans remains controversial. In their new work the Yang team performed experiments confirming that pig retroviruses can infect human cells—just as another retrovirus, HIV, does with people. In a lab dish the pig viruses infected human cells, and those infected cells were able to infect other human cells that had not been directly exposed to pig cells.
First pigs free of porcine endogenous retroviruses (PERVS)
Along with the potential to prevent some diseases, this technology also comes with complicated ethical questions, including what kind of gene edits would be acceptable and who could benefit or be harmed.
Professor Darren Griffin of the University of Kent, who was not involved in the study, said the finding represents “a significant step forward towards the possibility of making xenotransplantation a reality.”
“However, there are so many variables including ethical issues to resolve before xenotransplantation can take place,” Griffin cautioned.
Another outside expert, Professor Ian McConnell of the University of Cambridge, sounded a similarly cautionary note, calling this work “a promising first step.”
“It remains to be seen whether these results can be translated into a fully safe strategy in organ transplantation,” McConnell said. “Even if organs from these gene-edited pigs could be safely used to overcome virus transmission, there remain formidable obstacles in overcoming immunological rejection and physiological incompatibility of pig organs in humans.”