Before pig organs can be made safe for transplantation into human patients, two problems need to be solved. Both problems, ultimately, come down to features of the pig genome. First, the pig genome contains genes that give rise to proteins that the human immune system will not tolerate. Second, it contains retroviruses that could be transmissible to humans. The retrovirus problem, at least, may soon be tractable, thanks to the CRISPR gene-editing technique.
According to a newly published report, the CRISPR technique has been used to inactivate all 62 copies of a retrovirus in a pig cell line. CRISPR, then, has succeeded where other techniques—vaccines, RNA interference, and genetic editing by means of zinc finger nucleases and TAL effector nucleases—have failed. The feat is impressive even from a CRISPR-centric point of view. Never before have so many gene genes been modified in a single round of CRISPR editing. (Previously, the maximum number of genome sites to be edited simultaneously was no higher than six.)
The new work, from a team of scientists led by Harvard Medical School’s George Church, Ph.D., appeared October 11 in Science, in an article entitled, “Genome-wide inactivation of porcine endogenous retroviruses (PERVs).” The article described how the team eradicated all PERVs identified in a porcine kidney epithelial cell line.
“We first determined the PK15 PERV copy number to be 62,” wrote the authors. “Using CRISPR-Cas9, we disrupted all 62 copies of the PERV pol gene and demonstrated a >1,000-fold reduction in PERV transmission to human cells using our engineered cells.”
Critically, the CRISPR-Cas9 system did not appear to cause genomic rearrangements. That is, even though the genome was extensively edited, it remained functional. The cells remained viable.
Nonetheless, future work in pig embryonic cell lines will be needed if genetically modified pigs are to be developed that could serve as organ donors to human patients. “As no porcine embryonic stem cells exist,” the authors noted, “[our] system will need to be recapitulated in primary porcine cells and cloned into animals using somatic cell nuclear transfer.”
Another challenge—one not emphasized in the Science article—is developing a gene-editing approach that would encompass not only retroviral elements, but also the portions of the pig genome that would generate proteins objectionable to the human immune system. Each protein-coding gene that would need to be removed would probably pose a unique targeting problem, unlike the retroviral elements, which were all targetable via the pol gene they all contained. And so disabling or eradicating these genes all at once would be far more difficult than disrupting all of the retroviral elements.
Nonetheless, in an online news article in Science that highlighted the Church team’s research article, there were hints that research was already underway to identify and disrupt genomic targets, and so eliminate immunity issues for pig-to-human organ transplants. In addition, the Science news article indicates that the Church team “has successfully inactivated PERVs in cells from living pigs, and transferred the nuclei of those cells into pig embryos, but wouldn’t confirm that the PERVs were inactivated in the embryos.”
In the current study, Dr. Church indicated that he was affiliated not only with Harvard and the Wyss Institute, but also eGenesis Biosciences, a company that describes itself as “a life sciences company whose mission is to transform xenotransplantation into an everyday, lifesaving medical procedure.”