Gene Editing Could Help Pave the Way for Pig-to-Human Transplantations.

Circulation. 2017;136:2083–2084. DOI: 10.1161/CIRCULATIONAHA.117.032246 November 21, 2017 2083 Although much of the promise of CRISPR-Cas9 gene editing techniques has centered around correcting disease-causing gene mutations, the advance is also showing potential in the field of xenotransplantation, for example, for making porcine organs safe for transplantation into humans. In an important step in this direction, a team led by researchers at the biotechnology company eGenesis recently edited the pig genome to deactivate a family of retroviruses. Organs from pigs, which can grow to a convenient human size, could help alleviate the growing organ shortage if scientists continue to make strides in addressing their immunological incompatibility; however, the pig genome includes remnants of ancient viral infections called porcine endogenous retroviruses (PERVs), which can be passed on to other cells when cultured together. In contrast to human endogenous retroviruses, which are mostly defective and not replication competent, PERVs that are integrated in the pig genome can be released as particles that infect human cells. As reported with other retroviruses, PERV integration could potentially lead to immunodeficiency and tumorigenesis. There may be an especially high risk of PERV transmission in transplant recipients who often require lifelong treatment with immunosuppressive drugs to prevent organ rejection. Gene editing efforts by the eGenesis team have removed PERVs in cell lines but not live pigs. In their previous research published in Science, the investigators determined the PERV copy number in a porcine kidney epithelial cell line to be 62. Next, they used the CRISPR (clustered, regularly interspaced, palindromic repeats) technology, which is adapted from a bacterial defense system and uses the DNA-cutting enzyme Cas9 bound to a guide RNA molecule that targets a specific genetic sequence. CRISPR-Cas9 allowed them to disrupt all 62 copies of the PERV pol gene. This “multiplexability” was a significant accomplishment compared with simpler gene editing feats, highlighting the precise yet widespread genetic changes possible through CRISPRCas9 editing. The modifications led to a >1000-fold reduction in PERV transmission to human cells from the engineered cells. In the group’s latest study published in Science, cell culture experiments confirmed that PERVs in pig cells can be transmitted to human cells and went a step further to demonstrate that the viruses can pass from human cells to other human cells. Although it unclear whether PERVs infect humans in vivo, these findings substantiate the risk of cross-species viral transmission with xenotransplantation. As they did previously with kidney epithelial cells, the investigators mapped and characterized the PERVs present in the genome of porcine fetal fibroblast cells, this time identifying 25 viral copies. Next, they tailored the CRISPR-Cas9 technology to target the viral genomic sites they identified and to inactivate the PERVs within the fibroblasts. Despite the presence of highly modified cells in the population, none of the targeted cells could be grown with >90% PERV editing efficiency. The scientists suspected that the simultaneous DNA cuts by Cas9 Gene Editing Could Help Pave the Way for Pig-to-Human Transplantations