Extensive Porcine Germline Genome Engineering

Porcine xenotransplantation, the use of pig organs for human transplantation, has long been investigated as an alternative for patients suffering from organ failure. However, clinical application has been impeded by two main hurdles: 1) Molecular incompatibilities between donor pigs and humans and 2) Risk of transmission of porcine endogenous retroviruses (PERVs). We previously demonstrated the successful production of genome-modified pigs with PERV inactivated. In this study, we increased the scale of germline editing from targeting a single repetitive locus to engineering 13 different genes using multiple genome engineering methods. We engineered the pig genome at 42 alleles using a combination of CRISPR-Cas9 and transposon technology, producing pigs that carry PERV inactivation, the knock-out of 3 xeno-antigens, and the addition of 9 human transgenes. The engineered pigs exhibit normal physiology, fertility, and germline transmission of the edited alleles. In vitro assays have shown that, as with allogenic transplantation, cells from engineered pigs gain significant resistance to human humoral rejection, cell-mediated damage, and coagulation dysregulations. This represents a significant step towards safe and effective porcine xenotransplantation.