Inactivation of porcine endogenous retrovirus in pigs using CRISPR-Cas9

Abstract

Xenotransplantation is a promising strategy to alleviate the shortage of organs for human transplantation. In addition to the concern on pig-to-human immunological compatibility, the risk of cross-species transmission of porcine endogenous retroviruses (PERVs) has impeded the clinical application of this approach. Earlier, we demonstrated the feasibility of inactivating PERV activity in an immortalized pig cell line. Here, we confirmed that PERVs infect human cells, and observed the horizontal transfer of PERVs among human cells. Using CRISPR-Cas9, we inactivated all the PERVs in a porcine primary cell line and generated PERV-inactivated pigs via somatic cell nuclear transfer. Our study highlighted the value of PERV inactivation to prevent cross-species viral transmission and demonstrated the successful production of PERV-inactivated animals to address the safety concern in clinical xenotransplantation.

Pancreas regeneration

“…Recent advances in genetic engineering have led to reconsideration of the possibility of using organs grown in pigs. Using CRISPR–Cas9 technology, a pool of 62 known pig retroviruses was deleted from pig skin cells 128,129 , which in principle could be used to make pig iPS cells, and subsequently, genetically ‘clean’ pigs as islet donors. Future clinical use of pig islets will depend on advances in encapsulation technology to protect the cells from human immune reactions while ensuring long-term survival and functionality.…”

Advances in the immunology of heart transplantation

“…Addressing one of the major concerns of the Food and Drug Administration with regard to xenotransplantation, namely the transmission of porcine endogenous retroviruses (PERVs) to humans, healthy pigs have been generated in which all 62 PERV loci were simultaneously inactivated using the CRISPR/Cas9 systems (Figure 2). 73 Not only was this a gene editing “tour de force,” but the availability of pigs with a PERV-free genome makes moot any argument to limit clinical xenotransplantation based on the unlikely risk of pig-to-human viral transmission. 74 The goal now is using CRISPR/Cas9 to generate pigs with increased biologic compatibility with humans, perhaps using PERV-free pigs as a foundation strain.…”

Engineering Precision Medicine

“…[191] Although immune compatibility issues have not been addressed in these studies yet, the approach of generating humanized pig organs are promising for producing structurally complex organs for xenotransplantation. The other "top-down" approach that works toward directly growing artificial organs in animals to generate functional tissues is more straightforward.…”