Possible detrimental effects of beta‐2‐microglobulin knockout in pigs

Abstract

Background Despite major improvements in pig‐to‐primate xenotransplantation, long‐term survival of xenografts is still challenging. The major histocompatibility complex (MHC) class I, which is crucial in cellular immune response, is an important xenoantigen. Abrogating MHC class I expression on xenografts might be beneficial for extending graft survival beyond current limits. Methods In this study, we employed the CRISPR/Cas9 system to target exon 2 of the porcine beta‐2‐microglobulin (B2M) gene to abrogate SLA class I expression on porcine cells. B2M‐KO cells served as donor cells for somatic cell nuclear transfer, and cloned embryos were transferred to three recipient sows. The offspring were genotyped for mutations at the B2M locus, and blood samples were analyzed via flow cytometry for the absence of SLA class I molecules. Results Pregnancies were successfully established and led to the birth of seven viable piglets. Genomic sequencing proved that all piglets carried biallelic modifications at the B2M locus leading to a frameshift, a premature stop codon, and ultimately a functional knockout. However, survival times of these animals did not exceed 4 weeks due to unexpected disease processes. Conclusion Here, we demonstrate the feasibility of generating SLA class I knockout pigs by targeting the porcine beta‐2‐microglobulin gene using the CRISPR/Cas9 system. Additionally, our findings indicate for the first time that this genetic modification might have a negative impact on the viability of the animals. These issues need to be solved to unveil the real value for xenotransplantation in the future.

Triple (GGTA1, CMAH, B2M) modified pigs expressing an SLA class Ilow phenotype—Effects on immune status and susceptibility to human immune responses

“…Thus, targeting B2M affected health and immune status of pigs in various ways. Presumably, the massive perturbations of the immune system in B2M ‐targeted pigs are a major reason for the development of acute feverish conditions and subsequent death, which occurred in 3‐ to 4‐week‐old pigs in recent experiments . These animals were kept under standard housing conditions.…”

“…Presumably, the massive perturbations of the immune system in B2M-targeted pigs are a major reason for the development of acute feverish conditions and subsequent death, which occurred in 3-to 4-week-old pigs in recent experiments. 29 These animals were kept under standard housing conditions. In contrast, the B2M low piglets reported here were kept F I G U R E 6 Analysis of swine leukocyte antigen (SLA) class I expression in porcine tissues.…”

“…SLA class I–deficient pigs have already been generated, either by targeting directly the genes encoding for the heavy α‐chain or by knocking out B2M . Phenotypic studies on the consequences of B2M/SLA class I α‐chain targeting in pigs have already been described.…”

“…8,27 SLA class I-deficient pigs have already been generated, either by targeting directly the genes encoding for the heavy α-chain 28 or by knocking out B2M. 8,29 Phenotypic studies on the consequences of B2M/SLA class I α-chain targeting in pigs have already been described. However, so far it is not known to what extent porcine cells and tissues are protected against human immune responses by the elimination of SLA class I molecules.…”

Xenotransplantation literature update, November/December 2019

“…Although we are able to create new genetic modifications of the porcine genome (over 40 genetic variants to date), reliable long‐term survival of the xenograft in non‐human primates is still challenging due to the need for harsh immunosuppressive therapy . To eliminate the need of immunosuppressive regimen, Sake et al abrogated MHC class I expression on xenografts, in an approach to silence T‐cell and NK cell‐mediated cell lysis . They did this by using CRISPR/Cas9 system to target exon 2 of the porcine beta‐2‐microglobulin (B2M) gene to abrogate SLA class I expression on porcine cells and hence break the immune cell connection.…”