The human leukocyte antigen G1 (HLA-G1), a non-classical class I major histocompatibility complex (MHC-I) protein, is a potent immunomodulatory molecule at the maternal/fetal interface and other environments to regulate the cellular immune response. We created GGTA1-/HLAG1+ pigs to explore their use as organ and cell donors that may extend xenograft survival and function in both preclinical nonhuman primate (NHP) models and future clinical trials. In the present study, HLA-G1 was expressed from the porcine ROSA26 locus by homology directed repair (HDR) mediated knock-in (KI) with simultaneous deletion of α-1-3-galactotransferase gene (GGTA1; GTKO) using the clustered regularly interspersed palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9) (CRISPR/Cas9) gene-editing system. GTKO/HLAG1+ pigs showing immune inhibitory functions were generated through somatic cell nuclear transfer (SCNT). The presence of HLA-G1 at the ROSA26 locus and the deletion of GGTA1 were confirmed by next generation sequencing (NGS) and Sanger’s sequencing. Fibroblasts from piglets, biopsies from transplantable organs, and islets were positive for HLA-G1 expression by confocal microscopy, flow cytometry, or q-PCR. The expression of cell surface HLA-G1 molecule associated with endogenous β2-microglobulin (β2m) was confirmed by staining genetically engineered cells with fluorescently labeled recombinant ILT2 protein. Fibroblasts obtained from GTKO/HLAG1+ pigs were shown to modulate the immune response by lowering IFN-γ production by T cells and proliferation of CD4+ and CD8+ T cells, B cells and natural killer (NK) cells, as well as by augmenting phosphorylation of Src homology region 2 domain-containing phosphatase-2 (SHP-2), which plays a central role in immune suppression. Islets isolated from GTKO/HLA-G1+ genetically engineered pigs and transplanted into streptozotocin-diabetic nude mice restored normoglycemia, suggesting that the expression of HLA-G1 did not interfere with their ability to reverse diabetes. The findings presented here suggest that the HLA-G1+ transgene can be stably expressed from the ROSA26 locus of non-fetal maternal tissue at the cell surface. By providing an immunomodulatory signal, expression of HLA-G1+ may extend survival of porcine pancreatic islet and organ xenografts.

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Damage from hypoxia is a significant hurdle for effect islet transplantation. Past studies have shown that porcine islet cells are protected from hypoxic damage when cultured with human mesenchymal stem cells (MSCs). To better understand how MSCs protect islet cells, Nie et al evaluated the contribution of MSC-secreted factors. MSC-conditioned media (CM) and exosomes reduced cellular apoptosis leading to enhanced cell survival. 1 Additionally, CM and exosomes activated transcriptional programs in islet cells associated with hypoxia tolerance, including hypoxia-inducible factor 1 alpha (Hif1-alpha). The review article by Smith et al 2 focuses on cellular encapsulation to protect recipients from antigenic donor grafts. They discuss new discoveries, issues, and strategies for wrapping up the donor cells, including an overview of device characteristics. 2 of 2 | TAYLOR eT AL. Non-Gal antigens in porcine cells have long been of interest for engineering an optimal porcine donor graft to use in clinical xenotransplantation. The review written by Byrne et al provides detailed and supportive information on a critical non-Gal antigen, B4GalNT2, and illustrates that when deleted, porcine cells are targeted less by the immune system. 10 2 | IMMUNE SUPPRE SS ION Corneal transplants using porcine tissues are a promising solution to a global need for corneal tissue to correct blindness. Descemet's membrane endothelial keratoplasty (DMEK) is an innovative transplantation technique that benefits from low rejection rates and requisite immunosuppression and results in superior vision recovery compared to other surgical techniques. Indeed, in current issue, Kim et al 11 demonstrate that immunosuppression with the common agent tacrolimus can lead to asymptomatic thrombotic microangiopathy that can lead to a number of problems including organ damage. Minimizing the need for immunosuppression by improved graft quality, genetics, and preparation may therefore enhance tissue survival long term. Liu et al 12 tested the feasibility of porcine xenografts using the DMEK technique. Here, the group used two techniques, mechanical stripping and liquid bubble, to prepare the ultra-thin DMEK prior to transplantation. The data, including a comparison of endothelial growth, were promising and indicate that corneal xenografts using DMEK are a viable future option. The authors plan next to test the xeno-DMEK in a non-human primate model. Successful xenotransplantation currently requires immunosuppression. Unfortunately, the immunosuppression regimen may be lethal, by a yet unidentified mechanism, though often linked with anti-CD154 antibody treatment. Ock et al 13 used a non-human primate model (cynomolgus macaques) to evaluate the transcriptional changes prior to and after the standard immunosuppression protocol for porcine cardiac xenotransplantation (alpha-1,3-galactosyltransferase KO pigs). The regimen included cobra venom factor, anti-thymocyte globulin, and rituximab, in the presence or absence of anti-CD154 antibodies. The authors identified a...

Background Genetically engineered porcine donors are a potential solution for the shortage of human organs for transplantation. Incompatibilities between humans and porcine donors are largely due to carbohydrate xenoantigens on the surface of porcine cells, provoking an immune response which leads to xenograft rejection. Materials and Methods Multiplex genetic knockout of GGTA1, β4GalNT2, and CMAH is predicted to increase the rate of xenograft survival, as described previously for GGTA1. In this study, the clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeats–associated protein 9 system was used to target genes relevant to xenotransplantation, and a method for highly efficient editing of multiple genes in primary porcine fibroblasts was described. Results Editing efficiencies greater than 85% were achieved for knockout of GGTA1, β4GalNT2, and CMAH. Conclusion The high-efficiency protocol presented here reduces scale and cost while accelerating the production of genetically engineered primary porcine fibroblast cells for in vitro studies and the production of animal models.