Background: Clinical xenotransplantation is not possible because humans possess antibodies that recognize antigens on the surface of pig cells. and N-glycolylneuraminic acid (Neu5Gc) are two known xenoantigens. Methods: We report the homozygous disruption of the a1, 3-galactosyltransferase (GGTA1) and the cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH) genes in liver-derived female pig cells using zinc-finger nucleases (ZFNs). Somatic cell nuclear transfer (SCNT) was used to produce healthy cloned piglets from the genetically modified liver cells. Antibody-binding and antibody-mediated complement-dependent cytotoxicity assays were used to examine the immunoreactivity of pig cells deficient in Neu5Gc and Gal. Results: This approach enabled rapid production of a pig strain deficient in multiple genes without extensive breeding protocols. Immune recognition studies showed that pigs lacking both CMAH and GGTA1 gene activities reduce the humoral barrier to xenotransplantation, further than pigs lacking only GGTA1. Conclusions: This technology will accelerate the development of pigs for xenotransplantation research.
Pigs are emerging as important large animal models for biomedical research, and may represent a source of organs for xenotransplantation. The MHC is pivotal to the function of the immune system in health and disease, and is particularly important in infection and transplant rejection. Pigs deficient in class I MHC could serve as important reagents to study viral immunity, allograft and xenograft rejection. We report the creation and characterization of class I MHC knockout pigs using the Cas9 nuclease and gRNAs. Pig fetal fibroblasts were genetically engineered using Cas9 and gRNAs, and class I MHC negative cells were then used as nuclear donors for somatic cell nuclear transfer. We produced 3 piglets devoid of all cell surface class I proteins. Though these animals have reduced levels of CD4−CD8+ T cells in peripheral blood, the pigs appear healthy and are developing normally. These pigs are a promising reagent for immunological research.
The CRISPR/Cas system allows targeting of multiple genes in a single reaction with the potential to create pigs of one genetic strain or multiple genetic modifications in a single pregnancy. The application of this phenotypic selection strategy with multiplexed sgRNA and the Cas9 nuclease has accelerated our ability to produce and evaluate pigs important to xenotransplantation.
Background
The lethal thrombocytopenia that accompanies liver xenotransplantation is a barrier to clinical application. Human platelets are bound by the asialoglycoprotein receptor (ASGR) on pig sinusoidal endothelial cells and phagocytosed. Inactivation of the ASGR1 gene in donor pigs may prevent xenotransplantation-induced thrombocytopenia.
Methods
Transcription activator-like effector nucleases (TALENs) were targeted to the ASGR1 gene in pig liver derived cells. ASGR1 deficient pig cells were used for somatic cell nuclear transfer (SCNT). ASGR1 knock out (ASGR1−/−) fetal fibroblasts were used to produce healthy ASGR1 knock out piglets. Human platelet uptake was measured in ASGR1 and ASGR1−/− livers.
Results
Targeted disruption of the ASGR1 gene with TALENs eliminated expression of the receptor. ASGR1−/− livers phagocytosed fewer human platelets than domestic porcine livers during perfusion.
Conclusions
The use of TALENs in liver-derived cells followed by SCNT enabled the production of healthy homozygous ASGR1 knock out pigs. Livers from ASGR1−/− pigs exhibit decreased human platelet uptake. Deletion of the ASGR1 gene is a viable strategy to diminish platelet destruction in pig-to-human xenotransplantation.
Background
A profound thrombocytopenia limits hepatic xenotransplantation in the pig-to-primate model. Porcine livers also have shown the ability to phagocytose human platelets in the absence of immune-mediate injury. Recently, inactivation of the porcine ASGR1 gene has been shown to decrease this phenomenon. Inactivating GGTA1 and CMAH genes has reduced the antibody-mediated barrier to xenotransplantation; herein we describe the effect that these modifications have on xenogeneic consumption of human platelets in the absence of immune-mediated graft injury.
Methods
WT, ASGR1−/−, GGTA1−/−, and GGTA1−/−CMAH−/− knockout pigs were compared for their xenogeneic hepatic consumption of human platelets. An in vitro assay was established to measure the association of human platelets with liver sinusoidal endothelial cells (LSECs) by immunohistochemistry. Perfusion models were used to measure human platelet uptake in livers from WT, ASGR1−/−, GGTA1−/−, and GGTA1−/− CMAH−/− pigs.
Results
GGTA1−/−, CMAH−/− LSECs exhibited reduced levels of human platelet binding in vitro, when compared to GGTA1−/− and WT LSECs. In a continuous perfusion model, GGTA1−/− CMAH−/− livers consumed fewer human platelets than GGTA1−/− and WT livers. GGTA1−/− CMAH−/− livers also consumed fewer human platelets than ASGR1−/− livers in a single pass model.
Conclusions
Silencing the porcine carbohydrate genes necessary to avoid antibody-mediated rejection in a pig-to-human model also reduces the xenogeneic consumption of human platelets by the porcine liver. The combination of these genetic modifications may be an effective strategy to limit the thrombocytopenia associated with pig-to-human hepatic xenotransplantation.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.