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Review
. 2024 Jul 1;104(3):1409-1459.
doi: 10.1152/physrev.00041.2023. Epub 2024 Mar 22.

Physiological basis for xenotransplantation from genetically modified pigs to humans

Leigh Peterson  1 , Magdi H Yacoub  2 , David Ayares  1 , Kazuhiko Yamada  3 , Daniel Eisenson  3 , Bartley P Griffith  4 , Muhammad M Mohiuddin  4 , Willard Eyestone  1 , J Craig Venter  5 , Ryszard T Smolenski  6 , Martine Rothblatt  1
Affiliations
Review

Physiological basis for xenotransplantation from genetically modified pigs to humans

Leigh Peterson et al. Physiol Rev. .

Abstract

The collective efforts of scientists over multiple decades have led to advancements in molecular and cellular biology-based technologies including genetic engineering and animal cloning that are now being harnessed to enhance the suitability of pig organs for xenotransplantation into humans. Using organs sourced from pigs with multiple gene deletions and human transgene insertions, investigators have overcome formidable immunological and physiological barriers in pig-to-nonhuman primate (NHP) xenotransplantation and achieved prolonged pig xenograft survival. These studies informed the design of Revivicor's (Revivicor Inc, Blacksburg, VA) genetically engineered pigs with 10 genetic modifications (10 GE) (including the inactivation of 4 endogenous porcine genes and insertion of 6 human transgenes), whose hearts and kidneys have now been studied in preclinical human xenotransplantation models with brain-dead recipients. Additionally, the first two clinical cases of pig-to-human heart xenotransplantation were recently performed with hearts from this 10 GE pig at the University of Maryland. Although this review focuses on xenotransplantation of hearts and kidneys, multiple organs, tissues, and cell types from genetically engineered pigs will provide much-needed therapeutic interventions in the future.

Keywords: genetically modified pigs; xenotransplantation.

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Conflict of interest statement

L.P., D.A., W.E., and M.R. are employees of United Therapeutics. M.Y. is on the scientific advisory board of United Therapeutics. J.H.U. and U.M.B. have scientific research agreements with United Therapeutics. None of the other authors has any conflicts of interest, financial or otherwise, to disclose.

Figures

None
Graphical abstract
FIGURE 1.
FIGURE 1.
Mechanisms of rejection during xenotransplantation. A: hyperacute rejection (HAR). Preformed anti-pig antibodies in the recipient’s blood recognize xenoantigens on vascular endothelial cells in the pig graft and activate complement, leading to endothelial injury, inflammation, interstitial hemorrhage, thrombus formation, and ischemia. B: delayed (cellular and antibody mediated) xenograft rejection (DXR) with cellular infiltration and production of elicited antibodies and cytokines. Porcine major histocompatibility complex (MHC) class I [swine leukocyte antigen (SLA I)] molecules may not sufficiently interact with the inhibitory receptors on primate NK cells, leaving the porcine xenograft vulnerable to NK cell cytotoxicity. Porcine CD47 is not sufficient to inhibit primate SIRP-α, which leaves the porcine xenograft susceptible to macrophage phagocytosis. C: chronic rejection with chronic inflammation and recurring antibody- and cellular-mediated rejection events within the graft vascular endothelium result in thrombotic microangiopathy, proliferation of endothelial cells, vessel narrowing, and interstitial fibrosis. D: T-cell activation by costimulatory signaling provided by T-cell receptor (TCR) recognition of the antigen/MHC and the CD28 interaction with CD80/86; inhibition of T-cell activation via the cytotoxic T lymphocyte-associated protein 4 (CTLA-4)/CD80/86 interaction; B-cell activation by costimulatory signaling provided by TCR recognition of the antigen/MHC class II complex presented by the B cell and the CD40/CD40L (CD154) interaction. ADCC, antibody-dependent cellular cytotoxicity; AMR, antibody-mediated rejection; APC, antigen-presenting cell; MAC, membrane attack complex; NK, natural killer; RBC, red blood cell.
FIGURE 2.
FIGURE 2.
Wild-type pig heart after transplantation into baboon recipient. A: whole heart before reperfusion. B: whole heart after reperfusion demonstrating hyperacute rejection (HAR). C: normal heart tissue before transplant. D: heart tissue after transplant demonstrating thrombus formation. E: heart tissue after transplant demonstrating interstitial hemorrhage. F: heart tissue after transplant demonstrating myocyte loss and fibrosis. Image by Muhammad Mohiuddin.
FIGURE 3.
FIGURE 3.
Wild-type pig kidney after transplantation into baboon recipient. A: whole kidney 5 min after reperfusion. B: whole kidney 1 h after reperfusion demonstrating hyperacute rejection (HAR). Image by Kazuhiko Yamada.
FIGURE 4.
FIGURE 4.
Generation of transgenic pigs. Transgenic pigs are produced by injecting a DNA vector containing the transgene and a promoter into the pronuclei of fertilized eggs. The transgene integrates into the genome as head-to-tail DNA concatemers as a result of homologous recombination. Embryos are transferred into the oviduct of a surrogate mother to complete gestation. Approximately 10% of live births result in transgenic animals.
FIGURE 5.
FIGURE 5.
Targeted disruption of the α1,3GT gene locus in cultured cells. A targeting vector that includes a portion of the target gene plus adjacent 5′ and 3′ sequences (“homology arms”) with the neomycin resistance gene (neoR) inserted into the coding sequence to disrupt the gene. Electroporation to facilitate entry of the DNA into the cell and homologous recombination (HR) with the endogenous DNA results in cells with a heterozygous knockout of the endogenous α1,3GT gene and resistance to the antibiotic G418 (a neomycin analog).
FIGURE 6.
FIGURE 6.
Genetically altering pigs with gene targeting and somatic cell nuclear transfer. The somatic cell nuclear transfer (SCNT) methodology involves removing the metaphase II chromosomes from mature oocytes and placing a donor cell under the zona pellucida next to each enucleated oocyte. Application of an electric current fuses the cytoplast of the donor cell and oocyte and activates cell division. Reconstructed embryos are injected into the oviduct of a surrogate mother to complete gestation.
FIGURE 7.
FIGURE 7.
Clustered regularly interspaced short palindromic repeats (CRISPR)-facilitated gene editing. The CRISPR/CRISPR associated (Cas) system consists of a guide RNA with homology to the endogenous target site, a nuclease-binding domain, and a Cas nuclease. The guide RNA directs the Cas nuclease to the target genomic site, where it creates a double-strand break (DSB). Cellular machinery repairs the DSB, which involves the deletion and/or insertion of several nucleotides (indels) and nonhomologous end joining (NHEJ). Indels that occur within an exon can result in a frameshift, leading to generation of a stop codon and translation of truncated, nonfunctional protein. Gene-targeting vectors are used to generate insertions and point mutations at prespecified locations in the genome via homology-directed repair (HDR). PAM, protospacer adjacent motif; sgRNA, single guide RNA.
FIGURE 8.
FIGURE 8.
Generation of multiple genetic modifications in the pig. Fetal fibroblasts are collected from midgestation fetuses, cultured, and transfected with a targeting vector for gene editing. Cells are screened for the intended genetic modification and appropriate protein expression (or lack thereof) by polymerase chain reaction (PCR) and DNA sequencing and Western blot and flow cytometry, respectively. Cells with the intended genotype and phenotype are used for somatic cell nuclear transfer (SCNT) to generate cloned piglets. Once the genotype and phenotype of the cloned piglets are confirmed, they supply cells for additional rounds of genetic engineering. FITC-A, fluorescein isothiocyanate. See glossary for additional abbreviations.
FIGURE 9.
FIGURE 9.
Design of bi- and multicistronic targeting vectors to link transgenes. A: bicistronic transgene vector in which 2 transgenes are linked by a 2A sequence to permit expression by a single promoter. B: tetracistronic transgene vector composed of 2 bicistrons. The first 2 transgenes are linked by a 2A sequence and expressed by a single promoter. Downstream is a second bicistron containing 2 transgenes linked by a 2A sequence and driven by a single promoter. Each group of 2 transgenes ends with a poly A tail. pA, polyadenylation.
FIGURE 10.
FIGURE 10.
Design of multicistronic targeting vector for targeting to specific “landing pads.” Tetracistronic transgene vector composed of 2 bicistrons. The first 2 transgenes are linked by a 2A sequence and expressed by a single promoter. Downstream is a second bicistron containing 2 transgenes linked by a 2A sequence and driven by a single promoter. Each group of 2 transgenes ends with a poly A tail. This vector is flanked with homology arms to facilitate targeted insertion into landing pads at a specific genomic locus by homology-directed repair (HDR). pA, polyadenylation.
FIGURE 11.
FIGURE 11.
Expression of human complement inhibitors CD46 and CD55 in α1,3GT (Gal) knockout (KO) cells provides protection from cell lysis. Image-based complement-dependent cytotoxicity (CDC) assay using porcine aortic endothelial cells (pAECs) incubated with 30% pooled human serum (N = 3) followed by exposure to 5% rabbit complement for 120 min. Dead cells were stained with Cytotox Red Reagent (IncuCyte), and total cell counts were determined by high-contrast brightfield imaging with a Cytation cell imager (BioTek) to determine % cytotoxicity. Data from 3 replicates are expressed as the % cytotoxicity after 90 min of incubation and compared by ANOVA. WT, wild type. Figure adapted from Ref. , with permission from Springer International.
FIGURE 12.
FIGURE 12.
Example of consumptive coagulopathy in a Gal KO.hCD46 kidney recipient. Nonhuman primate (NHP) serum creatinine levels and platelet count after transplantation. Consumptive coagulopathy is indicated by a sudden decrease in platelets followed by an increase in serum creatinine. Image from Revivicor (unpublished) and reprinted with permission from Massachusetts Medical Society.
FIGURE 13.
FIGURE 13.
Expression of human anticoagulant transgenes in Gal knockout (KO) cells activates protein C to inhibit coagulation. The bioactivity of human thrombomodulin (hTHBD) in porcine aortic endothelial cells, with and without human endothelial protein C receptor (hEPCR), was evaluated by testing its ability to complex with human thrombin and activate human Protein C in vitro. Activated Protein C cleaves a colorimetric substrate that is quantified by absorbance. Figure adapted from Ref. , with permission from Springer International.
FIGURE 14.
FIGURE 14.
Inhibition of staurosporin-induced apoptosis in cells expressing human heme oxygenase 1 (hHO1). Porcine aortic endothelial cells (pAECs) from pigs expressing hHO1 and pAECs from Gal knockout (KO) control pigs that do not express hHO1 were treated with 1 µM staurosporin for 10 h to induce apoptosis. Apoptosis was assessed with a real-time Caspase 3 assay. Figure adapted from Ref. , with permission from Springer International.
FIGURE 15.
FIGURE 15.
Inhibition of phagocytosis of porcine cells expressing human CD47 (hCD47). Porcine aortic endothelial cells (pAECs) transfected with a hCD47 expression vector, pAECs from pigs expressing hCD47, and pAECs from control pigs that do not express hCD47 were transfected with a constitutive green fluorescent protein marker. Transfected cells were then cocultured with human macrophages tagged with red and blue fluorescent antibodies to major histocompatibility complex (MHC) class II and CD14, respectively. After 4 h, cells displaying all 3 fluorescent markers were counted as having undergone phagocytosis. Image from Revivicor (unpublished) and reprinted with permission from Massachusetts Medical Society.
FIGURE 16.
FIGURE 16.
Reduced human serum antibody binding to porcine vascular endothelial cells with disruptions in genes for synthesis of additional major carbohydrate antigens. Porcine vascular endothelial cells were incubated with sera from human donors (N = 3), probed with anti-IgG secondary antibody, and counted by flow cytometry. Results are expressed as % immunoglobulin bound relative to wild type. FITC-A, fluorescein isothiocyanate; Ig, immunoglobulin. See glossary for additional abbreviations. Image from Revivicor (unpublished) and reprinted with permission from Massachusetts Medical Society.
FIGURE 17.
FIGURE 17.
Reduced cytotoxicity with additional carbohydrate deletions. Image-based complement-dependent cytotoxicity (CDC) assay using pAECs incubated with pooled human serum (N = 3) followed by exposure to rabbit complement. Dead cells were stained, and total cell counts were determined by high-contrast brightfield imaging. See glossary for abbreviations. Figure adapted from Ref. , with permission from Springer International.
FIGURE 18.
FIGURE 18.
Growth hormone receptor gene knockout results in reduced growth of the pig. GHr knockout (KO) pig (left) and wild-type pig (right). Image from Revivicor.
FIGURE 19.
FIGURE 19.
Targeting vector designs used to generate the 10 GE pig. A: bicistronic transgene vector in which human (h)CD46 and hCD55 are linked by a 2A sequence to permit expression by a single CAG promoter. This vector is flanked with homology arms to facilitate targeted insertion by homology-directed repair (HDR) into a landing pad directed to the α1,3GT locus. The second allele of the α1,3GT gene was inactivated by a neoR insertion via homologous recombination. B: tetracistronic transgene vector composed of 2 bicistrons. In the first, human thrombomodulin (hTHBD) and human endothelial protein C receptor (hEPCR) are linked by a 2A sequence and expressed by a single porcine THBD promoter. Linked downstream to this is a second bicistron containing hCD47 and hHO1, linked by a 2A sequence and driven by a single CAG promoter. This vector is flanked with homology arms to facilitate targeted insertion into landing pads on the cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH) locus by HDR. The second allele of the CMAH gene was inactivated by CRISPR/Cas9-mediated NHEJ. C: inactivation of both alleles of the β4GalNT2 gene by CRISPR/Cas9-mediated nonhomologous end joining (NHEJ). D: inactivation of both alleles of the GHr gene by CRISPR/Cas9-mediated NHEJ. Chr, chromosome; HO1, heme oxygenase-1; NeoR, neomycin resistance. See glossary for additional abbreviations.
FIGURE 20.
FIGURE 20.
Representative phenotypic identity of 10 GE pigs. A–C: flow cytometry confirming the absence of αGal (A), Sda (B), and Neu5Gc (C). D: Western blot of human transgene expression in pig tissue. E: serum IGF-1 levels in GHr KO donors vs. wild-type pigs. F: immunohistochemical detection of human transgene products in pig tissue. FITC-A, fluorescein isothiocyanate. See glossary for additional abbreviations. Figure adapted from Ref. , with permission from Xenotransplantation.
FIGURE 21.
FIGURE 21.
Transplantation of thymic tissue as a composite thymus and kidney. A: pig thymokidney in baboon soon after xenotransplantation. B: histology of a pig thymokidney prepared 8 wk before procurement. Image by Kazuhiko Yamada.
FIGURE 22.
FIGURE 22.
Genetically engineered pig cardiac xenograft transplanted into the first human recipient. A: adaptation of computed tomography (CT) 3-dimensional reconstruction imaging 7 days after xenotransplantation showing left and right atria, pulmonary artery, and aortic anastomoses. B: view of pig and human structures during transplantation, showing size discrepancies between pig and human atria, pulmonary artery, and aorta. A wedge of tissue from the roof of recipient’s left atrium was excised to accommodate size mismatch.
FIGURE 23.
FIGURE 23.
Clinical details and test results during the postoperative course. A: mean fluorescence intensity (MFI) values were normalized to a positive control (assigned a value of 100%). B: global longitudinal strain is expressed as the absolute value; strain is typically reported as a negative percentage. C: cell free DNA analyses of serum samples from the patient during the course of his survival. ECMO, extracorporeal membrane oxygenation; G-CSF, granulocyte colony-stimulating factor; IVIG, intravenous immune globulin; LVEF, left ventricular ejection fraction. Image from Ref. , reprinted with permission from Massachusetts Medical Society.
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