-
Current Opinion in Organ Transplantation Apr 2012Cardiac xenotransplantation (CXTx) remains a promising approach to alleviate the chronic shortage of donor hearts. This review summarizes recent results of heterotopic... (Review)
Review
PURPOSE OF REVIEW
Cardiac xenotransplantation (CXTx) remains a promising approach to alleviate the chronic shortage of donor hearts. This review summarizes recent results of heterotopic and orthotopic CXTx, highlights the role of non-Gal antibody in xenograft rejection, and discusses challenges to clinical orthotopic CXTx.
RECENT FINDINGS
Pigs mutated in the α 1,3 galactosyltransferase gene (GTKO pigs) are devoid of the galactose α1,3 galactose (αGal) carbohydrate antigen. This situation effectively eliminates any role for anti-Gal antibody in GTKO cardiac xenograft rejection. Survival of heterotopic GTKO cardiac xenografts in nonhuman primates continues to increase. GTKO graft rejection commonly involves vascular antibody deposition and variable complement deposition. Non-Gal antibody responses to porcine antigens associated with inflammation, complement, and hemostatic regulation and to new carbohydrate antigens have been identified. Their contribution to rejection remains under investigation. Orthotopic CXTx is limited by early perioperative cardiac xenograft dysfunction (PCXD). However, hearts affected by PCXD recover full cardiac function and orthotopic survival up to 2 months without rejection has been reported.
SUMMARY
CXTx remains a promising technology for treating end-stage cardiac failure. Genetic modification of the donor and refinement of immunosuppressive regimens have extended heterotopic cardiac xenograft survival from minutes to in excess of 8 months.
Topics: Animals; Galactosyltransferases; Gene Expression; Graft Rejection; Heart Failure; Heart Transplantation; Primates; Swine; Thrombosis; Tissue Donors; Transplantation, Heterologous; Transplants
PubMed: 22327911
DOI: 10.1097/MOT.0b013e3283509120 -
Current Opinion in Organ Transplantation Oct 2008To review the latest development in cardiac xenotransplantation in small and large animal models and related in-vitro studies. (Review)
Review
PURPOSE OF REVIEW
To review the latest development in cardiac xenotransplantation in small and large animal models and related in-vitro studies.
RECENT FINDINGS
With the recent introduction of alpha1,3-galactosyltransferase gene-knockout (GT-KO) pig organs for xenotransplantation, improved cardiac graft survival has been obtained. However, this experience has demonstrated the importance of pig antigens other than Galalpha1,3Gal (Gal) antigens (so-called nonGal antigens) as targets for primate anti-pig antibodies. Several in-vitro studies have confirmed that, although the incidence and levels of anti-nonGal antibodies in nonhuman primates and humans are significantly less when compared with total anti-pig antibodies (i.e., anti-Gal + anti-nonGal), they can result in complement-mediated lysis of GT-KO pig cells. More recently, it has been demonstrated that regulatory T cells suppress the cellular xenogeneic response, thus potentially preventing or reducing T-cell-mediated rejection. The importance of thrombotic microangiopathy as a feature of the immune/inflammatory response and incompatibilities between the coagulation-anticoagulation systems of pig and primate are receiving increasing attention. Development of GT-KO pigs transgenic for one or more 'antithrombotic' genes, for example, CD39 or tissue factor pathway inhibitor, may contribute to overcoming these problems.
SUMMARY
Although GT-KO pigs have provided an advance over wild-type pigs as a source of organs for transplantation into primates, further genetic modification of GT-KO pigs is required to overcome the remaining immune barriers before a clinical trial of cardiac xenotransplantation can be contemplated.
Topics: Animals; Animals, Genetically Modified; Antibodies; Blood Coagulation; Cell Transplantation; Complement Activation; Galactosyltransferases; Gene Knockout Techniques; Graft Rejection; Graft Survival; Heart Transplantation; Humans; Swine; T-Lymphocytes, Regulatory; Transplantation, Heterologous
PubMed: 19060538
DOI: 10.1097/MOT.0b013e32830fdf89 -
Xenotransplantation 2011Xenotransplantation could provide an unlimited and elective supply of grafts, once mechanisms of graft loss and vascular injury are better understood. The development of... (Review)
Review
Xenotransplantation could provide an unlimited and elective supply of grafts, once mechanisms of graft loss and vascular injury are better understood. The development of α-1,3-galactosyltransferase gene-knockout (GalT-KO) swine with the removal of a dominant xeno-antigen has been an important advance; however, delayed xenograft and acute vascular reaction in GalT-KO animals persist. These occur, at least in part, because of humoral reactions that result in vascular injury. Intrinsic molecular incompatibilities in the regulation of blood clotting and extracellular nucleotide homeostasis between discordant species may also predispose to thrombophilia within the vasculature of xenografts. Although limited benefits have been achieved with currently available pharmacological anti-thrombotics and anti-coagulants, the highly complex mechanisms of platelet activation and thrombosis in xenograft rejection also require potent immunosuppressive interventions. We will focus on recent thromboregulatory approaches while elucidating appropriate anti-platelet mechanisms. We will discuss potential benefits of additional anti-thrombotic interventions that are possible in transgenic swine and review recent developments in pharmacological anti-platelet therapy.
Topics: Animals; Animals, Genetically Modified; Fibrinolytic Agents; Galactosyltransferases; Gene Knockout Techniques; Graft Rejection; Humans; Platelet Activation; Platelet Aggregation Inhibitors; Swine; Thrombosis; Transplantation, Heterologous
PubMed: 21496115
DOI: 10.1111/j.1399-3089.2011.00628.x -
Current Opinion in Organ Transplantation Apr 2011Deletion of the α1,3-galactosyltransferase (GalT) gene in pigs has removed a major xenoantigen but has not eliminated the problem of dysregulated coagulation and... (Review)
Review
PURPOSE OF REVIEW
Deletion of the α1,3-galactosyltransferase (GalT) gene in pigs has removed a major xenoantigen but has not eliminated the problem of dysregulated coagulation and vascular injury. Rejecting GalT knockout organ xenografts almost invariably show evidence of thrombosis and platelet sequestration, and primate recipients frequently develop consumptive coagulopathy. This review examines recent findings that illuminate potential mechanisms of this current barrier to successful xenotransplantation.
RECENT FINDINGS
The coagulation response to xenotransplantation differs depending on the type of organ and quite likely the distinct vasculatures. Renal xenografts appear more likely to initiate consumptive coagulopathy than cardiac xenografts, possibly reflecting differential transcriptional responses. Liver xenografts induce rapid and profound thrombocytopenia resulting in recipient death within days due to bleeding; ex-vivo data suggest that liver endothelial cells and hepatocytes are responsible for platelet consumption by a coagulation-independent process.It has been proposed that expression of recipient tissue factor on platelets and monocytes is an important trigger of consumptive coagulopathy. Finally, pigs transgenic for human anticoagulants and antithrombotics are slowly but surely coming on line, but have not yet been rigorously tested to date.
SUMMARY
Successful control of coagulation dysregulation in xenotransplantation may require different combinatorial pharmacological and genetic strategies for different organs.
Topics: Animals; Blood Coagulation; Blood Coagulation Disorders; Galactosyltransferases; Gene Deletion; Graft Rejection; Humans; Immunity, Innate; Swine; Transplantation Tolerance; Transplantation, Heterologous
PubMed: 21415824
DOI: 10.1097/MOT.0b013e3283446c65 -
Scientific Reports Jun 2022Pig-to-human organ transplantation is a feasible solution to resolve the shortage of organ donors for patients that wait for transplantation. To overcome immunological...
Pig-to-human organ transplantation is a feasible solution to resolve the shortage of organ donors for patients that wait for transplantation. To overcome immunological rejection, which is the main hurdle in pig-to-human xenotransplantation, various engineered transgenic pigs have been developed. Ablation of xeno-reactive antigens, especially the 1,3-Gal epitope (GalT), which causes hyperacute rejection, and insertion of complement regulatory protein genes, such as hCD46, hCD55, and hCD59, and genes to regulate the coagulation pathway or immune cell-mediated rejection may be required for an ideal xenotransplantation model. However, the technique for stable and efficient expression of multi-transgenes has not yet been settled to develop a suitable xenotransplantation model. To develop a stable and efficient transgenic system, we knocked-in internal ribosome entry sites (IRES)-mediated transgenes into the α 1,3-galactosyltransferase (GGTA1) locus so that expression of these transgenes would be controlled by the GGTA1 endogenous promoter. We constructed an IRES-based polycistronic hCD55/hCD39 knock-in vector to target exon4 of the GGTA1 gene. The hCD55/hCD39 knock-in vector and CRISPR/Cas9 to target exon4 of the GGTA1 gene were co-transfected into white yucatan miniature pig fibroblasts. After transfection, hCD39 expressed cells were sorted by FACS. Targeted colonies were verified using targeting PCR and FACS analysis, and used as donors for somatic cell nuclear transfer. Expression of GalT, hCD55, and hCD39 was analyzed by FACS and western blotting. Human complement-mediated cytotoxicity and human antibody binding assays were conducted on peripheral blood mononuclear cells (PBMCs) and red blood cells (RBCs), and deposition of C3 by incubation with human complement serum and platelet aggregation were analyzed in GGTA1 knock-out (GTKO)/CD55/CD39 pig cells. We obtained six targeted colonies with high efficiency of targeting (42.8% of efficiency). Selected colony and transgenic pigs showed abundant expression of targeted genes (hCD55 and hCD39). Knocked-in transgenes were expressed in various cell types under the control of the GGTA1 endogenous promoter in GTKO/CD55/CD39 pig and IRES was sufficient to express downstream expression of the transgene. Human IgG and IgM binding decreased in GTKO/CD55/CD39 pig and GTKO compared to wild-type pig PBMCs and RBCs. The human complement-mediated cytotoxicity of RBCs and PBMCs decreased in GTKO/CD55/CD39 pig compared to cells from GTKO pig. C3 was also deposited less in GTKO/CD55/CD39 pig cells than wild-type pig cells. The platelet aggregation was delayed by hCD39 expression in GTKO/CD55/CD39 pig. In the current study, knock-in into the GGTA1 locus and GGTA1 endogenous promoter-mediated expression of transgenes are an appropriable strategy for effective and stable expression of multi-transgenes. The IRES-based polycistronic transgene vector system also caused sufficient expression of both hCD55 and hCD39. Furthermore, co-transfection of CRISPR/Cas9 and the knock-in vector not only increased the knock-in efficiency but also induced null for GalT by CRISPR/Cas9-mediated double-stranded break of the target site. As shown in human complement-mediated lysis and human antibody binding to GTKO/CD55/CD39 transgenic pig cells, expression of hCD55 and hCD39 with ablation of GalT prevents an effective immunological reaction in vitro. As a consequence, our technique to produce multi-transgenic pigs could improve the development of a suitable xenotransplantation model, and the GTKO/CD55/CD39 pig developed could prolong the survival of pig-to-primate xenotransplant recipients.
Topics: Animals; Animals, Genetically Modified; CD55 Antigens; Complement System Proteins; Galactosyltransferases; Gene Knockout Techniques; Humans; Leukocytes, Mononuclear; Swine; Swine, Miniature; Transplantation, Heterologous
PubMed: 35688851
DOI: 10.1038/s41598-022-13536-z -
Xenotransplantation Sep 2018Analysis of non-Gal antibody induced after pig-to-baboon cardiac xenotransplantation identified the glycan produced by porcine... (Review)
Review
Analysis of non-Gal antibody induced after pig-to-baboon cardiac xenotransplantation identified the glycan produced by porcine beta-1,4-N-acetyl-galactosaminyltransferase 2 (B4GALNT2) as an immunogenic xenotransplantation antigen. The porcine B4GALNT2 enzyme is homologous to the human enzyme, which synthesizes the human SDa blood group antigen. Most humans produce low levels of anti-SDa IgM which polyagglutinates red blood cells from rare individuals with high levels of SDa expression. The SDa glycan is also present on GM2 gangliosides. Clinical GM2 vaccination studies for melanoma patients suggest that a human antibody response to SDa can be induced. Expression of porcine B4GALNT2 in human HEK293 cells results in increased binding of anti-SDa antibody and increased binding of Dolichos biflorus agglutinin (DBA), a lectin commonly used to detect SDa. In pigs, B4GALNT2 is expressed by vascular endothelial cells and endothelial cells from a wide variety of pig backgrounds stain with DBA, suggesting that porcine vascular expression of B4GALNT2 is not polymorphic. Mutations in B4GALNT2 have been engineered in mice and pigs. In both species, the B4GALNT2-KO animals are apparently normal and no longer show evidence of SDa antigen expression. Pig tissues with a mutation in B4GALNT2, added to a background of alpha-1,3-galactosyltransferase deficient (GGTA1-KO) and cytidine monophosphate-N-acetylneuraminic acid hydroxylase deficient (CMAH-KO), show reduced antibody binding, confirming the presence of B4GALNT2-dependent antibodies in both humans and non-human primates. Preclinical xenotransplantation using B4GALNT2-deficient donors has recently been reported. Elimination of this source of immunogenic pig antigen should minimize acute injury by preformed anti-pig antibody and eliminate an induced clinical immune response to this newly appreciated xenotransplantation antigen.
Topics: Animals; Animals, Genetically Modified; Galactosyltransferases; Gene Knockout Techniques; Graft Rejection; Heterografts; Humans; Transplantation, Heterologous
PubMed: 29604134
DOI: 10.1111/xen.12394 -
Xenotransplantation 2012The increasing availability of genetically engineered pigs is steadily improving the results of pig organ and cell transplantation in non-human primates (NHPs). Current... (Comparative Study)
Comparative Study
BACKGROUND
The increasing availability of genetically engineered pigs is steadily improving the results of pig organ and cell transplantation in non-human primates (NHPs). Current techniques offer knockout of pig genes and/or knockin of human genes. Knowledge of normal values of hematologic, biochemical, coagulation, and other parameters in healthy genetically engineered pigs and NHPs is important, particularly following pig organ transplantation in NHPs. Furthermore, information on parameters in various NHP species may prove important in selecting the optimal NHP model for specific studies.
METHODS
We have collected hematologic, biochemical, and coagulation data on 71 α1,3-galactosyltransferase gene-knockout (GTKO) pigs, 18 GTKO pigs additionally transgenic for human CD46 (GTKO.hCD46), four GTKO.hCD46 pigs additionally transgenic for human CD55 (GTKO.hCD46.hCD55), and two GTKO.hCD46 pigs additionally transgenic for human thrombomodulin (GTKO.hCD46.hTBM).
RESULTS
We report these data and compare them with similar data from wild-type pigs and the three major NHP species commonly used in biomedical research (baboons, cynomolgus, and rhesus monkeys) and humans, largely from previously published reports.
CONCLUSIONS
Genetic modification of the pig (e.g., deletion of the Gal antigen and/or the addition of a human transgene) (i) does not result in abnormalities in hematologic, biochemical, or coagulation parameters that might impact animal welfare, (ii) seems not to alter metabolic function of vital organs, although this needs to be confirmed after their xenotransplantation, and (iii) possibly (though, by no means certainly) modifies the hematologic, biochemical, and coagulation parameters closer to human values. This study may provide a good reference for those working with genetically engineered pigs in xenotransplantation research and eventually in clinical xenotransplantation.
Topics: Animals; Animals, Genetically Modified; Blood Coagulation; Galactosyltransferases; Gene Knockout Techniques; Genetic Engineering; Graft Rejection; Humans; Primates; Swine; Transplantation, Heterologous
PubMed: 23145497
DOI: 10.1111/xen.12007 -
Biochimica Et Biophysica Acta.... Jun 2023Nucleotide sugar transporters (NSTs) are multitransmembrane proteins, localized in the Golgi apparatus and/or endoplasmic reticulum, which provide glycosylation enzymes...
Nucleotide sugar transporters (NSTs) are multitransmembrane proteins, localized in the Golgi apparatus and/or endoplasmic reticulum, which provide glycosylation enzymes with their substrates. It has been demonstrated that NSTs may form complexes with functionally related glycosyltransferases, especially in the N-glycosylation pathway. However, potential interactions of NSTs with enzymes mediating the biosynthesis of mucin-type O-glycans have not been addressed to date. Here we report that UDP-galactose transporter (UGT; SLC35A2) associates with core 1 β-1,3-galactosyltransferase 1 (C1GalT1; T-synthase). This provides the first example of an interaction between an enzyme that acts exclusively in the O-glycosylation pathway and an NST. We also found that SLC35A2 associated with the C1GalT1-specific chaperone Cosmc, and that the endogenous Cosmc was localized in both the endoplasmic reticulum and Golgi apparatus of wild-type HEK293T cells. Furthermore, in SLC35A2-deficient cells protein levels of C1GalT1 and Cosmc were decreased and their Golgi localization was less pronounced. Finally, we identified SLC35A2 as a novel molecular target for the antifungal agent itraconazole. Based on our findings we propose that NSTs may contribute to the stabilization of their interaction partners and help them to achieve target localization in the cell, most likely by facilitating their assembly into larger functional units.
Topics: Humans; HEK293 Cells; Molecular Chaperones; Galactosyltransferases; Glycosylation; Golgi Apparatus
PubMed: 36933771
DOI: 10.1016/j.bbamcr.2023.119462 -
International Journal of Molecular... Jan 2016The α-Gal epitope (Galα1,3Galα1,4GlcNAc‑R) is ubiquitously presented in non-primate mammals, marsupials and New World Monkeys, but it is absent in humans, apes and... (Review)
Review
The α-Gal epitope (Galα1,3Galα1,4GlcNAc‑R) is ubiquitously presented in non-primate mammals, marsupials and New World Monkeys, but it is absent in humans, apes and Old World monkeys. However, the anti-Gal antibody (~1% of immunoglobulins) is naturally generated in human, and is found as the immunoglobulin G (IgG), IgM and IgA isotypes. Owing to the specific binding of the anti‑Gal antibody with the α‑Gal epitope, humans have a distinct anti‑α‑gal reactivity, which is responsible for hyperacute rejection of organs transplanted from α‑gal donors. In addition, the α1,3 galactosyltransferases (α1,3GT) can catalyze the synthesis of the α‑Gal epitope. Therefore, the α1,3GT gene, which encodes the α1,3GT, is developed profoundly. The distributions of the α‑Gal epitope and anti‑Gal antibody, and the activation of α1,3GT, reveal that the enzyme of α1,3GT in ancestral primates is ineffective. Comparison of the nucleotide sequence of the human α1,3‑GT pseudogene to the corresponding different species sequence, and according to the evolutionary tree of different species, the results of evolutionary inactivation of the α1,3GT gene in ancestral primates attribute to the mutations under a stronger selective pressure. However, on the basis of the structure, the mechanism and the specificity of the α‑Gal epitope and anti‑Gal antibody, they can be applied to clinical exploitation. Knocking out the α1,3GT gene will eliminate the xenoantigen, Gal(α1,3)Gal, so that the transplantation of α1,3GT gene knockout pig organ into human becomes a potential clinically acceptable treatment for solving the problem of organ shortage. By contrast, the α‑Gal epitope expressed through the application of chemical, biochemical and genetic engineering can be exploited for the clinical use. Targeting anti‑Gal‑mediated autologous tumor vaccines, which express α‑Gal epitope to antigen‑presenting cells, would increase their immunogenicity and elicit an immune response, which will be potent enough to eradicate the residual tumor cells. For tumor vaccines, the way of increasing immunogenicity of certain viral vaccines, including flu vaccines and human immunodeficiency virus vaccines, can also be used in the elderly. Recently, α‑Gal epitope nanoparticles have been applied to accelerate wound healing and further directions on regeneration of internally injured tissues.
Topics: Animals; Antibodies; Base Sequence; Biological Evolution; Epitopes; Galactosyltransferases; Gene Knockout Techniques; Graft Rejection; Humans; Immunotherapy; Molecular Sequence Data; Neoplasms; Regeneration; Transplantation, Heterologous; Trisaccharides; Wound Healing
PubMed: 26531137
DOI: 10.3892/ijmm.2015.2397 -
Proceedings of the Japan Academy.... 2015It has been well established that structural changes in glycans attached to proteins and lipids are associated with malignant transformation of cells. We focused on... (Review)
Review
It has been well established that structural changes in glycans attached to proteins and lipids are associated with malignant transformation of cells. We focused on galactose residues among the sugars since they are involved in the galectin-mediated biology, and many carbohydrate antigens are frequently expressed on this sugar. We found changes in the expression of the β4-galactosyltransferase (β4GalT) 2 and 5 genes in cancer cells: decreased expression of the β4GalT2 gene and increased expression of the β4GalT5 gene. The growth of mouse melanoma cells showing enhanced expression of the β4GalT2 gene or reduced expression of the β4GalT5 gene is inhibited remarkably in syngeneic mice. Tumor growth inhibition is probably caused by the induction of apoptosis, inhibition of angiogenesis, and/or reduced MAPK signals. Direct transduction of human β4GalT2 cDNA together with the adenovirus vector into human hepatocellular carcinoma cells grown in SCID mice results in marked growth retardation of the tumors. β4GalT gene-transfer appears to be a potential tool for cancer therapy.
Topics: Animals; Antigens, Neoplasm; Cell Proliferation; Galactosyltransferases; Genetic Therapy; Humans; Neoplasms; Substrate Specificity
PubMed: 25743061
DOI: 10.2183/pjab.91.1