Growth hormone receptor knockout: Relevance to xenotransplantation.

Xenotransplantation research has made considerable progress in recent years, largely through the increasing availability of pigs with multiple genetic modifications, effective immunosuppressive therapy, and anti-inflammatory therapy to protect pig tissues from the primate immune and inflammatory responses and correct molecular incompatibilities. Further study is required regarding identification and investigation of physiological incompatibilities. Although the exact cause remains uncertain, we and others have observed relatively rapid growth of kidney xenografts after transplantation into nonhuman primates (NHPs). There has also been some evidence of growth, or at least ventricular hypertrophy, of the pig heart after orthotopic transplantation into NHPs. Rapid growth could be problematic, particularly with regard to the heart within the relatively restricted confines of the chest. It has been suggested that the problem of rapid growth of the pig organ after transplantation could be resolved by growth hormone receptor (GHR) gene knockout in the pig. The GHR, although most well-known for regulating growth, has many other biological functions, including regulating metabolism and controlling physiological processes. Genetically modified GHRKO pigs have recently become available. We provide data on their growth compared to comparable pigs that do not include GHRKO, and we have reviewed the literature regarding the effect of GHRKO, and its relevance to xenotransplantation.

B-Cell-Deficient and CD8 T-Cell-Depleted Gnotobiotic Pigs for the Study of Human Rotavirus Vaccine-Induced Protective Immune Responses.

Genetically modified pigs have become available recently. In this study, we established the gnotobiotic pig model of human rotavirus (HRV) infection using cloned pigs with homozygous disruption in the gene encoding immunoglobulin heavy chain (HCKO), which totally impairs B-cell development. To clarify importance of B cells and cytotoxic T cells in rotavirus immunity, CD8 cells in a subset of the pigs were depleted by injecting antipig CD8 antibodies and the immune phenotypes of all pigs were examined. HCKO pigs, CD8 cell-depleted HCKO pigs, and wild-type (WT) pigs were vaccinated with an attenuated HRV vaccine and challenged with virulent HRV. Protection against HRV infection and diarrhea was assessed postchallenge and detailed T-cell subset responses were determined pre- and postchallenge. Significantly longer duration of virus shedding was seen in vaccinated HCKO pigs than in WT pigs, indicating the importance of B cells in vaccine-induced protective immunity. Vaccinated HCKO/CD8(-) pigs shed significantly higher number of infectious virus than WT pigs and non-CD8-depleted HCKO pigs, indicating the importance of CD8 T cells in controlling virus replication. Therefore, both B cells and CD8 T cells play an important role in the protection against rotavirus infection. HCKO and HCKO/CD8(-) pigs did not differ significantly in diarrhea and virus shedding postchallenge; increased CD4 and CD8(-) γδ T-cell responses probably compensated partially for the lack of CD8 T cells. This study demonstrated that HCKO pigs can serve as a valuable model for dissection of protective immune responses against viral infections and diseases.

Gene expression of Dnmt1 isoforms in porcine oocytes, embryos, and somatic cells.

In the mouse, the dynamics of genomic methylation and the initial events of gametic imprinting are controlled by the activity of an oocyte isoform of the DNA methyltransferase-1 (Dnmt1o) enzyme. The objectives of this study were to identify the alternative splicing variants of Dnmt1 in porcine oocytes and determine the gene expression pattern of the different Dnmt1 isoforms during embryo development. A rapid amplification of cDNA ends (RACE ) system was used to amplify the 5' cDNA end of Dnmt1 isoforms in porcine oocytes. RNA levels of the Dnmt1 isoforms were analyzed in porcine oocytes and embryos. DNMT1 protein expression of oocytes and somatic cells were analyzed by western blot and immunostaining. Two new Dnmt1o RNA isoforms were identified--Dnmt1o1 and Dnmt1o2. The previously reported somatic Dnmt1 isoform (Dnmt1s) was expressed at low but constant levels in oocytes and embryos from the two-cell to the blastocyst stage. Abundant RNA levels of Dnmt1o1 and Dnmt1o2 were detected in oocytes and embryos from the two- to the eight- to 16-cell stage. Levels of these Dnmt1o transcripts were low at the morula and blastocyst stages. Although Dnmt1s was present in all the somatic cell types analyzed, Dnmt1o1 and Dnmt1o2 were not detected in any somatic tissues. As predicted by the RNA sequence and verified by western blot analysis, Dnmt1o1 and Dnmt1o2 RNAs translate one DNMT1o enzyme. Western blot analysis confirmed that both the oocyte and the somatic forms of DNMT1 protein are present in porcine oocytes and early embryos, whereas somatic cells produce only DNMT1s protein. DNMT1o is localized mainly in the nuclei of oocytes and early embryos, whereas DNMT1s is expressed in the ooplasm cortex of oocytes and cytoplasm of early embryos.

Comparison of hematologic, biochemical, and coagulation parameters in α1,3-galactosyltransferase gene-knockout pigs, wild-type pigs, and four primate species.

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.

Production of transgenic and knockout pigs by somatic cell nuclear transfer.

Xenotransplantation is one alternative to transplantation of human organs which has been investigated. It is generally accepted that the pig represents the most logical choice of animals to serve as organ donors for xenotransplantation. Moreover, the implementation of cloning by somatic cell nuclear transfer (SCNT) and transgenic techniques have resulted in the production of numerous transgenic pigs than can be used for xenotransplantation purposes as well as models for human diseases.

Initial in vitro investigation of the human immune response to corneal cells from genetically engineered pigs.

PURPOSE: To compare the in vitro human humoral and cellular immune responses to wild-type (WT) pig corneal endothelial cells (pCECs) with those to pig aortic endothelial cells (pAECs). These responses were further compared with CECs from genetically engineered pigs (α1,3-galactosyltransferase gene-knockout [GTKO] pigs and pigs expressing a human complement-regulatory protein [CD46]) and human donors. METHODS: The expression of Galα1,3Gal (Gal), swine leukocyte antigen (SLA) class I and class II on pCECs and pAECs, with or without activation by porcine IFN-γ, was tested by flow cytometry. Pooled human serum was used to measure IgM/IgG binding to and complement-dependent cytotoxicity (CDC) to cells from WT, GTKO, and GTKO/CD46 pigs. The human CD4(+) T-cell response to cells from WT, GTKO, GTKO/CD46 pigs and human was tested by mixed lymphocyte reaction (MLR). RESULTS: There was a lower level of expression of the Gal antigen and of SLA class I and II on the WT pCECs than on the WT pAECs, resulting in less antibody binding and reduced human CD4(+) T-cell proliferation. However, lysis of the WT pCECs was equivalent to that of the pAECs, suggesting more susceptibility to injury. There were significantly weaker humoral and cellular responses to the pCECs from GTKO/CD46 pigs compared with the WT pCECs, although the cellular response to the GTKO/CD46 pCECs was greater than to the human CECs. CONCLUSIONS: These data provide the first report of in vitro investigations of CECs from genetically engineered pigs and suggest that pig corneas may provide an acceptable alternative to human corneas for clinical transplantation.

Insulin secretion and glucose metabolism in alpha 1,3-galactosyltransferase knock-out pigs compared to wild-type pigs.

BACKGROUND: Xenotransplantation of porcine islets could be a valuable alternative to the shortage of human islets for transplantation. To overcome the immunological obstacle of antibody-mediated rejection, pigs homozygous for alpha1,3-galactosyltransferase gene knock-out (GT-KO) have been produced. The effect of this mutation on glucose metabolism is unknown. METHODS: Glucose, insulin, C-peptide and glucagon levels were studied in eight adult pigs (four wild-type [WT] and four GT-KO) during intravenous glucose tolerance test (IVGTT), arginine stimulation test (AST), and insulin tolerance test (ITT). Morphological analysis of the pancreata was also performed. The in vitro insulin response to a high glucose concentration and theophylline were studied in a dynamic perfusion system with isolated islets. RESULTS: Basal and stimulated blood glucose levels were similar in WT and GT-KO pigs. Basal insulin, C-peptide and glucagon were higher in GT-KO pigs. C-peptide and insulin responses to arginine and glucose were also higher in GT-KO animals. The reduction in blood glucose during ITT and IVGTT was similar in WT and GT-KO pigs. The extent of staining for insulin and glucagon in the pancreata were similar. The basal insulin secretion of isolated islets was higher in GT-KO pigs, while stimulation indexes for glucose and theophylline were similar to WT. CONCLUSIONS: GT-KO pigs demonstrated differences in glucose metabolism compared to WT pigs, the cause for which remains uncertain. It is unlikely that these differences would in any way affect the outcome of GT-KO porcine islet xenotransplantation.

Production of transgenic pigs that express porcine endogenous retrovirus small interfering RNAs.

BACKGROUND: The presence of multiple copies of porcine endogenous retrovirus (PERV) within the pig genome, and the demonstration that replication competent PERV, that infect human cells in culture, can be isolated from pig cells, directly impacts the drive towards the development of pigs for xenotransplantation. The development of technology to produce pigs that do not propagate PERV has the potential to facilitate the development of xenotransplantation products for human use, and as such, is the focus of this investigation. The shear number of PERV loci, most of which are defective or pseudogenes, renders conventional gene targeting impractical, if not impossible, to inactivate all PERV provirus within the pig genome, including potential replication competent PERV arising from spontaneous recombination. The recently developed RNA interference (RNAi) technology to knockdown/silence post-transcriptional gene expression, offers a promising alternative to achieving this goal. METHODS: Here, the combination of nuclear transfer cloning and RNAi technology was used to produce pigs that may not propagate PERV. Small interfering RNAs (siRNA) were expressed as short hairpin RNAs (shRNA) against the gag and pol PERV genes, respectively, under the control of a RNA polymerase III (pol III), or a pol II promoter. PERV gag and pol model-genes, in combination with a Green Fluorescent Protein (GFP) reporter system, were developed to assess in vitro PERV target knockdown. Two shRNAs were selected, and transgenic pigs were produced that expressed the anti-gag and -pol shRNAs, in tandem, under the control of a ubiquitous pol II promoter. RESULTS: The anti-gag and -pol shRNAs, effectively knocked down expression of the PERV model-genes, and also endogenous PERV within cells in vitro. PERV knockdown was achieved whether the shRNA was expressed under the control of a RNA pol III, or a pol II promoter. Three litters of cloned pigs were produced. The shRNA construct was expressed by all the transgenic cloned animals, and within all the tissues of transgenic animals tested. PERV expression at the mRNA and PERV particulate levels in the pigs was virtually undetectable, compared with the infectious levels expressed by the positive control PK15 cell line in vitro. Immunofluorescence and Western blotting, with an anti-PERV-envelope antibody, did not detect PERV in pig tissues or cells whether activated or not, as compared to the positive control on PK15 cells. CONCLUSIONS: The stable long-term expression of anti-PERV siRNAs was shown to be effective in knocking down PERV expression in cells. However, the very low (sometimes undetectable), and variable levels of expression of PERV in normal pigs make it difficult to obtain suitable control animals for comparison, to assess knockdown of PERV in vivo. This was demonstrated by the observation that even cloned non-transgenic littermates, express levels of PERV as low as that of some of their siRNA transgenic littermates. Further analysis is required to conclusively quantitate in vivo effects in the shRNA transgenic pigs.

Production and characterization of transgenic pigs expressing porcine CTLA4-Ig.

BACKGROUND: Inhibition of the T-cell-mediated immune response is a necessary component of preventing rejection following xenotransplantation with pig alpha1,3-galactosyltransferase gene-knockout (GTKO) organs. Cytotoxic T lymphocyte-associated antigen (CTLA4) is a co-stimulatory molecule that inhibits T-cell activity and may be useful in prolonging graft rejection. METHODS: An expression vector was built containing the extracellular coding region of porcine (p) CTLA4 fused to the hinge and CH2/CH3 regions of human IgG1 (pCTLA4-Ig). Pigs transgenic for pCTLA4-Ig, on either a GTKO or wild-type (WT) genetic background, were produced by nuclear transfer and characterized using Western blot analysis, immunofluorescence, ELISA, and necropsy. RESULTS: Fifteen pCTLA4-Ig-transgenic piglets resulted from five pregnancies produced by nuclear transfer. All transgenic pigs exhibited robust expression of the pCTLA4-Ig protein and most expressed the transgene in all organs analyzed, with significant levels in the blood as well. Despite initial good health, these pigs exhibited diminished humoral immunity, and were susceptible to infection, which could be managed for a limited time with antibiotics. CONCLUSIONS: Viable pigs exhibiting robust and ubiquitous expression of pCTLA4-Ig were produced on both a WT and GTKO background. Expression of pCTLA4-Ig resulted in acute susceptibility to opportunistic pathogens due at least in part to a significantly compromised humoral immune status. As this molecule is known to have immunosuppressive activity, high levels of pCTLA4-Ig expression in the blood, as well as defective development related to exposure to pCTLA4-Ig in utero, may contribute to this reduced immune status. Prophylactic treatment with antibiotics may promote survival of disease-free transgenic pigs to a size optimal for organ procurement for transplantation. Additional genetic modifications and/or tightly regulated expression of pCTLA4Ig may reduce the impact of this transgene on the humoral immune system.

The pig-to-primate immune response: relevance for xenotransplantation.

BACKGROUND: The allotransplantation of some solid organs can be associated with a graft-vs.-host (GVH) response from the activity of donor B or T cells. We have investigated whether there is a risk of a GVH response following pig-to-primate organ xenotransplantation. METHODS: The responses of 16 pigs (six farm-housed wild-type and five wild-type housed under high herd health conditions [all designated WT], and 5 alpha1,3-galactosyltransferase gene-knockout [GT-KO] housed under high herd health conditions) to human (n = 6) and baboon (n = 6) peripheral blood mononuclear cells (PBMC) were determined. Assays included flow cytometry, complement-dependent cytotoxicity, and mixed lymphocyte reaction. RESULTS: Anti-primate cytotoxic IgM antibodies were detected in the sera of all pigs, but anti-primate IgG antibodies were minimal. All pigs demonstrated a cellular proliferative response to primate PBMC that was equivalent to, or greater than, the allo response. The strength of the pig-to-primate GVH responses was proportional to the health status of the pigs, those from a high health status herd, particularly from a specific pathogen-free herd maintained under clean husbandry conditions, where colonization of the gastrointestinal tract may be reduced, having lower responses. CONCLUSIONS: After pig organ transplantation in a primate, if the organ is from an early-weaned, early-segregated GT-KO pig, the strength of a GVH response is likely to be relatively weak. Although not investigated here, any GVH response is likely to be suppressed by the immunosuppressive therapy administered to the recipient to suppress the anti-donor immune response.

Antibodies directed to pig non-Gal antigens in naïve and sensitized baboons.

BACKGROUND: As pigs homozygous for alpha1,3-galactosyltransferase gene-knockout (GT-KO) are available, primate antibodies to pig non-Gal antigens can be studied. METHODS: Sera from 56 baboons were tested for binding of IgM and IgG to peripheral blood mononuclear cells (PBMC) from both wild-type (WT) and GT-KO pigs by flow cytometry. Complement-dependent cytotoxicity was measured in 39 sera. Antibody and cytotoxicity responses were measured in two baboons exposed to a GT-KO pig heart, one not immunosuppressed and one that received only cobra venom factor. RESULTS: IgM and IgG bound to 95% and 79% of WT PBMC, and 32% and 9% GT-KO PBMC, respectively (WT vs. GT-KO, P<0.01). Whereas 97% of sera were cytotoxic to WT PBMC, only 64% were cytotoxic to GT-KO PBMC, and the level of cytotoxicity was less (mean 60% vs. 25% lysis, P<0.05). In the two baboons exposed to GT-KO hearts, anti-non-Gal antibodies increased markedly, peaking after 2 (IgM) and 3 (IgG) weeks, associated with an increase in lysis of GT-KO PBMC. CONCLUSIONS: Two-thirds of baboon sera demonstrated cytotoxicity to GT-KO PBMC. After GT-KO organ transplantation, if an elicited antibody response develops, it is likely to cause rapid graft rejection.

Allosensitized humans are at no greater risk of humoral rejection of GT-KO pig organs than other humans.

BACKGROUND: The availability of pigs homozygous for alpha1,3-galactosyltransferase gene-knockout (GT-KO) has enabled study of the incidence and cytotoxicity of primate antibodies directed to antigens other than Galalpha1,3Gal (Gal), termed non-Gal antigens. METHODS: Sera from 27 healthy humans and 31 patients awaiting renal allotransplantation, who were either unsensitized [panel reactive antibodies (PRA) < 10%] or allosensitized (PRA > 70%), were tested by flow cytometry for binding of immunoglobulin M (IgM) and IgG to peripheral blood mononuclear cells (PBMC) from both wild-type (WT) and GT-KO pigs. Complement-dependent cytotoxicity to WT and GT-KO PBMC was also measured. RESULTS: IgM and IgG from all 27 (100%) healthy human sera bound to WT PBMC, while 78% and 63% of these sera had IgM and IgG that bound to GT-KO PBMC, respectively. Mean binding to WT PBMC was significantly greater than GT-KO PBMC. Whereas 100% of sera were cytotoxic to WT PBMC, only 61% were cytotoxic to GT-KO PBMC, and the extent of lysis was significantly less. Neither mean binding of IgM and IgG nor cytotoxicity of unsensitized and allosensitized sera to WT and GT-KO PBMC was significantly different to that of healthy sera. CONCLUSIONS: More than half of the healthy humans tested had cytotoxic antibodies to GT-KO PBMC, but allosensitized patients will be at no greater risk of rejecting a pig xenograft by a humoral mechanism.

Incidence and cytotoxicity of antibodies in cynomolgus monkeys directed to nonGal antigens, and their relevance for experimental models.

The recent availability of pigs homozygous for alpha1,3-galactosyltransferase gene-knockout (GT-KO) has enabled the study of incidence and cytotoxicity of antibodies of cynomolgus monkeys directed to antigens other than Galalpha1,3Gal (Gal), termed nonGal antigens. To this aim, sera from 21 cynomolgus monkeys were tested by flow cytometry for binding of IgM and IgG to peripheral blood mononuclear cells (PBMC) from wild-type (WT) and GT-KO pigs. The sera were also tested for complement-dependent cytotoxicity to WT and GT-KO PBMC. Anti-WT IgM and IgG were found in 100% and 95%, respectively, and anti-GT-KO IgM and IgG in 76% and 66%, respectively, in the sera of the monkeys tested (P < 0.01). Whereas 100% of sera were cytotoxic to WT PBMC, only 76% were cytotoxic to GT-KO PBMC, and the level of cytotoxicity was significantly less (P < 0.01). Although the incidence and cytotoxicity of antibodies in monkey sera to GT-KO pig PBMC are significantly less than to WT PBMC, approximately three-quarters of the monkeys tested had cytotoxic antibodies to GT-KO PBMC. This incidence of cytotoxicity is significantly higher than that found in baboons and humans, suggesting the baboon may be an easier and possibly more suitable model to study antibody-mediated rejection of transplanted GT-KO pig organs and cells.

Production of alpha 1,3-galactosyltransferase-deficient pigs.

The enzyme alpha1,3-galactosyltransferase (alpha1,3GT or GGTA1) synthesizes alpha1,3-galactose (alpha1,3Gal) epitopes (Galalpha1,3Galbeta1,4GlcNAc-R), which are the major xenoantigens causing hyperacute rejection in pig-to-human xenotransplantation. Complete removal of alpha1,3Gal from pig organs is the critical step toward the success of xenotransplantation. We reported earlier the targeted disruption of one allele of the alpha1,3GT gene in cloned pigs. A selection procedure based on a bacterial toxin was used to select for cells in which the second allele of the gene was knocked out. Sequencing analysis demonstrated that knockout of the second allele of the alpha1,3GT gene was caused by a T-to-G single point mutation at the second base of exon 9, which resulted in inactivation of the alpha1,3GT protein. Four healthy alpha1,3GT double-knockout female piglets were produced by three consecutive rounds of cloning. The piglets carrying a point mutation in the alpha1,3GT gene hold significant value, as they would allow production of alpha1,3Gal-deficient pigs free of antibiotic-resistance genes and thus have the potential to make a safer product for human use.

Targeted disruption of the alpha1,3-galactosyltransferase gene in cloned pigs.

Galactose-alpha1,3-galactose (alpha1,3Gal) is the major xenoantigen causing hyperacute rejection in pig-to-human xenotransplantation. Disruption of the gene encoding pig alpha1,3-galactosyltransferase (alpha1,3GT) by homologous recombination is a means to completely remove the alpha1,3Gal epitopes from xenografts. Here we report the disruption of one allele of the pig alpha1,3GT gene in both male and female porcine primary fetal fibroblasts. Targeting was confirmed in 17 colonies by Southern blot analysis, and 7 of them were used for nuclear transfer. Using cells from one colony, we produced six cloned female piglets, of which five were of normal weight and apparently healthy. Southern blot analysis confirmed that these five piglets contain one disrupted pig alpha1,3GT allele.