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Author(s): Irina A. Polejaeva (corresponding author) [1]; Shu-Hung Chen [1]; Todd D. Vaught [1]; Raymond L. Page [1]; June Mullins [1]; Suyapa Ball [1]; Yifan Dai [1]; Jeremy Boone [1]; [...]

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. [ABSTRACT FROM AUTHOR]

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 α1,3-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. [ABSTRACT FROM AUTHOR]

The enzyme α1,3-galactosyltransferase (α1,3GT or CGTA1) synthesizes a1,3galactose (α1,3Gal) epitopes (Galα1,3Galβ1,4GlcNAc-R), which are the major xenoantigens causing hyperacute rejection in pig-to-human xenotransplantation. Complete removal of α1,3Gal from pig organs is the critical step toward the success of xenotransplantation. We reported earlier the targeted disruption of one allele of the α1,3GT 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 α1,3GT gene was caused by a T-to-G single point mutation at the second base of exon 9, which resulted in inactivation of the α1,3GT protein. Four healthy α1,3GT double-knockout female piglets were produced by three consecutive rounds of cloning. The piglets carrying a point mutation in the α1,3GT gene hold significant value, as they would allow production of α1,3Gal-deficient pigs free of antibiotic-resistance genes and thus have the potential to make a safer product for human use. [ABSTRACT FROM AUTHOR]

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.