The enzyme α1,3-galactosyltransferase (α1,3GT or GCTA1) synthesizes α1,3-galactose (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.The enzyme α1,3-galactosyltransferase (α1,3GT or GGTA1) synthesizes α1,3Gal epitopes (Galα1,3Galβ1,4GlcNAc-R) on the cell surface of almost all mammals with the exception of humans, apes, and Old World monkeys (1). α1,3Gal epitopes are the major xenoantigens causing hyperacute rejection (HAR) in pig-to-human xenotransplantation (2-4). Many reports have also indicated that α1,3Gal epitopes are involved in acute vascular rejection (AVR) of xenografts (4-6). Piglets with α1,3GT heterozygous knockout have been cloned Copyright © 2003 by our group (7) and another team (8) in the last year. To produce homozygous α1,3GT knockout piglets by natural breeding, assuming both male and female heterozygous knockout pigs are available at the same time and are fertile, is feasible but takes up to 12 months. However, by using a second-round knockout and cloning strategy, we could save up to 6 months and all cloned piglets would be α1,3GT double knockout (DKO). We have selected and enriched for α1,3GT DKO cells by using a bacterial toxin, toxin A from Clostridium difficile, which binds with high affinity to α1,3Gal epitopes and produces a cytotoxic effect on cells that are α1,3Gal-positive (9). Toxin A uses α1,3Gal epitopes as a cell surface receptor and causes "rounding" and lifting of the α1,3Gal-positive cells from the surface of the growth vessel (10, 11).Heterozygous α1,3GT knockout fetal fibroblasts, 657A-I11 1-6 cells, were isolated from a day-32 pregnancy as described in (7). To avoid using a second antibiotic-resistance gene as a selection marker, we constructed an ATG (start codon)-targeting α1,3GT knockout vector, pPL680 (12), which also contains a neo gene, to knock out the second allele of the α1,3GT gene. 657A-I11 1-6 cells were transfected by electroporation with pPL680 and selected for the α1,3Gal-negative phenotype with purified C. difficile toxin A (13). One colony (680B1) was isolated and expanded af...

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.

Preventing xenograft rejection is one of the greatest challenges of transplantation medicine. Here, we describe a reproducible, long-term survival of cardiac xenografts from alpha 1-3 galactosyltransferase gene knockout pigs, which express human complement regulatory protein CD46 and human thrombomodulin (GTKO.hCD46.hTBM), that were transplanted into baboons. Our immunomodulatory drug regimen includes induction with anti-thymocyte globulin and αCD20 antibody, followed by maintenance with mycophenolate mofetil and an intensively dosed αCD40 (2C10R4) antibody. Median (298 days) and longest (945 days) graft survival in five consecutive recipients using this regimen is significantly prolonged over our recently established survival benchmarks (180 and 500 days, respectively). Remarkably, the reduction of αCD40 antibody dose on day 100 or after 1 year resulted in recrudescence of anti-pig antibody and graft failure. In conclusion, genetic modifications (GTKO.hCD46.hTBM) combined with the treatment regimen tested here consistently prevent humoral rejection and systemic coagulation pathway dysregulation, sustaining long-term cardiac xenograft survival beyond 900 days.

Background Genetically-engineered pigs could provide a source of kidneys for clinical transplantation. The two longest kidney graft survivals reported to date have been 136 days and 310 days, but graft survival >30 days has been unusual until recently. Methods Donor pigs (n=4) were on an α1,3-galactosyltransferase gene-knockout (GTKO)/human complement-regulatory protein (CD46) background (GTKO/CD46). In addition, the pigs were transgenic for at least one human coagulation-regulatory protein. Two baboons received a kidney from a 6-gene pig (Group A) and two from a 3-gene pig (Group B). Immunosuppressive therapy was identical in all 4 cases, and consisted of anti-thymoglobulin (ATG) + anti-CD20mAb (induction) and anti-CD40mAb + rapamycin + corticosteroids (maintenance). Anti-TNF-α and anti-IL-6R mAbs were administered to reduce the inflammatory response. Baboons were followed by clinical/laboratory monitoring of immune/coagulation/inflammatory/physiological parameters. At biopsy or euthanasia, the grafts were examined by microscopy. Results The two Group A baboons remained healthy with normal renal function >7 and >8 months, respectively, but then developed infectious complications. However, no features of a consumptive coagulopathy, e.g., thrombocytopenia, reduction of fibrinogen, or of a protein-losing nephropathy were observed. There was no evidence of an elicited anti-pig antibody response, and histology of biopsies taken at approximately 4, 6, and 7 months and at necropsy showed no significant abnormalities. In contrast, both Group B baboons developed features of a consumptive coagulopathy and required euthanasia on day 12. Conclusions The combination of (i) a graft from a specific 6-gene genetically-modified pig, (ii) an effective immunosuppressive regimen, and (iii) anti-inflammatory therapy prevented immune injury, a protein-losing nephropathy, and coagulation dysfunction for >7 months. Although the number of experiments is very limited, our impression is that expression of human endothelial protein C receptor (+/− CD55) in the graft is important if coagulation dysregulation is to be avoided.

The longest survival of a nonhuman primate with a life-supporting kidney graft to date has been 90 days, though graft survival >30 days has been unusual. A baboon received a kidney graft from an α1,3-galactosyltransferase gene-knockout pig transgenic for two human complement- and three human coagulation- regulatory proteins (though only one was expressed in the kidney). Immunosuppressive therapy was with ATG+anti-CD20mAb (induction) and anti-CD40mAb+rapamycin+corticosteroids (maintenance). Anti-TNF-α and anti-IL-6R were administered. The baboon survived 136 days with a generally stable serum creatinine (0.6–1.6mg/dL) until terminally. No features of a consumptive coagulopathy (e.g., thrombocytopenia, decreased fibrinogen) or of a protein-losing nephropathy were observed. There was no evidence of an elicited anti-pig antibody response. Death was from septic shock (Myroides spp). Histology of a biopsy on day 103 was normal, but by day 136 the kidney showed features of glomerular enlargement, thrombi, and mesangial expansion. The combination of (i) a graft from a specific genetically-engineered pig, (ii) an effective immunosuppressive regimen, and (iii) anti-inflammatory agents prevented immune injury and a protein-losing nephropathy, and delayed coagulation dysfunction. This outcome encourages us that clinical renal xenotransplantation may become a reality.