Effects of human TFPI and CD47 expression and selectin and integrin inhibition during GalTKO.hCD46 pig lung perfusion with human blood.

BACKGROUND: Loss of barrier function when GalTKO.hCD46 porcine lungs are perfused with human blood is associated with coagulation pathway dysregulation, innate immune system activation, and rapid sequestration of human formed blood elements. Here, we evaluate whether genetic expression of human tissue factor pathway inhibitor (hTFPI) and human CD47 (hCD47), alone or with combined selectin and integrin adhesion pathway inhibitors, delays GalTKO.hCD46 porcine lung injury or modulates neutrophil and platelet sequestration. METHODS: In a well-established paired ex vivo lung perfusion model, GalTKO.hCD46.hTFPI.hCD47 transgenic porcine lungs (hTFPI.hCD47, n = 7) were compared to GalTKO.hCD46 lungs (reference, n = 5). All lung donor pigs were treated with a thromboxane synthase inhibitor, anti-histamine, and anti-GPIb integrin-blocking Fab, and were pre-treated with Desmopressin. In both genotypes, one lung of each pair was additionally treated with PSGL-1 and GMI-1271 (P- and E-selectin) and IB4 (CD11b/18 integrin) adhesion inhibitors (n = 6 hTFPI.hCD47, n = 3 reference). RESULTS: All except for two reference lungs did not fail within 480 min when experiments were electively terminated. Selectin and integrin adhesion inhibitors moderately attenuated initial pulmonary vascular resistance (PVR) elevation in hTFPI.hCD47 lungs. Neutrophil sequestration was significantly delayed during the early time points following reperfusion and terminal platelet activation was attenuated in association with lungs expressing hTFPI.hCD47, but additional adhesion pathway inhibitors did not show further effects with either lung genotype. CONCLUSION: Expression of hTFPI.hCD47 on porcine lung may be useful as part of an integrated strategy to prevent neutrophil adhesion and platelet activation that are associated with xenograft injury. Additionally, targeting canonical selectin and integrin adhesion pathways reduced PVR elevation associated with hTFPI.hCD47 expression, but did not significantly attenuate neutrophil or platelet sequestration. We conclude that other adhesive mechanisms mediate the residual sequestration of human formed blood elements to pig endothelium that occurs even in the context of the multiple genetic modifications and drug treatments tested here.

hEPCR.hTBM.hCD47.hHO-1 with donor clodronate and DDAVP treatment improves perfusion and function of GalTKO.hCD46 porcine livers perfused with human blood.

INTRODUCTION: Platelet sequestration, inflammation, and inappropriate coagulation cascade activation are prominent in liver xenotransplant models and are associated with poor outcomes. Here, we evaluate a cassette of six additional genetic modifications to reduce anti-pig antibody binding (α-1,3-galactosyl transferase knockout [GalTKO]) and target coagulation dysregulation (human endothelial protein C receptor [hEPRC] and thrombomodulin [hTBM]), complement pathway regulation (human membrane cofactor protein, hCD46), inflammation heme oxygenase 1 [hHO-1]), and a self-recognition receptor (integrin-associated protein [hCD47]), as well as donor pharmacologic treatments designed to blunt these phenomena. METHODS: Livers from GaltKO.hCD46 pigs ("2-gene," n = 3) and GalTKO.hCD46 pigs also transgenic for hEPRC, hTBM, hCD47, and hHO-1 ("6-gene," n = 4) were perfused ex vivo with whole human blood. Six-gene pigs were additionally pretreated with desmopressin (DDAVP) and clodronate liposomes to deplete vWF and kupffer cells, respectively. RESULTS: The average perfusion times increased from 304 (±148) min in the 2-gene group to 856 (±61) min in the 6-gene group (p = .010). The average heparin administration was decreased from 8837 U/h in the 2-gene to 1354 U/h in the 6-gene group (p = .047). Platelet sequestration tended to be delayed in the 6-gene group (p = .070), while thromboxane B2 (TXB2, a platelet activation marker) levels were lower over the first hour (p = .044) (401 ± 124 vs. 2048 ± 712 at 60 min). Thrombin production as measured by F1+2 levels tended to be lower in the 6-gene group (p = .058). CONCLUSIONS: The combination of the hEPCR.hTBM.hCD47.hHO-1 cassette along with donor pig DDAVP and clodronate liposome pretreatment was associated with prolonged function of xenoperfused livers, reduced coagulation pathway perturbations, and decreased TXB2 elaboration, and reflects significant progress to modulate liver xenograft injury in a pig to human model.

Progressive genetic modifications of porcine cardiac xenografts extend survival to 9 months.

We report orthotopic (life-supporting) survival of genetically engineered porcine cardiac xenografts (with six gene modifications) for almost 9 months in baboon recipients. This work builds on our previously reported heterotopic cardiac xenograft (three gene modifications) survival up to 945 days with an anti-CD40 monoclonal antibody-based immunosuppression. In this current study, life-supporting xenografts containing multiple human complement regulatory, thromboregulatory, and anti-inflammatory proteins, in addition to growth hormone receptor knockout (KO) and carbohydrate antigen KOs, were transplanted in the baboons. Selective "multi-gene" xenografts demonstrate survival greater than 8 months without the requirement of adjunctive medications and without evidence of abnormal xenograft thickness or rejection. These data demonstrate that selective "multi-gene" modifications improve cardiac xenograft survival significantly and may be foundational for paving the way to bridge transplantation in humans.

Stable expression of the human thrombomodulin transgene in pig endothelial cells is associated with a reduction in the inflammatory response.

BACKGROUND: Xenotransplantation is associated with an inflammatory response. The proinflammatory cytokine, TNF-α, downregulates the expression of thrombomodulin (TBM), and induces coagulation dysfunction. Although human (h) TBM-transgenic pigs (p) have been developed to reduce coagulation dysfunction, the effect of TNF-α on the expression of hTBM and its functional activity has not been fully investigated. The aims of this study were to investigate (i) whether the expression of hTBM on pig (p) cells is down-regulated during TNF-α stimulation, and (ii) whether cells from hTBM pigs regulate the inflammatory response. METHODS: TNF-α-producing T, B, and natural killer cells in blood from baboons with pig heart or kidney xenografts were investigated by flow cytometry. TNF-α staining in the grafts was detected by immunohistochemistry. Aortic endothelial cells (AECs) from GTKO/CD46 and GTKO/CD46/hTBM pigs were stimulated by hTNF-α, and the expression of the inflammatory/coagulation regulatory protein, TBM, was investigated. RESULTS: After pig organ xenotransplantation, there was a trend to increases in TNF-α-producing T and natural killer cells in the blood of baboons. In vitro observations demonstrated that after hTNF-α stimulation, there was a significant reduction in the expression of endogenous pTBM on pAECs, and a significant increase in the expression of inflammatory molecules. Blocking of NF-κB signaling significantly up-regulated pTBM expression, and suppressed the inflammatory response induced by hTNF-α in pAECs. Whereas the expression of pTBM mRNA was significantly reduced by hTNF-α stimulation, hTBM expression on the GTKO/CD46/hTBM pAECs was not affected. Furthermore, after hTNF-α stimulation, there was significant suppression of expression of inflammatory molecules on GTKO/CD46/hTBM pAECs compared to GTKO/CD46 pAECs. CONCLUSIONS: The stable expression of hTBM in pig cells may locally regulate the inflammatory response. This will help suppress the inflammatory response and prevent coagulation dysregulation after xenotransplantation.

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.

TALEN-mediated modification of the bovine genome for large-scale production of human serum albumin.

As an initial step towards creating genetically modified cattle as a biopharming source of recombinant human serum albumin (rHSA), we report modification of the bovine albumin (bA) locus by transcription activator-like effector nuclease (TALEN)-stimulated homology-directed repair (HDR). Pedigreed bovine fibroblasts were co-transfected with TALENs and an 11.5-kb human serum albumin (HSA) minigene donor construct, designed to simultaneously disrupt and replace bovine serum albumin (BSA) expression with controlled rHSA expression in both the liver and the milk. Targeted integration of the HSA minigene was confirmed in transfected fibroblasts at a frequency of approximately 11% and transgenic bovine embryos were produced from targeted fibroblasts using somatic cell nuclear transfer (SCNT). The research delineated here lays the foundation for the future generation of transgenic rHSA cattle with the potential to provide a large-scale, reliable, and quality-controlled source of rHSA.