Expression of human thrombomodulin by GalTKO.hCD46 pigs modulates coagulation cascade activation by endothelial cells and during ex vivo lung perfusion with human blood.

Thrombomodulin is important for the production of activated protein C (APC), a molecule with significant regulatory roles in coagulation and inflammation. To address known molecular incompatibilities between pig thrombomodulin and human thrombin that affect the conversion of protein C into APC, GalTKO.hCD46 pigs have been genetically modified to express human thrombomodulin (hTBM).

Increased human complement pathway regulatory protein gene dose is associated with increased endothelial expression and prolonged survival during ex-vivo perfusion of GTKO pig lungs with human blood.

Introduction: Expression of human complement pathway regulatory proteins (hCPRP's) such as CD46 or CD55 has been associated with improved survival of pig organ xenografts in multiple different models. Here we evaluate the hypothesis that an increased human CD46 gene dose, through homozygosity or additional expression of a second hCPRP, is associated with increased protein expression and with improved protection from injury when GTKO lung xenografts are perfused with human blood.

Methods: Twenty three GTKO lungs heterozygous for human CD46 (GTKO.

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.

Humanized von Willebrand factor reduces platelet sequestration in ex vivo and in vivo xenotransplant models.

The transplantation of organs across species offers the potential to solve the shortage of human organs. While activation of human platelets by human von Willebrand factor (vWF) requires vWF activation by shear stress, contact between human platelets and porcine vWF (pvWF) leads to spontaneous platelet adhesion and activation. This non-physiologic interaction may contribute to the thrombocytopenia and coagulation pathway dysregulation often associated with xenotransplantation of pig organs in nonhuman primates.

Pig-to-baboon lung xenotransplantation: Extended survival with targeted genetic modifications and pharmacologic treatments.

Galactosyl transferase knock-out pig lungs fail rapidly in baboons. Based on previously identified lung xenograft injury mechanisms, additional expression of human complement and coagulation pathway regulatory proteins, anti-inflammatory enzymes and self-recognition receptors, and knock-down of the β4Gal xenoantigen were tested in various combinations. Transient life-supporting GalTKO.

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.

Thromboxane and histamine mediate PVR elevation during xenogeneic pig lung perfusion with human blood.

Background: Elevated pulmonary vascular resistance (PVR), platelet adhesion, coagulation activation, and inflammation are prominent features of xenolung rejection. Here, we evaluate the role of thromboxane and histamine on PVR, and their contribution to other lung xenograft injury mechanisms.

Methods: GalTKO.

Synthetic liver function is detectable in transgenic porcine livers perfused with human blood.

In addition to immune barriers, molecular incompatibilities between species are predicted to limit pig liver survival in primate xenotransplantation models. Assessment and measurement of synthetic function of genetically modified porcine livers after ex vivo perfusion with human blood have not previously been described. Eight porcine livers from α1,3-galactosyl transferase knockout and human membrane cofactor (GalTKO.

Immunological and physiological observations in baboons with life-supporting genetically engineered pig kidney grafts.

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 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).

Transgenic expression of human leukocyte antigen-E attenuates GalKO.hCD46 porcine lung xenograft injury.

Background: Lung xenografts remain susceptible to loss of vascular barrier function within hours in spite of significant incremental advances based on genetic engineering to remove the Gal 1,3-αGal antigen (GalTKO) and express human membrane cofactor protein (hCD46). Natural killer cells rapidly disappear from the blood during perfusion of GalTKO.hCD46 porcine lungs with human blood and presumably are sequestered within the lung vasculature.

Chimeric 2C10R4 anti-CD40 antibody therapy is critical for long-term survival of GTKO.hCD46.hTBM pig-to-primate cardiac xenograft.

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.

The role of genetically engineered pigs in xenotransplantation research.

There is a critical shortage in the number of deceased human organs that become available for the purposes of clinical transplantation. This problem might be resolved by the transplantation of organs from pigs genetically engineered to protect them from the human immune response. The pathobiological barriers to successful pig organ transplantation in primates include activation of the innate and adaptive immune systems, coagulation dysregulation and inflammation.

Pig kidney graft survival in a baboon for 136 days: longest life-supporting organ graft survival to date.

The longest survival of a non-human primate with a life-supporting kidney graft to date has been 90 days, although graft survival > 30 days has been unusual. A baboon received a kidney graft from an α-1,3-galactosyltransferase gene-knockout pig transgenic for two human complement-regulatory proteins and three human coagulation-regulatory proteins (although only one was expressed in the kidney). Immunosuppressive therapy was with ATG+anti-CD20mAb (induction) and anti-CD40mAb+rapamycin+corticosteroids (maintenance).

Pig-to-baboon heterotopic heart transplantation--exploratory preliminary experience with pigs transgenic for human thrombomodulin and comparison of three costimulation blockade-based regimens.

Background: Three costimulation blockade-based regimens have been explored after transplantation of hearts from pigs of varying genetic backgrounds to determine whether CTLA4-Ig (abatacept) or anti-CD40mAb+CTLA4-Ig (belatacept) can successfully replace anti-CD154mAb.

Methods: All pigs were on an α1,3-galactosyltransferase gene-knockout/CD46 transgenic (GTKO.CD46) background.

Meta-analysis of the independent and cumulative effects of multiple genetic modifications on pig lung xenograft performance during ex vivo perfusion with human blood.

Background: Genetically modified pigs are a promising potential source of lung xenografts. Ex vivo xenoperfusion is an effective platform for testing the effect of new modifications, but typical experiments are limited by testing of a single genetic intervention and small sample sizes. The purpose of this study was to analyze the individual and aggregate effects of donor genetic modifications on porcine lung xenograft survival and injury in an extensive pig lung xenoperfusion series.

Glucose metabolism in pigs expressing human genes under an insulin promoter.

Background: Xenotransplantation of porcine islets can reverse diabetes in non-human primates. The remaining hurdles for clinical application include safe and effective T-cell-directed immunosuppression, but protection against the innate immune system and coagulation dysfunction may be more difficult to achieve. Islet-targeted genetic manipulation of islet-source pigs represents a powerful tool to protect against graft loss.

Regulation of human platelet aggregation by genetically modified pig endothelial cells and thrombin inhibition.

Background: Coagulation disorders remain barriers to successful pig-to-primate organ xenotransplantation. In vitro, we investigated the impact of pig genetic modifications on human platelet aggregation in response to pig aortic endothelial cells (pAEC).

Methods: In comparison to human (h)AEC and wild-type (WT) pAEC, the expression of human complement- (CD46, CD55) or coagulation (thrombomodulin [TBM], endothelial protein C receptor [EPCR]) -regulatory proteins on pAEC from WT or α1,3-galactosyltransferase gene-knockout (GTKO) pigs was studied by flow cytometry.

The potential of genetically-engineered pigs in providing an alternative source of organs and cells for transplantation.

There is a critical shortage of organs, cells, and corneas from deceased human donors worldwide. There are also shortages of human blood for transfusion. A potential solution to all of these problems is the transplantation of organs, cells, and corneas from a readily available animal species, such as the pig, and the transfusion of red blood cells from pigs into humans.

New concepts of immune modulation in xenotransplantation.

The shortage of human organs for transplantation has focused research on the possibility of transplanting pig organs into humans. Many factors contribute to the failure of a pig organ graft in a primate. A rapid innate immune response (natural anti-pig antibody, complement activation, and an innate cellular response; e.

Human dominant-negative class II transactivator transgenic pigs - effect on the human anti-pig T-cell immune response and immune status.

Swine leucocyte antigen (SLA) class II molecules on porcine (p) cells play a crucial role in xenotransplantation as activators of recipient human CD4(+) T cells. A human dominant-negative mutant class II transactivator (CIITA-DN) transgene under a CAG promoter with an endothelium-specific Tie2 enhancer was constructed. CIITA-DN transgenic pigs were produced by nuclear transfer/embryo transfer.

Clinical lung xenotransplantation--what donor genetic modifications may be necessary?

Barriers to successful lung xenotransplantation appear to be even greater than for other organs. This difficulty may be related to several macro anatomic factors, such as the uniquely fragile lung parenchyma and associated blood supply that results in heightened vulnerability of graft function to segmental or lobar airway flooding caused by loss of vascular integrity (also applicable to allotransplants). There are also micro-anatomic considerations, such as the presence of large numbers of resident inflammatory cells, such as pulmonary intravascular macrophages and natural killer (NK) T cells, and the high levels of von Willebrand factor (vWF) associated with the microvasculature.

Relative efficiency of porcine and human cytotoxic T-lymphocyte antigen 4 immunoglobulin in inhibiting human CD4+ T-cell responses co-stimulated by porcine and human B7 molecules.

α1,3-Galactosyltransferase gene-knockout pigs transgenic for porcine cytotoxic T-lymphocyte antigen 4 immunoglobulin (pCTLA4-Ig) have been produced to reduce T-cell-mediated rejection following xenotransplantation. The level of soluble pCTLA4-Ig in their blood was greatly in excess of the therapeutic level in patients, rendering the pigs immune-incompetent. Soluble pCTLA4-Ig produced by these transgenic pigs was evaluated for binding to porcine and human (h) B7 molecules, and for its inhibitory effect on allogeneic and xenogeneic human T-cell responses.