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BackgroundSurvival of vascularized xenografts is dependent on pre-emptive inhibition of the xenoantibody response against galactosyltransferase knockout (GTKO) porcine organs. Our analysis in multiple GTKO pig-to-primate models of xenotransplantation has demonstrated that the anti-non-gal-α-1,3-gal (anti-non-Gal) xenoantibody response displays limited structural diversity. This allowed our group to identify an experimental compound which selectively inhibited induced anti-non-Gal IgM xenoantibodies. However, because this compound had an unknown safety profile, we extended this line of research to include screening small molecules with known safety profiles allowing rapid advancement to large animal models.MethodsThe NIH clinical collections of small molecules were screened by ELISA for their ability to inhibit xenoantibody binding to GTKO pig endothelial cells. Serum collected from non-immunosuppressed rhesus monkeys at day 14 post-injection with GTKO pig endothelial cells was utilized as a source of elicited xenoantibody for initial screening. Virtual small molecule screening based on xenoantibody structure was used to assess the likelihood that the identified small molecules bound xenoantibody directly. As a proxy for selectivity, ELISAs against tetanus toxoid and the natural antigens laminin, thyroglobulin, and single-stranded DNA (ssDNA) were utilized to assess the ability of the identified reagents to inhibit additional antibody responses. The identified inhibitory small molecules were further tested for their ability to inhibit xenoantibody elicited in multiple settings, including rhesus monkeys pre-treated with an anti-non-Gal selective anti-idiotypic antibody, non-immunosuppressed rhesus monkeys immunized with wild-type fetal pig isletlike cell clusters, and non-immunosuppressed baboons transplanted with GTKO multiple transgenic pig kidneys.ResultsFour clinically relevant small molecules inhibited anti-non-Gal IgM binding to GTKO pig endothelial cells in vitro. Three of these drugs displayed a limited region of structural similarity suggesting they may inhibit xenoantibody by a similar mechanism. One of these, the anti-hypertensive agent clonidine, displayed only minimal inhibition of antibodies elicited by vaccination against tetanus toxoid or pre-existing natural antibodies against laminin, thyroglobulin, or ssDNA. Furthermore, clonidine inhibited elicited anti-non-Gal IgM from all animals that demonstrated a xenoantibody response in each experimental setting.ConclusionsClinically relevant small molecule drugs with known safety profiles can inhibit xenoantibody elicited against non-Gal antigens in diverse experimental xenotransplantation settings. These molecules are ready to be tested in large animal models. However, it will first be necessary to optimize the timing and dosing required to inhibit xenoantibodies in vivo.

BackgroundB-cell depletion significantly extends survival of α-1,3-galactosyltranferase knockout (GTKO) porcine organs in pig-to-primate models. Our previous work demonstrated that the anti-non-Gal xenoantibody response is structurally restricted. Selective inhibition of xenoantigen/xenoantibody interactions could prolong xenograft survival while preserving B-cell-mediated immune surveillance.MethodsThe anti-idiotypic antibody, B4N190, was selected from a synthetic human phage display library after enrichment against a recombinant anti-non-Gal xenoantibody followed by functional testing in vitro. The inhibitory small molecule, JMS022, was selected from the NCI diversity set III using virtual screening based on predicted xenoantibody structure. Three rhesus monkeys were pre-treated with anti-non-Gal-specific single-chain anti-idiotypic antibody, B4N190. A total of five monkeys, including two untreated controls, were then immunized with GTKO porcine endothelial cells to initiate an anti-non-α-1,3-Gal (non-Gal) xenoantibody response. The efficacy of the inhibitory small molecule specific for anti-non-Gal xenoantibody, JMS022, was tested in vitro.ResultsAfter the combination of in vivo anti-id and in vitro small molecule treatments, IgM xenoantibody binding to GTKO cells was reduced to pre-immunization levels in two-thirds of animals; however, some xenoantibodies remained in the third animal. Furthermore, when treated with anti-id alone, all three experimental animals displayed a lower anti-non-Gal IgG xenoantibody response compared with controls. Treatment with anti-idiotypic antibody alone reduced IgM xenoantibody response intensity in only one of three monkeys injected with GTKO pig endothelial cells. In the one experimental animal, which displayed reduced IgM and IgG responses, select B-cell subsets were also reduced by anti-id therapy alone. Furthermore, natural antibody responses, including anti-laminin, anti-ssDNA, and anti-thyroglobulin antibodies were intact despite targeted depletion of anti-non-Gal xenoantibodies in vivo indicating that selective reduction of xenoantibodies can be accomplished without total B-cell depletion.ConclusionsThis preliminary study demonstrates the strength of approaches designed to selectively inhibit anti-non-Gal xenoantibody. Both anti-non-Gal-specific anti-idiotypic antibody and small molecules can be used to selectively limit xenoantibody responses.

Background: Pig islet xenotransplantation is a potential treatment for type 1 diabetes. We have shown that maintenance immunosuppression is required to protect genetically modified (GM) porcine islet xenografts from T cell‐mediated rejection in baboons. Local expression of a depleting anti‐CD2 monoclonal antibody (mAb) by the xenograft may provide an alternative solution. We have previously reported the generation of GGTA1 knock‐in transgenic pigs expressing the chimeric anti‐CD2 mAb diliximab under an MHC class I promoter (MHCIP). In this study, we generated GGTA1 knock‐in pigs in which MHCIP was replaced by the β‐cell‐specific porcine insulin promoter (PIP), and compared the pattern of diliximab expression in the two lines. Methods: A PIP‐diliximab knock‐in construct was prepared and validated by transfection of NIT‐1 mouse insulinoma cells. The construct was knocked into GGTA1 in wild type (WT) porcine fetal fibroblasts using CRISPR, and knock‐in cells were used to generate pigs by somatic cell nuclear transfer (SCNT). Expression of the transgene in MHCIP‐diliximab and PIP‐diliximab knock‐in pigs was characterised at the mRNA and protein levels using RT‐qPCR, flow cytometry, ELISA and immunohistochemistry. Islets from MHCIP‐diliximab and control GGTA1 KO neonatal pigs were transplanted under the kidney capsule of streptozotocin‐diabetic SCID mice. Results: NIT‐1 cells stably transfected with the PIP‐diliximab knock‐in construct secreted diliximab into the culture supernatant, confirming correct expression and processing of the mAb in β cells. PIP‐diliximab knock‐in pigs showed a precise integration of the transgene within GGTA1. Diliximab mRNA was detected in all tissues tested (spleen, kidney, heart, liver, lung, pancreas) in MHCIP‐diliximab pigs, but was not detectable in PIP‐diliximab pigs. Likewise, diliximab was present in the serum of MHCIP‐diliximab pigs, at a mean concentration of 1.8 μg/mL, but was not detected in PIP‐diliximab pig serum. An immunohistochemical survey revealed staining for diliximab in all organs of MHCIP‐diliximab pigs but not of PIP‐diliximab pigs. Whole genome sequencing (WGS) of a PIP‐diliximab pig identified a missense mutation in the coding region for the dixilimab light chain. This mutation was also found to be present in the fibroblast knock‐in clone used to generate the PIP‐diliximab pigs. Islet xenografts from neonatal MHCIP‐diliximab pigs restored normoglycemia in diabetic immunodeficient mice, indicating no overt effect of the transgene on islet function, and demonstrated expression of diliximab in situ. Conclusion: Diliximab was widely expressed in MHCIP‐diliximab pigs, including in islets, consistent with the endogenous expression pattern of MHC class I. Further investigation is required to determine whether the level of expression in islets from the MHCIP‐diliximab pigs is sufficient to prevent T cell‐mediated islet xenograft rejection. The unexpected absence of diliximab expression in the islets of PIP‐diliximab pigs was probably due to a mutation in the transgene arising during the generation of the knock‐in cells used for SCNT. [ABSTRACT FROM AUTHOR]

Xenotransplantation using porcine donors is rapidly approaching clinical applicability as an alternative therapy for treatment of many end-stage diseases including type 1 diabetes. Porcine neonatal islet cell clusters (NICC) have normalised blood sugar levels for relatively short periods in the preclinical diabetic rhesus model but have met with limited success in the stringent baboon model. Here we report that NICC from genetically modified (GM) pigs deleted for αGal and expressing the human complement regulators CD55 and CD59 can cure diabetes long-term in immunosuppressed baboons, with maximum graft survival exceeding 22 months. Five diabetic baboons were transplanted intraportally with 9,673 – 56,913 islet equivalents (IEQ) per kg recipient weight. Immunosuppression consisted of T cell depletion with an anti-CD2 mAb, tacrolimus for the first 4 months, and maintenance with belatacept and anti-CD154; no anti-inflammatory treatment or cytomegalovirus (CMV) prophylaxis/treatment was given. This protocol was well tolerated, with all recipients maintaining or gaining weight. Recipients became insulin-independent at a mean of 87 ± 43 days post-transplant and remained insulin-independent for 397 ± 174 days. Maximum graft survival was 675 days. Liver biopsies showed functional islets staining for all islet endocrine components, with no evidence of the inflammatory blood-mediated inflammatory reaction (IBMIR) and minimal leukocytic infiltration. The costimulation blockade-based immunosuppressive protocol prevented an anti-pig antibody response in all recipients. In conclusion, we demonstrate that genetic modification of the donor pig enables attenuation of early islet xenograft injury, and in conjunction with judicious immunosuppression provides excellent long-term function and graft survival in the diabetic baboon model. [ABSTRACT FROM AUTHOR]

Introduction: After orthotopic cardiac xenotransplantation, the combination of both the inflammatory responses to the exposure of a recipient to the xenogeneic organ and the use of cardiopulmonary bypass has been assumed to cause detrimental side effects. These have been described not only to affect the transplanted organ (heart) itself, but also the recipient's lungs. In this article, we summarize how these possible detrimental processes can be minimized or even avoided. Methods: Data from eight pig‐to‐baboon orthotopic cardiac xenotransplantation experiments were analyzed with a special focus on early (within the first week) postoperative organ dysfunction and systemic inflammatory responses. Non‐ischemic heart preservation and the careful management of the heart‐lung machine were deemed essential to guarantee not only the immediate function of the transplanted xenogeneic organ but also the prompt recovery of the recipient. Results: After weaning from cardiopulmonary bypass, very low catecholamine amounts were needed to ensure an adequate pump function and cardiac output. Central venous oxygen saturation and serum lactate levels remained within normal ranges. All animals were successfully weaned from ventilation within the first postoperative hours. Serum parameters of the transplants and native kidneys and livers were initially slightly elevated or always normal, as were hemoglobin, LDH, and platelet measurements. Markers of systemic inflammation, C‐reactive protein, and IL‐6 were slightly elevated, but the reactions caused no lasting damage. Conclusion: Consistent short‐term and long‐term results were achieved after orthotopic cardiac pig‐to‐baboon transplantation without detrimental inflammatory responses or signs of multiorgan failure. In comparison to allogeneic procedures, non‐ischemic heart preservation was important for successful immediate organ function, as was the management of the heart‐lung machine. Thus, we believe that genetically modified porcine hearts are ready for use in the clinical setting. [ABSTRACT FROM AUTHOR]

A number of reviews have been written recently celebrating the 25th anniversary of the birth of Dolly the cloned sheep and the effect this breakthrough has had on various fields of research. However, arguably the biggest impact Dolly has had is on the field of xenotransplantation, described here based on our own experience and that of others. [ABSTRACT FROM AUTHOR]

In this commentary, we present a brief history of the development of a national regulatory framework for xenotransplantation clinical research in Australia, including the reasons behind the imposition of a 5‐year moratorium in 2005 and its subsequent lifting. We conclude with a summary of current relevant guidelines and standards. [ABSTRACT FROM AUTHOR]

Background: Endothelial protein C receptor (EPCR) plays an anticoagulant and anti‐inflammatory role by promoting the activation of protein C by thrombin bound to thrombomodulin (TBM). Incompatibility between pig TBM and human/primate thrombin is thought to contribute to dysregulated coagulation in pig‐to‐primate organ xenografts, and expression of human TBM (hTBM) in pigs has shown benefit in preclinical models. However, it is not known whether there are incompatibilities—or molecular barriers—between endogenous pig EPCR (pEPCR) and transgenically expressed human TBM. Aim: To clone and express pEPCR, and determine its function in the human protein C pathway in vitro. Methods: Pig endothelial protein C receptor cDNA was generated from pig lung RNA by RT‐PCR. Primate COS‐7 transfectants expressing various combinations of human and pig TBM and EPCR were incubated with human thrombin and human protein C, and tested for TBM cofactor activity. Results: The predicted protein sequence of pEPCR shared 72.3% amino acid sequence identity with hEPCR, and residues critical for protein C binding were conserved. COS‐7 cells transfected with hEPCR, pEPCR or vector showed minimal TBM cofactor activity (0.13 ± 0.04, 0.13 ± 0.02 and 0.14 ± 0.06 U, respectively). The cofactor activity of hTBM‐transfected cells (1.18 ± 0.29 U) was 8‐fold higher than vector‐transfected cells (P =.004) and further increased 4‐fold and 3‐fold by co‐transfection with hEPCR (5.01 ± 1.12 U, P =.004) or pEPCR (3.73 ± 0.65 U, P =.003), respectively. Conclusions: Our data show that pEPCR is largely compatible with the human TBM/thrombin complex, when expressed on COS‐7 cells in vitro, promoting the activation of human protein C. These findings suggest that endogenous pEPCR will enhance the activity of transgenic hTBM in the xenograft setting. [ABSTRACT FROM AUTHOR]

Abstract: The last few years have seen significant progress in xenotransplantation. Porcine xenograft survival in preclinical models continues to improve, accompanied by the adjustment of immunosuppression to more clinically realistic levels. The rapid uptake of CRISPR/Cas9 technology has accelerated the generation of new knock‐in and knockout pigs, including animals null for the endogenous retrovirus PERV. This brief review presents a personal view of recent developments in the field. [ABSTRACT FROM AUTHOR]

The International Xenotransplantation Association has updated its original 'Consensus Statement on Conditions for Undertaking Clinical Trials of Porcine Islet Products in Type 1 Diabetes,' which was published in Xenotransplantation in 2009. This update is timely and important in light of scientific progress and changes in the regulatory framework pertinent to islet xenotransplantation. Except for the chapter on 'informed consent,' which has remained relevant in its 2009 version, all other chapters included in the initial consensus statement have been revised for inclusion in this update. These chapters will not provide complete revisions of the original chapters; rather, they restate the key points made in 2009, emphasize new and under-appreciated topics not fully addressed in 2009, suggest relevant revisions, and communicate opinions that complement the consensus opinion. Chapter 1 provides an update on national regulatory frameworks addressing xenotransplantation. Chapter 2 a, previously Chapter 2, suggests several important revisions regarding the generation of suitable source pigs from the perspective of the prevention of xenozoonoses. The newly added Chapter 2b discusses conditions for the use of genetically modified source pigs in clinical islet xenotransplantation. Chapter 3 reviews porcine islet product manufacturing and release testing. Chapter 4 revisits the critically important topic of preclinical efficacy and safety data required to justify a clinical trial. The main achievements in the field of transmission of all porcine microorganisms, the rationale for more proportionate recipient monitoring, and response plans are reviewed in Chapter 5. Patient selection criteria and circumstances where trials of islet xenotransplantation would be both medically and ethically justified are examined in Chapter 6 in the context of recent advances in available and emerging alternative therapies for serious and potentially life-threatening complications of diabetes. It is hoped that this first update of the International Xenotransplantation Association porcine islet transplant consensus statement will assist the islet xenotransplant scientific community, sponsors, regulators, and other stakeholders actively involved in the clinical translation of islet xenotransplantation. [ABSTRACT FROM AUTHOR]

Dysregulation of coagulation and disordered hemostasis are frequent complications in the pig-to-nonhuman primate preclinical xenotransplantation model. The most extreme manifestations are the systemic development of a life-threatening consumptive coagulopathy, characterized by thrombocytopenia and bleeding, which is balanced at the opposite extreme by local complications of graft loss due to thrombotic microangiopathy. The contributing mechanisms include inflammation, vascular injury, heightened innate, humoral and cellular immune responses, and molecular incompatibilities affecting the regulation of coagulation. There also appear to be organ-specific factors that have been linked to vascular heterogeneity. As examples, liver xenografts rapidly induce thrombocytopenia by sequestering human/primate platelets; renal xenografts cause a broader coagulopathy, linked in some cases to reactivation of porcine CMV, whereas cardiac xenografts often succumb to microvascular thrombosis without associated systemic coagulopathy but with local perturbations in fibrinolysis. Overcoming coagulation dysfunction will require a combination of genetic and pharmacological strategies. Deletion of the xenoantigen αGal, transgenic expression of human complement regulatory proteins, and refinement of immunosuppression to blunt the antibody response have all had some impact, without providing a complete solution. More recently, the addition of approaches specifically targeted at coagulation have produced promising results. As an example, heterotopic cardiac xenografts from donors expressing human thrombomodulin have survived for more than a year in immunosuppressed baboons, with no evidence of thrombotic microangiopathy or coagulopathy. [ABSTRACT FROM AUTHOR]

Background Xenotransplantation of porcine organs holds promise of solving the human organ donor shortage. The use of α-1,3-galactosyltransferase knockout (GTKO) pig donors mitigates hyperacute rejection, while delayed rejection is currently precipitated by potent immune and hemostatic complications. Previous analysis by our laboratory suggests that clotting factor VIII (FVIII) inhibitors might be elicited by the structurally restricted xenoantibody response which occurs after transplantation of either pig GTKO/ hCD55/ hCD59/ hHT transgenic neonatal islet cell clusters or GTKO endothelial cells. Methods A recombinant xenoantibody was generated using sequences from baboons demonstrating an active xenoantibody response at day 28 after GTKO/ hCD55/ hCD59/ hHT transgenic pig neonatal islet cell cluster transplantation. Rhesus monkeys were immunized with GTKO pig endothelial cells to stimulate an anti-non-Gal xenoantibody response. Serum was collected at days 0 and 7 after immunization. A two-stage chromogenic assay was used to measure FVIII cofactor activity and identify antibodies which inhibit FVIII function. Molecular modeling and molecular dynamics simulations were used to predict antibody structure and the residues which contribute to antibody-FVIII interactions. Competition ELISA was used to verify predictions at the domain structural level. Results Antibodies that inhibit recombinant human FVIII function are elicited after non-human primates are transplanted with either GTKO pig neonatal islet cell clusters or endothelial cells. There is an apparent increase in inhibitor titer by 15 Bethesda units (Bu) after transplant, where an increase greater than 5 Bu can indicate pathology in humans. Furthermore, competition ELISA verifies the computer modeled prediction that the recombinant xenoantibody, H66K12, binds the C1 domain of FVIII. Conclusions The development of FVIII inhibitors is a novel illustration of the potential impact the humoral immune response can have on coagulative dysfunction in xenotransplantation. However, the contribution of these antibodies to rejection pathology requires further evaluation because 'normal' coagulation parameters after successful xenotransplantation are not fully understood. [ABSTRACT FROM AUTHOR]

Background Promising developments in porcine islet xenotransplantation could resolve the donor pancreas shortage for patients with type 1 diabetes. Using α1,3-galactosyltransferase gene knockout (GTKO) donor pigs with multiple transgenes should extend xenoislet survival via reducing complement activation, thrombus formation, and the requirement for exogenous immune suppression. Studying the xenoantibody response to GTKO/ hCD55/ hCD59/ hHT islets in the pig-to-baboon model, and comparing it with previously analyzed responses, would allow the development of inhibitory reagents capable of targeting conserved idiotypic regions. Methods We generated IgM heavy and light chain gene libraries from 10 untreated baboons and three baboons at 28 days following transplantation of GTKO/ hCD55/ hCD59/ hHT pig neonatal islet cell clusters with immunosuppression. Flow cytometry was used to confirm the induction of a xenoantibody response. IgM germline gene usage was compared pre- and post-transplant. Homology modeling was used to compare the structure of xenoantibodies elicited after transplantation of GTKO/ hCD55/ hCD59/ hHT pig islets with those induced by GTKO and wild-type pig endothelial cells without further genetic modification. Results IgM xenoantibodies that bind to GTKO pig cells and wild-type pig cells were induced after transplantation. These anti-non-Gal antibodies were encoded by the IGHV3-66*02 (Δ28%) and IGKV1-12*02 (Δ25%) alleles, for the immunoglobulin heavy and light chains, respectively. IGHV3-66 is 86.7% similar to IGHV3-21 which was elicited by rhesus monkeys in response to GTKO endothelial cells. Heavy chain genes most similar to IGHV3-66 were found to utilize the IGHJ4 gene in 85% of V-D regions analyzed. However, unlike the wild-type response, a consensus complementary determining region 3 was not identified. Conclusions Additional genetic modifications in transgenic GTKO pigs do not substantially modify the structure of the restricted group of anti-non-Gal xenoantibodies that mediate induced xenoantibody responses with or without immunosuppression. The use of this information to develop new therapeutic agents to target this restricted response will likely be beneficial for long-term islet cell survival and for developing targeted immunosuppressive regimens with less toxicity. [ABSTRACT FROM AUTHOR]

Xenotransplantation using pigs as donors offers the possibility of eliminating the chronic shortage of donor kidneys, but there are several obstacles to be overcome before this goal can be achieved. Preclinical studies have shown that, while porcine renal xenografts are broadly compatible physiologically, they provoke a complex rejection process involving preformed and elicited antibodies, heightened innate immune cell reactivity, dysregulated coagulation, and a strong T cell-mediated adaptive response. Furthermore, the susceptibility of the xenograft to proinflammatory and procoagulant stimuli is probably increased by cross-species molecular defects in regulatory pathways. To balance these disadvantages, xenotransplantation has at its disposal a unique tool to address particular rejection mechanisms and incompatibilities: genetic modification of the donor. This review focuses on the pathophysiology of porcine renal xenograft rejection, and on the significant genetic, pharmacological, and technical progress that has been made to prolong xenograft survival. [ABSTRACT FROM AUTHOR]

Background We investigated whether graft produced anti-human CD2, mediated by adenovirus (Adv) transduction of pig neonatal islet cell clusters ( pNICC), would protect xenografts in a humanized mouse model from immune attack and whether such immunosuppression would remain local. Methods A mouse anti-human CD2 Ab (CD2hb11) previously generated by us was genetically engineered to produce chimeric and humanized versions. The three forms of CD2hb11 were named dilimomab (mouse), diliximab (chimeric) and dilizumab (humanized). All 3 forms of CD2hb11 Ab were tested for their ability to bind CD3+ human T cells and to inhibit a human anti-pig xenogeneic mixed lymphocyte reaction (MLR). They were administered systemically in a humanized mouse model in order to test their ability to deplete human CD3+ T cells and whether they induced a cytokine storm. An adenoviral vector expressing diliximab was generated for transduction of p NICC. Humanized mice were transplanted with either control-transduced p NICC or diliximab-transduced p NICC and human T cells within grafts and spleens were enumerated by flow cytometry. Results Dilimomab and diliximab inhibited a human anti-pig xenogeneic response but dilizumab did not. All 3 forms of CD2hb11 Ab bound human T cells in vitro though dilimomab and diliximab exhibited 300-fold higher avidity than dilizumab. All 3 anti-CD2 Abs could deplete human CD3+ T cells in vivo in a humanized mouse model without inducing upregulation of activation markers or significant release of cytokines. Humanized mice transplanted with diliximab-transduced p NICC afforded depletion of CD3+ T cells at the graft site leaving the peripheral immune system intact. Conclusions Local production of a single Ab against T cells can reduce graft infiltration at the xenograft site and may reduce the need for conventional, systemic immunosuppression. [ABSTRACT FROM AUTHOR]

Background Activation of the clotting cascade is central in acute xenograft rejection ( AHXR) that occurs when pig organs are transplanted into primates. The coagulopathy reported in this model is a very complex process that involves simultaneously coagulation factors, platelets and phospholipid-bearing cells (i.e., leukocytes, red blood cells, and endothelial cells). Choosing whole blood for coagulation analysis theoretically appears more favorable compared with plasma. Whole blood rotation thromboelastometry ( ROTEM®) is a point-of-care global coagulation analyzer able to evaluate the characteristics of clot formation and lysis by dynamic monitoring. The aim of this study was to record thromboelastographic profiles, performed by ROTEM®, in a series of immunosuppressed nephrectomized primates that received a life-supporting kidney. Methods Of the eight primates, n = 4 received a pig kidney transgenic for human decay-accelerating factor ( hDAF/ Gal+); n = 2, an α 1,3-galactosyltransferase gene-knockout ( GT- KO) pig kidney transgenic for human CD39, CD55, CD59 and fucosyltransferase ( HTF); and n = 2, a GT- KO pig kidney transgenic for hDAF. Blood samples were collected before and at least once per week after transplantation till euthanasia. Intrinsic ( INTEM) and extrinsic ( EXTEM) coagulation pathways and the function of fibrinogen ( FIBTEM) were evaluated. Thromboelastographic parameters considered were clotting time ( CT, seconds) and clot formation time ( CFT, seconds) in INTEM and EXTEM and maximum clot firmness ( MCF, mm) in FIBTEM. The correlations between CT in INTEM and activated partial thromboplastin time ( aPTT), CT in EXTEM and PT, CFT in INTEM and EXTEM, and platelet counts and MCF in FIBTEM and fibrinogen plasma levels were also considered. Results In all animals, thromboelastographic profiles showed progressive prolongation of CT (activation of coagulative cascade) in INTEM. A close correspondence was observed between (i) the prolongation of the CFT values (propagation of clot formation), both in INTEM and EXTEM, and the decrease in platelet counts; (ii) the reduction in MCF values (clot firmness) in FIBTEM and the decrease in fibrinogen plasma levels. No concordance between CT in INTEM and aPTT and between CT in EXTEM and PT was observed. Conclusions Our study demonstrated that ROTEM® analyzer could be a useful and complementary tool to study the consumptive coagulopathy, either 'compensated' or 'non-compensated,' that takes place when transgenic pig kidneys are transplanted into primates. Larger and prospective studies are needed to confirm our results and to evaluate the role of ROTEM® to guide the management of consumptive coagulopathy in order to prolong the survival of the transplanted organ. [ABSTRACT FROM AUTHOR]

Schmelzle M, Cowan PJ, Robson SC. Which anti-platelet therapies might be beneficial in xenotransplantation? Xenotransplantation 2011; 18: 79-87. © 2011 John Wiley & Sons A/S. Xenotransplantation could provide an unlimited and elective supply of grafts, once mechanisms of graft loss and vascular injury are better understood. The development of α-1,3-galactosyltransferase gene-knockout (GalT-KO) swine with the removal of a dominant xeno-antigen has been an important advance; however, delayed xenograft and acute vascular reaction in GalT-KO animals persist. These occur, at least in part, because of humoral reactions that result in vascular injury. Intrinsic molecular incompatibilities in the regulation of blood clotting and extracellular nucleotide homeostasis between discordant species may also predispose to thrombophilia within the vasculature of xenografts. Although limited benefits have been achieved with currently available pharmacological anti-thrombotics and anti-coagulants, the highly complex mechanisms of platelet activation and thrombosis in xenograft rejection also require potent immunosuppressive interventions. We will focus on recent thromboregulatory approaches while elucidating appropriate anti-platelet mechanisms. We will discuss potential benefits of additional anti-thrombotic interventions that are possible in transgenic swine and review recent developments in pharmacological anti-platelet therapy. [ABSTRACT FROM AUTHOR]

Fisicaro N, Londrigan SL, Brady JL, Salvaris E, Nottle MB, O'Connell PJ, Robson SC, d'Apice AJF, Lew AM, Cowan PJ. Versatile co-expression of graft-protective proteins using 2A-linked cassettes. Xenotransplantation 2011; 18: 121-130. © 2011 John Wiley & Sons A/S. Expression of multiple graft-protective proteins targeted to different locations (i.e., intracellular, cell surface, and secreted) has become an increasingly important goal in xenotransplantation. The 2A 'ribosome skip' signal is used as a linker to enable transgene co-expression, but some studies have shown that post-translational modification and trafficking of 2A-linked proteins may be adversely affected depending on their position relative to 2A. We tested whether several relevant proteins, subject to a range of processing and localization mechanisms, could be efficiently co-expressed using the 2A system. Six expression cassettes were constructed, each containing up to four 2A-linked open reading frames, encoding combinations of human CD55, thrombomodulin (TBM), CD39, CTLA4-Ig and hygromycin resistance. Each linker incorporated a furin cleavage site to remove the carboxy-terminal extension that remains on upstream proteins after 2A processing. The cassettes were used to produce vectors for transfection, adenoviral transduction and transgenesis. Expression was detected by flow cytometry and/or Western blotting. All proteins were expressed in the appropriate location following transient transfection of COS-7 cells, irrespective of the number of linked genes. The percentage of stable transfectants expressing a linked gene was increased 10-fold (from 4-5% to 58-67%) by incorporating the hygromycin resistance gene into the cassette. Stable transfection of transgenic GalT KO pig fibroblasts with a hygromycin- TBM-CD39 construct resulted in surface expression of both TBM and CD39 by the majority of hygromycin-resistant cells. Expression was maintained after flow cytometric sorting and expansion. Adenoviral transduction of NIT-1 mouse insulinoma cells with a TBM-CD39 construct resulted in strong expression of both genes on the cell surface. Mice transgenic for 3-gene (CD55- TBM-CD39) or 4-gene (CD55- TBM-CTLA4Ig-CD39) constructs expressed all genes except CD55. These results confirm the versatility of the 2A system, and demonstrate that careful construct design can minimize potential problems with post-translational modification and trafficking. In addition, incorporation of a selection marker into the 2A-linked chain can dramatically increase the proportion of stable transfectants expressing proteins of interest. This provides a powerful method for the rapid modification of existing genetically modified pigs. [ABSTRACT FROM AUTHOR]

The article focuses on the manipulation of the xenotransplantation method to pigs wherein unmatched, untested and immunosuppressing possibilities in the operation are overcome. It notes the potential genetic modification of the pig using the employed technology which include removing particular pig characteristics and adding human characteristics to the pig tissue. It mentions the manipulation of the power of genetic engineering making the pig donor tissue to look like human allograft.

Xenotransplantation using porcine donors holds tremendous promise for alleviating the chronic shortage of human organ donors. However, transplantation from pigs into higher primates triggers a vigorous immune response involving complement activation and thrombosis. Hyperacute rejection can be prevented by using donors in which GalT, the gene responsible for the predominant target of anti‐pig natural antibodies, has been deleted. Unfortunately, the adaptive response to GalT knockout (KO) organs has been difficult to immunosuppress. The focus has shifted to identifying additional genetic modifications that will extend xenograft survival beyond the several months currently achievable. Which gene(s) should be added to the GalT KO background is open to debate. Overexpression of a complement regulatory factor(s) seems a logical first choice, supported by recent in vitro data but not yet validated in preclinical models. The next obvious candidate would be an anticoagulant protein(s) to deal with the dysregulated coagulation that is almost invariably associated with xenograft rejection. Several potentially useful molecules have been identified, although this study is yet to be translated to the pig. Our understanding of the mechanisms of GalT KO xenograft rejection continues to grow, providing a rational basis for tailoring further genetic modification of the donor and immunosuppression of the recipient. [ABSTRACT FROM AUTHOR]

Background: Despite overcoming xenograft hyperacute rejection (HAR), Gal (galactose-α1,3-galactose) expression may not be completely eliminated from the α1,3-galactosyltransferase gene knockout (Gal KO) pig because of alternative galactosyltransferases. Whether low levels of “residual” Gal are still susceptible to either complement fixing or non-complement fixing antibody beyond the HAR barrier remains unknown. Furthermore, it would be impossible to analyze the immune response specific to low-level Gal in a xenograft setting given the multitude of xenoantigens that could induce a recipient response. To investigate this question, we therefore used a skin graft model in BALB/c mice where the sole difference between donor and recipient was the expression of Gal, where rejection is caused by passively administered anti-Gal monoclonal antibody and where HAR does not occur. Methods: Gal expression over time was examined by immunohistochemistry in wildtype-to-Gal KO skin grafts. Graft rejection in response to passively administered anti-Gal monoclonal antibody at early and late time points was studied to determine changes in susceptibility to antibody. To independently test the effect of reduced Gal expression on antibody-mediated rejection, we used two separate lines of α1,2-fucosyltransferase transgenic mice as skin donors in the model. These mice have known reduced but different levels of Gal as determined by flow cytometry on peripheral blood leukocytes. Results: Gal expression on skin grafts diminished with time with a corresponding reduction in susceptibility to antibody-mediated rejection. Skin grafts at day 30 (n = 7) and 150 (n = 11) had a rejection rate of 100% and 45% respectively in response to non-complement fixing anti-Gal antibody administered to the recipient. Similar results were demonstrated with a complement fixing anti-Gal antibody. When α1,2-fucosyltransferase transgenic mice skin was used in the model, the line with lowest level of Gal expression was resistant to antibody-induced rejection with a rate 0% (n = 9) vs. 60% (n = 5) in the alternative line with relatively more Gal expressed but still much less than normal mice. Conclusions: Resistance to anti-Gal antibody-mediated damage in the model was observed in skin grafts 100 to 150 days post-grafting but not earlier and was associated with a reduction in Gal expression. It is possible that below a threshold level of Gal expression, the grafts were not susceptible to anti-Gal antibody. [ABSTRACT FROM AUTHOR]

We report here our experience regarding the production of double or homozygous Gal knockout (Gal KO) pigs by breeding and somatic cell nuclear transfer (SCNT). Large White × Landrace female heterozygous Gal KO founders produced using SCNT were mated with Hampshire or Duroc males to produce a F1 generation. F1 heterozygous pigs were then bred to half-sibs to produce a F2 generation which contained Gal KO pigs. To determine the viability of mating Gal KO pigs with each other, one female F2 Gal KO pig was bred to a half-sib and subsequently a full-sib Gal KO. F1 and F2 heterozygous females were also mated to F2 Gal KO males. All three types of matings produced Gal KO pigs. To produce Gal KO pigs by SCNT, heterozygous F1s were bred together and F2 fetuses were harvested to establish primary cultures of Gal KO fetal fibroblasts. Gal KO embryos were transferred to five recipients, one of which became pregnant and had a litter of four piglets. Together our results demonstrate that Gal KO pigs can be produced by breeding with each other and by SCNT using Gal KO fetal fibroblasts. [ABSTRACT FROM AUTHOR]

There is considerable interest in the viral modification of insulin-producing islets, including porcine islets, in the context of islet xenotransplantation to treat type 1 diabetes. Adenovirus (Adv) gene delivery offers the potential to modify pre-transplant islets for enhanced survival. Modifications include transfer of cytoprotective molecules to ensure islet survival immediately post-transplant, and molecules to dampen the immune system and prevent chronic islet graft rejection. In this study, we compared different promoters (three promiscuous and two tissue-specific promoters) for their efficiency in driving gene expression in neonatal pig islet tissue after Adv delivery. We also compared the efficiency of these promoters in adult islets from mouse and human pancreata. We observed that the promiscuous cytomegalovirus promoter was the most potent, eliciting high luciferase expression in neonatal pig islets, as well as in human and mouse islets. In contrast, the mammalian EF1- α promoter educed comparatively intermediate gene expression. The mouse major histocompatibility complex class I promoter H-2Kb and the pancreatic-specific promoters insulin and human pdx-1 (area II) performed poorly in islets from all three species. This has important implications for the generation of modified neonatal pig islets for transplantation into humans. [ABSTRACT FROM AUTHOR]

Acute humoral rejection remains the major barrier to long-term pig-to-primate xenograft survival, and microvascular thrombosis is a critical element of the rejection process. It appears that persistent endothelial cell activation and injury, by even low levels of anti-graft antibodies, eventually overwhelm the cellular anticoagulant defences and promote the development of thrombotic microangiopathy. Porcine endothelium may be particularly vulnerable because of cross-species molecular incompatibilities affecting the function of thrombomodulin and possibly TFPI. Recent data from small animal models suggest that transgenic overexpression of anti-thrombotic molecules on xenograft endothelium is capable of inhibiting intravascular thrombosis and preventing acute humoral rejection. In conjunction with existing genetic modifications (e.g. Gal KO, hDAF), this is a promising strategy to move xenotransplantation to the clinic. [ABSTRACT FROM AUTHOR]

Abstract: It is highly likely that successful pig-to-human xenotransplantation of vascularized organs will require genetic modification of the donor pig, and in particular of donor vascular endothelium. Promoters are generally tested in transgenic mice before generating transgenic pigs. Several promoters have been used to drive endothelial cell-specific expression in mice but none have yet been tested in pigs. We compared the promoters of three human genes that are predominantly expressed in vascular endothelium: intercellular adhesion molecule 2 (ICAM-2), platelet endothelial cell adhesion molecule 1 (PECAM-1) and endoglin. Expression of human complement regulatory proteins (hCRPs), directed by each of the promoters in mice, was largely restricted to vascular endothelium and leukocyte subpopulations. However, expression from the PECAM-1 promoter was weak in liver and non-uniform in the small vessels of heart, kidney, and lung. Conversely, expression from the endoglin promoter was consistently strong in the small vessels of these organs but was absent in larger vessels. The ICAM-2 promoter, which produced strong and uniform endothelial expression in all organs examined, was therefore used to generate hCRP transgenic pigs. Leukocytes from 57 pigs containing at least one intact transgene were tested for transgene expression by flow cytometry. Forty-seven of these transgenic pigs were further analyzed by immunohistochemical staining of liver biopsies, and 18 by staining of heart and kidney sections. Only two of the pigs showed expression, which appeared to be restricted to vascular endothelium in heart and kidney but was markedly weaker than in transgenic mice produced with the same batch of DNA. Thus, in this case, promoter performance in mice and pigs was not equivalent. The weak expression driven by the human ICAM-2 promoter in pigs relative to mice suggests the need for additional regulatory elements to achieve species-specific gene expression in pigs. [ABSTRACT FROM AUTHOR]

Xenotransplantation offers a potential solution to the shortfall in donor organs for human transplantation. This review describes the barriers to xenotransplantation and the progress that has been made towards making it a clinical reality. Data from preclinical pig-to-primate cardiac and pulmonary xenografts are highlighted. (Heart, Lung and Circulation 2002; 11: 32-41). [ABSTRACT FROM AUTHOR]

Gene editing using clustered regularly interspaced short palindromic repeats/Cas9 has great potential for improving the compatibility of porcine organs with human recipients. However, the risk of detrimental off‐target mutations in gene‐edited pigs remains largely undefined. We have previously generated GGTA1 knock‐in pigs for xenotransplantation using FokI‐dCas9, a variant of Cas9 that is reported to reduce the frequency of off‐target mutagenesis. In this study, we used whole genome sequencing (WGS) and optimized bioinformatic analysis to assess the fidelity of FokI‐dCas9 editing in the generation of these pigs. Genomic DNA was isolated from porcine cells before and after gene editing and sequenced by WGS. The genomic sequences were analyzed using GRIDSS variant‐calling software to detect putative structural variations (SVs), which were validated by PCR of DNA from knock‐in and wild‐type pigs. Platypus variant‐calling software was used to detect single‐nucleotide variations (SNVs) and small insertions/deletions (indels). GRIDSS analysis confirmed the precise integration of one copy of the knock‐in construct in the gene‐edited cells. Three additional SVs were detected by GRIDSS: deletions in intergenic regions in chromosome 6 and the X chromosome and a duplication of part of the CALD1 gene on chromosome 18. These mutations were not associated with plausible off‐target sites, and were not detected in a second line of knock‐in pigs generated using the same pair of guide RNAs, suggesting that they were the result of background mutation rather than off‐target activity. Platypus identified 1375 SNVs/indels after quality filtering, but none of these were located in proximity to potential off‐target sites, indicating that they were probably also spontaneous mutations. This is the first WGS analysis of pigs generated from FokI‐dCas9‐edited cells. Our results demonstrate that FokI‐dCas9 is capable of high‐fidelity gene editing with negligible off‐target or undesired on‐target mutagenesis. [ABSTRACT FROM AUTHOR]

The article discusses the third World Health Organization (WHO) Global conference on regulatory requirements for Xenotransplantation clinical trials that was held in Changsha, China on 12–14 December, 2018. It mentions recent advances in the development of technologies for solid organ xenotransplantation; and also mentions Dr. Megan Sykes reviewed the current technologies and studies in the development of tolerance and use of regulatory T cells.

In this article, the authors discuss the study on the production of galactosyltransferase knockout (Gal KO) pigs expressing multiple transgenes in one generation using porcine embryonic stem cells (pESCs) to advance xenotransplantation research. The authors say that they employed new approach in isolating pESCs by using expanded blastocysts and added growth factors and additives in place of foetal calf serum. Benefits of pESCs including removal of antibiotic resistance genes are also discussed.

The number of donated human organs and tissues for patients with terminal organ failure falls far short of the need. Alternative sources, such as organs and tissues from animals, are therefore urgently required. During the past few years, major progress has been made in the development of genetically multi-modified donor pigs, and their organs have been shown to be safe and efficacious in life-supporting transplantation models into non-human primates, paving the way to clinical xenotransplantation studies. Here, we summarize recent developments in pig genome engineering and discuss efforts to develop the optimum donor pig for xenotransplantation. In addition, we speculate on how many genetic modifications may be required for initial xenotransplantation clinical trials. [ABSTRACT FROM AUTHOR]

Background: Xenotransplantation could provide a solution to the donor shortage that is currently the major barrier to solid-organ transplantation. The ability to breed pigs with multiple genetic modifications provides a unique opportunity to explore the immunologic challenges of pulmonary xenotransplantation. Methods: Explanted lungs from wild-type and 3 groups of genetically modified pigs were studied: (i) α1,3-galactosyltransferase gene knockout (GTKO); (ii) GTKO pigs expressing the human complementary regulatory proteins CD55 and CD59 (GTKO/CD55-59); and (iii) GTKO pigs expressing both CD55-59 and CD39 (GTKO/CD55-59/CD39). The physiologic, immunologic and histologic properties of porcine lungs were evaluated on an ex vivo rig after perfusion with human blood. Results: Lungs from genetically modified pigs demonstrated stable pulmonary vascular resistance and better oxygenation of the perfusate, and survived longer than wild-type lungs. Physiologic function was inversely correlated with the degree of platelet sequestration into the xenograft. Despite superior physiologic profiles, lungs from genetically modified pigs still showed evidence of intravascular thrombosis and coagulopathy after perfusion with human blood. CONCLUSIONS: The ability to breed pigs with multiple genetic modifications, and to evaluate lung physiology and histology in real-time on an ex vivo rig, represent significant advances toward better understanding the challenges inherent to pulmonary xenotransplantation. [ABSTRACT FROM AUTHOR]