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Genetic modification of the source pig offers the opportunity to improve the engraftment and survival of islet xenografts. The type of modification can be tailored to the transplant setting; for example, intraportal islet xenografts have been shown to benefit from the expression of anticoagulant and anti-inflammatory transgenes, whereas cytoprotective transgenes are probably more relevant for encapsulated islets. The rapid development of pig genetic engineering, particularly with the introduction of genome editing techniques such as CRISPR-Cas, has accelerated the generation of new pig lines with multiple modifications. With pre-clinical testing in progress, it is an opportune time to consider any implications of genetic modification for the conditions for undertaking clinical trials. Obviously, the stringent requirements to fulfill designated pathogen-free status that are applied to wild-type pigs will apply equally to genetically modified ( GM) source pigs. In addition, it is important from a safety perspective that the genetic modifications are characterized at the molecular level (e.g., integration site, absence of off-target mutations), the phenotypic level (e.g., durability and stability of transgene expression), and the functional level (e.g., protection of islets in vitro or in vivo, absence of detrimental effects on insulin secretion). The assessment of clinical trial protocols using GM pig islets will need to be performed on a case-by-case basis, taking into account a range of factors including the particular genetic modification(s) and the site and method of delivery. [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]

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]

Background Human corneal allografting is an established procedure to cure corneal blindness. However, a shortage of human donor corneas as well as compounding economic, cultural, and organizational reasons in many countries limit its widespread use. Artificial corneas as well as porcine corneal xenografts have been considered as possible alternatives. To date, all preclinical studies using de-cellularized pig corneas have shown encouraging graft survival results; however, relatively few studies have been conducted in pig to non-human primate ( NHP) models, and particularly using genetically engineered donors. Methods In this study, we assessed the potential benefit of using either hCTLA4-Ig transgenic or α1,3-Galactosyl Transferase (GT) Knock-Out (KO) plus transgenic hCD39/ hCD55/ hCD59/fucosyl-transferase pig lines in an anterior lamellar keratoplasty pig to NHP model. Results Corneas from transgenic animals expressing hCTLA4-Ig under the transcriptional control of a neuron-specific enolase promoter showed transgene expression in corneal keratocytes of the stroma and expression was maintained after transplantation. Although a first acute rejection episode occurred in all animals during the second week post-keratoplasty, the median final rejection time was 70 days in the hCTLA4-Ig group vs. 21 days in the wild-type (WT) control group. In contrast, no benefit for corneal xenograft survival from the GTKO/transgenic pig line was found. At rejection, cell infiltration in hCTLA4Ig transgenic grafts was mainly composed of macrophages with fewer CD3+ CD4+ and CD79+ cells than in other types of grafts. Anti-donor xenoantibodies increased dramatically between days 9 and 14 post-surgery in all animals. Conclusions Local expression of the hCTLA4-Ig transgene dampens rejection of xenogeneic corneal grafts in this pig-to-NHP lamellar keratoplasty model. The hCTLA4-Ig transgene seems to target T-cell responses without impacting humoral responses, the control of which would presumably require additional peripheral immunosuppression. [ABSTRACT FROM AUTHOR]

Background B-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. Methods The 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. Results After 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-ss DNA, 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. Conclusions This 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. [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 Immunological and histopathological features in pig-to-primate renal xenotransplantation are widely studied. Only limited data have been reported about clinicopathological findings in primate recipients of life-supporting renal xenografts. In human medicine, proteinuria represents a common complication in kidney transplantation and is associated with impaired graft survival. The detection of low molecular weight proteins of tubular origin is considered an early method for predicting potential graft rejection. In this study, the presence and the significance of quantitative and qualitative proteinuria were evaluated in xenotransplanted non-human primates in which kidney function was supported only by the transplanted organ. Methods Eight bilaterally nephrectomized cynomolgus monkeys ( Macaca fascicularis) were transplanted with a single kidney from α1,3-galactosyltransferase gene-knockout ( GTKO) pigs transgenic for human CD39, CD55, CD59, and α1,2-fucosyltransferase. In addition to hematological and biochemical analyses, quantitative and qualitative analysis of proteinuria was evaluated by urinary protein-to-creatinine ratio ( UPC ratio) and sodium dodecyl sulfate-agarose gel electrophoresis ( SDS- AGE), respectively. Results The main hematological and biochemical changes recorded after transplantation were a progressive anemia and a severe and progressive decrease in total proteins. In urine samples, the UPC ratio was low before transplantation and increased after transplantation. Similarly, SDS- AGE was negative before transplantation, but bands consistent with mixed (i.e., tubular and glomerular) proteinuria were observed in all samples collected post-transplantation. Conclusions The study of clinicopathological changes in cynomolgus monkey renal xenograft recipients provides a valid help in monitoring the health conditions in the post-transplant period. Moreover, the evaluation of UPC ratio and the use of SDS- AGE technique in urine samples of cynomolgus monkey renal xenograft recipients may be considered a valid, inexpensive, and less time-consuming method than more sophisticated techniques in monitoring proteinuria. Proteinuria and presence of low molecular weight ( LMW) proteins were consistently found in urine after transplantation, independent of fluctuations in renal function. [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]

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]

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.Immunology and Cell Biology (2009) 87, 203–208; doi:10.1038/icb.2008.107; published online 20 January 2009 [ABSTRACT FROM AUTHOR]

Rejected pig-to-primate organ xenografts almost invariably exhibit significant microvascular thrombosis, believed to be due in part to several molecular incompatibilities affecting the regulation of coagulation. In this study, we tested one such proposed incompatibility: whether there is, at least in part, a functional incompatibility in pig tissue factor pathway inhibitor (TFPI) that impedes binding of human factor Xa and regulation of human tissue factor-initiated coagulation. TFPIα cDNA was cloned from pig aortic endothelial cells and found to encode a 279-residue mature protein with 79% overall identity to human TFPIα, increasing to 88 to 90% in the functional Kunitz-1 and Kunitz-2 domains. Transfected primate cells expressing equivalent levels of GPI-linked pig or human TFPIα were assayed for binding of human factor Xa and inhibition of the human factor VIIa/tissue factor complex. The activity of the expressed pig anticoagulant was equivalent to that of the human protein in both measures of TFPI function in these systems. These data indicate that there are no apparent incompatibilities between recombinant pig TFPI and the human tissue factor pathway. Other factors must account for the thromboregulatory failure of pig endothelium and aberrant tissue factor activity in xenograft rejection. [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]

Pig-to-primate vascularized xenografts undergo hyperacute rejection (HAR). This results from pre-formed xenoreactive antibodies directed against galactose-α,3-galactose (αGal) in the donor organ and activation of the complement cascade. We describe an in vivo murine model of HAR using a BALB/c mice system devoid of histocompatibility or complement differences between donor and recipient to investigate in isolation, the effects of αGal epitope and anti-αGal antibody interactions in causing rejection of vascularized heart transplants. Gal KO mice were immunized with rabbit red blood cell membranes to induce high anti-αGal antibody titers that were predominantly IgM by ELISA (enzymelinked immunosorbent assay). When αGal-expressing mice hearts were transplanted heterotopically into these recipients (n=12), 67% of grafts rejected within 24 h, the majority within 16 h with histological features of HAR. In contrast, none of the grafts in the non-immunized Gal KO recipient control group (n=11) underwent HAR. Interestingly, approximately 50% of the remaining grafts in both the immunized and non-immunized Gal KO recipient group were rejected between 7 and 27 days by a rejection process characterized by a dense infiltrate of macrophage/monocytes, perivascular cuffing and tissue destruction similar to recent descriptions of delayed xenograft rejection (DXR). In addition, some grafts (21.5%) continued to survive in the immunized Gal KO recipients despite the presence of anti-αGal antibody and normal complement activity and these showed well-preserved myocardium when harvested whilst still functioning well at days 30 or 90. No rejection was seen when Gal KO donors were used in this system (n=4), nor when αGal-expressing BALB/c hearts were transplanted into αGal-expressing BALB/c recipients (n=5). This in vivo small animal model offers the opportunity to test a variety of strategies to overcome HAR prior to more resource... [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]

Examines the effects of combined CTLA14-crystallized fragments and anti-CD40L monoclonal antibody treatment on islet xenograft tolerance. Comparison of rat islet and fetal pig pancreas xenotransplantation survival; Measurement of T cell response after xenografting.