Improved Xenograft Survival Research Articles

The Time Has Come: The Case for Initiating Pilot Clinical Trials of Pig Kidney Xenotransplantation.

In vitro studies indicate that kidney transplantation from gene-edited pigs in which expression of all three of the known glycan xenoantigens has been deleted may be more challenging in nonhuman primates (NHPs) than it will be in human recipients. Furthermore, pig-to-human xenotransplantation offers several other advantages - (i) the patient can communicate with the surgical team; (ii) recipient microbiological monitoring and environment will be clinical-grade; and (iii) sophisticated graft monitoring and imaging techniques, (v) therapeutic interventions, e.g., dialysis, plasmapheresis, and (v) intensive care can be deployed that are not easily available in NHP laboratory models. We suggest, therefore, that progress to develop safe, informative human clinical trials will be accelerated if pilot clinical cases are initiated. The selection of patients for kidney xenotransplantation can include those who are at high risk of dying imminently, e.g., those experiencing increasing vascular access challenges with no realistic alternative therapy available, and those who have been accepted onto the waitlist for an allograft, but who are unlikely ever to receive one. Patients with an increased risk of dying include those with (i) age >60 years, (ii) blood groups O or B, and (iii) diabetic nephropathy. UNOS data indicate that an average of 25 patients on the kidney waitlist in the USA die or are removed from the list every day (i.e., >9,000 each year). Given the improved xenograft survival observed in preclinical studies, we suggest that it is time to plan a small pilot clinical trial for healthy dialysis patients who understand the risks and potential benefits of kidney xenotransplantation.

Co-expression of HLA-E and HLA-G on genetically modified porcine endothelial cells attenuates human NK cell-mediated degranulation.

Natural killer (NK) cells play an important role in immune rejection in solid organ transplantation. To mitigate human NK cell activation in xenotransplantation, introducing inhibitory ligands on xenografts via genetic engineering of pigs may protect the graft from human NK cell-mediated cytotoxicity and ultimately improve xenograft survival. In this study, non-classical HLA class I molecules HLA-E and HLA-G were introduced in an immortalized porcine liver endothelial cell line with disruption of five genes (GGTA1, CMAH, β4galNT2, SLA-I α chain, and β-2 microglobulin) encoding three major carbohydrate xenoantigens (αGal, Neu5Gc, and Sda) and swine leukocyte antigen class I (SLA-I) molecules. Expression of HLA-E and/or HLA-G on pig cells were confirmed by flow cytometry. Endogenous HLA-G molecules as well as exogenous HLA-G VL9 peptide could dramatically enhance HLA-E expression on transfected pig cells. We found that co-expression of HLA-E and HLA-G on porcine cells led to a significant reduction in human NK cell activation compared to the cells expressing HLA-E or HLA-G alone and the parental cell line. NK cell activation was assessed by analysis of CD107a expression in CD3-CD56+ population gated from human peripheral blood mononuclear cells. CD107a is a sensitive marker of NK cell activation and correlates with NK cell degranulation and cytotoxicity. HLA-E and/or HLA-G on pig cells did not show reactivity to human sera IgG and IgM antibodies. This in vitro study demonstrated that co-expression of HLA-E and HLA-G on genetically modified porcine endothelial cells provided a superior inhibition in human xenoreactive NK cells, which may guide further genetic engineering of pigs to prevent human NK cell mediated rejection.

Upregulation of CD80 on glomerular podocytes plays an important role in development of proteinuria following pig-to-baboon xeno-renal transplantation - an experimental study.

We have previously reported that co-transplantation of the kidney with vascularized donor thymus from α-1,3-galactosyltransferase gene knockout pigs with an anti-CD154 with rituximab-based regimen led to improved xenograft survival in baboons with donor-specific unresponsiveness. However, nephrotic syndrome emerged as a complication in which the glomeruli showed mild mesangial expansion with similarities to minimal change disease (MCD) in humans. Since MCD is associated with CD80 expression in glomeruli and elevated urinary excretion, we evaluated a potential role for CD80 in xenograft nephropathy. Study 1 confirmed high urinary CD80 excretion in nephrotic animals with renal xenografts showing CD80 expression in glomeruli. In Study 2, baboons receiving xenografts received CTLA4-Ig once a week from the second postoperative week or no CTLA4-Ig. The non-CTLA4-Ig group developed severe proteinuria with modest mesangial expansion with high urinary excretion of CD80 and documented CD80 expression in glomerular podocytes. All of the recipients in non-CTLA4-Ig groups had to be euthanized before POD 60. In contrast, CTLA4-Ig group showed a marked reduction in proteinuria and survived significantly longer, up to 193 days. These results demonstrate that anti-CD80 targeted therapy represents a promising strategy for reduction of proteinuria following renal xeno-transplantation with improved survival.

Encouraging experience using multi-transgenic xenografts in a pig-to-baboon cardiac xenotransplantation model.

Innovations in transgenic technology have facilitated improved xenograft survival. Additional gene expression appears to be necessary to overcome the remaining immune and biologic incompatibilities. We report for the first time the novel use of six-gene modifications within a pig-to-baboon cardiac xenotransplantation model. Baboons (8-15kg) underwent heterotopic cardiac transplantation using xenografts obtained from genetically engineered pigs. Along with previously described modifications (GTKO, hCD46), additional expression of human transgenes for thromboregulation (endothelial protein C receptor, tissue factor pathway inhibitor, thrombomodulin), complement inhibition (decay accelerating factor), and cellular immune suppression (hCD39, hCD47) was used. Immunosuppression consisted of targeted T-cell and B-cell depletion and conventional anti-rejection agents. Heterotopic cardiac transplantations were performed without complication. Flow cytometry and immunohistochemistry on donor biopsies confirmed transgenic phenotype. In contrast to the prior three-gene generation, significant coagulopathy or consumptive thrombocytopenia has not been observed in the six-gene cohort. As a result, these recipients have experienced decreased bleeding-related complications. Pro-inflammatory responses also appear to be mitigated based on cytokine analysis. Baboons survived the critical 30-day post-operative period when mortality has historically been highest, with no evidence of graft rejection. The inclusion of additional human genes in genetically engineered pigs appears to confer superior xenograft outcomes. Introduction of these genes has not been associated with adverse outcomes. This multifactorial approach to genetic engineering furthers the prospect of long-term cardiac xenograft survival and subsequent clinical application.

Xenotransplantation: Where Are We with Potential Kidney Recipients? Recent Progress and Potential Future Clinical Trials.

Inter-species transplantation, xenotransplantation, is becoming a realistic strategy to solve the organ shortage crisis. Here we focus on seminal publications that have driven research in xenotransplantation, as well as recently published literature and future endeavors. Advances in gene editing technology have allowed for the efficient production of multi-transgenic porcine donors leading improved xenograft survival in baboons, up to 2-years following heterotopic heart xenotransplantation and from weeks to several months following life-supporting kidney xenotransplanation. As technology evolves, additional challenges have arisen, including the development of proteinuria, early graft loss associated with porcine CMV, disparities in organ growth between donors and recipients as well as high-dose continuous immunosuppression requirements. To address these issues, our laboratory developed a tolerance-inducing protocol which has allowed for >6 months survival of a life-supporting kidney with further approaches currently underway to address the challenges mentioned above. Our recent findings, reviewed in this article, led us to develop methods to overcome obstacles, which, in conjunction with the work of others, are promising for future clinical applications of xenotransplantation.

Regulation of Instant Blood Mediated Inflammatory Reaction (IBMIR) in Pancreatic Islet Xeno-Transplantation: Points for Therapeutic Interventions.

Xeno-transplantation of pancreatic islets represents a promising therapeutic alternative for the treatment of type 1 diabetes mellitus. However, potent innate immune responses induced shortly after the transplantation of donor islets to the recipient, comprising the Instant Blood Mediated Immune Reaction (IBMIR), exert detrimental actions on islet graft function. The coagulation and complement cascades together with the leukocyte and platelet populations are the major players in IBMIR. This innate immune attack affects dramatically islet integrity and leads to significant loss of function of the xenograft. In the present review, we focus on the mechanisms contributing to IBMIR components and address therapeutic intervention approaches to limit IBMIR by administering inhibitors in circulation, by coating the islet surface with inhibitors or by generating transgenic donor animals; these approaches could result in improved xenograft survival.

Suppression of NF‐kappaB p65 expression attenuates delayed xenograft rejection

Delayed xenograft rejection (DXR) involves type II vascular endothelial cell (VEC) activation including upregulation of pro-inflammatory genes, which contributes to infiltration into the graft and a complex process of cytokine production. Approaches to prevent DXR have shown limited success. In this study, we modified heart donors using siRNA in an attempt to attenuate DXR and to improve xenograft survival in the mouse-to-rat heterotopic heart transplant model. siRNA technology was used to inhibit NF-kappaB p65 gene expression in vivo in mice. After the donor was transfected with siRNA, the effects of NF-kappaB siRNA on DXR and expression of NF-kappaB and pro-inflammatory genes were evaluated in a concordant mouse-to-rat cardiac xenograft model. Treatment of NF-kappaB siRNA prolonged median heart graft survival time in the recipient rats from 1.7days in a PBS control group to 5.4days in the NF-kappaB siRNA-treated group (P<0.05). Compared with normal mouse hearts, the NF-kappaB p65 mRNA relative levels following siRNA injection in the donors decreased significantly (approximately 70% reduction) in grafts harvested 12h after transplantation. The mRNA levels of VCAM-1, ICAM-1, and interleukin-1 displayed a similar reduction. Histological evaluation using light and electron microscopy showed that damage of endothelial cells after NF-kappaB siRNA treament occured at a later time. Transfection of NF-kappaB p65 siRNA in donor animals can delay the emergence of DXR. This treatment may be used as part of strategies to minimize the complex and multi-faceted rejection responses in vascularized xenografts.

Mechanisms of Gal-knockout pig cell selection for somatic cell nuclear transfer

The Gal alpha (1,3) Gal epitope has been shown to play a significant role in mediating hyperacute rejection of end organs in xenotransplantation. For the past two decades, scientists have experimented with pig cells lacking the alpha-Gal antigen by knocking out the galactyltransferase gene, a method that has shown to improve xenograft survival in pig-to-primate transplants [1–6]. In a recently published article in the Journal of Surgical Research, Reyes et al. share their procedure by which they were able to efficiently select double Gal-knockout (KO) cells for somatic cell nuclear transfer (SCNT), through which they successfully produced healthy Gal-null piglets [7].

Molecular regulation of endothelial cell activation: novel mechanisms and emerging targets

To outline some recently described mechanisms that regulate endothelial cell function and vascular inflammation. A particular focus will be given to protective or adverse regulatory events, at molecular or cellular level, that could provide new therapeutic targets to control endothelial cell dysfunction in the xenotransplantation setting. Targeting protective gene expression to porcine endothelium by genetic modification of the donor is required to improve xenograft survival by controlling endothelial cell activation and death. Increasing evidences indicate the importance of microRNAs in the regulation of endothelial cell function and propose strategies to overcome endothelial cell activation, injury that may apply to xenotransplantation. Recent advances have been made in our understanding of endothelial cell repair, replacement and senescence that could impact on xenograft outcome. Finally, refinements to endothelial cell targeting in vivo also open the way to the design of effective gene transfer in vascularized organs. Xenograft survival in the pig-to-primate experimental model is still limited by humoral vascular xenograft rejection resulting from acute endothelial cell damage. Current knowledge in mechanisms regulating inflammatory gene transcription in endothelial cell such as microRNAs, endothelial cell infection by virus, endothelial cell repair, senescence and replacement processes may help to design new approaches targeting and protecting graft endothelial cell to avoid vascular injury and rejection.

Gene transfer of the adaptor Lnk (SH2B3) prevents porcine endothelial cell activation and apoptosis: implication for xenograft’s cytoprotection

Targeting protective gene expression to porcine endothelium by genetic modification of the donor could improve xenograft survival by controlling cell activation and death. We previously found that, in endothelial cells (EC), the molecular adaptor Lnk (SH2B3) is a negative regulator of cytokine signaling. We also have shown that Lnk is upregulated in pig EC (PAEC) in response to tumor necrosis factor-α (TNF) and xenoreactive natural antibodies (XNA) binding. The present study investigated whether ectopic expression of human Lnk using gene transfer may be efficient to control signaling pathways associated with inflammation and apoptosis in porcine aortic endothelial cells (PAEC). Endothelial cells cultures were established from WT and Gal(-/-) pigs and transduced with a recombinant adenovirus encoding human Lnk. Phenotype and functions of transduced PAEC expressing Lnk were analyzed by flow cytometry, western blot and XNA and complement-dependent assays. The regulatory functions of Lnk toward inflammation were assessed in TNF-activated EC, and the protective functions were tested toward TNF-induced apoptosis and anoïkis. Apoptosis assays included DNA content analysis and caspase-3/7 activity. First, we found that as a result of adenoviral transduction, human Lnk was efficiently and similarly expressed in EC from WT or Gal(-/-) pigs. Lnk expression or EC transduction caused no significant change in the binding of XNA (IgG and IgM) to PAEC and has no effect on complement activation and C5b-9 formation. We demonstrated that expression of human Lnk efficiently inhibits TNF signaling in PAEC and decreases VCAM-1 induction by 46.3 ± 1.2% compared to controls (n = 6, **P < 0.01). Furthermore, expression of Lnk was associated with a significant decrease in the percentage of caspase-3/7-dependent apoptosis caused by TNF in the presence of actinomycin D and also reduces cell death by anoïkis by 25.0 ± 1.9% compared to controls (n = 5, **P < 0.01). Together, these findings indicate that the signaling adaptor Lnk is effective to reduce PAEC activation and apoptosis. Thus, Lnk is a potential candidate for the modulation of signaling pathways to protect vascular EC from inflammation in xenotransplantation.

Aurintricarboxylic acid inhibits endothelial activation, complement activation, and von Willebrand factor secretion in vitro and attenuates hyperacute rejection in an ex vivo model of pig‐to‐human pulmonary xenotransplantation

In the xenotransplantation of vascularized organs, such as the lung, a large area of endothelial cell layer is a big hurdle to be overcome. We investigated the potential protective effect of aurintricarboxylic acid (ATA), a known inhibitor of platelet adhesion, on endothelial damage induced by xenogeneic serum. We also assessed its role in hyperacute xenograft rejection using a porcine ex vivo lung perfusion model. Porcine endothelial cells were incubated with human serum and other inflammatory stimuli. For the evaluation of von Willebrand factor (vWF) secretion and tissue factor (TF) expression, we used human endothelial cells. E-selectin expression, complement activation, TF expression and platelet activation were investigated by flow cytometry. In an ex vivo porcine lung perfusion model, the porcine lungs were perfused with fresh human whole blood: unmodified blood (n = 5), ATA-treated blood (n = 5), and ATA and lepirudin-treated blood (n = 5). Aurintricarboxylic acid significantly inhibited TNF-alpha- or lipopolysaccharide-induced endothelial E-selectin expression in a dose-dependent manner. ATA also prevented human serum induced-E-selectin expression and human monocytic cell adhesion to porcine endothelial cells. Moreover, ATA abolished thrombin-induced vWF secretion as well as complement activation. However, ATA induced endothelial TF expression and platelet activation in vitro. In ex-vivo experiments, ATA treatment improved pulmonary function and attenuated sequestration of leukocytes. Although ATA did not influence thrombin generation, we were able to minimize its activity by adding lepirudin to the blood with ATA. Our study demonstrated in vitro protective effect of ATA on the inhibition of endothelial activation and vWF secretion and confirmed detrimental effect of ATA on induction of endothelial TF and platelet activation. The combination of ATA and lepirudin may act beneficially by preventing coagulation perturbation while maintaining improved xenograft survival.

The effect of soluble complement receptor type 1 on acute humoral xenograft rejection in hDAF‐transgenic pig‐to‐primate life‐supporting kidney xenografts

In pig-to-nonhuman primate solid organ xenotransplantation using organs from donors transgenic for human decay-accelerating factor (hDAF), the main type of rejection is antibody-mediated (acute humoral xenograft rejection, AHXR). This occurs despite the complement-regulatory function of the transgene, neutralization of natural antibodies to Galalpha1-3Gal (Gal) using soluble glycoconjugates, and chronic immunosuppression. As complement components play a major role in graft destruction after antibody binding, we evaluated the efficacy of chronic complement inhibition by soluble complement receptor type 1 (TP10). Life-supporting hDAF-transgenic kidney transplantation was performed in cynomolgus monkeys, using cyclophosphamide induction, and maintenance immunosuppression with cyclosporin A, mycophenolate sodium, and tapering steroids. Rejection was treated with bolus steroid injections: if not successful animals were terminated. Three groups were studied: in group 1 (n=4) GAS914 (a soluble glycoconjugate comprising Gal on a poly-L-lysine backbone) was added before and after transplantation; group 2 (n=2) received GAS914 as in group 1 and in addition TP10 before and after transplantation; in group 3 (n=4) GAS914 was only given before transplantation and TP10 as in group 2. Monitoring included the regular assessment of anti-porcine antibodies, complement activity (soluble C5b-9), therapeutic drug monitoring, and graft histology. Survival in group 1 was 6, 12, 31 and 37 days, respectively, and in all four cases graft histology showed AHXR. The two animals in groups 2 survived 3 and 15 days, respectively, and similarly showed AHXR in graft histology. In group 3 two animals showed AHXR (10 and 37 days survival, respectively), and two others did not show AHXR (20 and 32 days survival, respectively). The diagnosis AHXR included the deposition of complement activation products in the graft, which were present at lower intensity in animals treated with TP10. In all animals GAS914 effectively neutralized circulating anti-Gal antibody. Antibodies were detectable in the circulation of all animals using porcine erythrocytes in a hemolytic assay, although at lower levels than before transplantation. Soluble C5b-9 was not detectable in the circulation of animals receiving TP10, and circulating TP10 concentrations in these animals were in a presumed pharmacologically active range. The inclusion of TP10 in the immunosuppressive protocol does not clearly lead to improved xenograft survival. Despite effective neutralization of anti-Gal antibodies and effective inhibition of systemic complement activity, AHXR was apparent in four of six animals under chronic TP10 treatment, including deposits of complement activation products in the graft. Apparently, effective systemic complement inhibition by TP10 in combination with local complement regulation by the hDAF transgene product does not necessarily result in effective inhibition of complement activation at locations in the xenograft upon binding of anti-porcine antibodies to the grafted endothelium.

Phase-Directed Therapy and Cardiac Xenograft Survival

Xenotransplant rejection is facilitated not only by T cell upregulation but also by endothelial activation and B cell/antibody mechanisms, which standard immunosuppression is unable to overcome and xenorejection ensues. However, therapy directed specifically at each phase of xenorejection may improve xenograft survival. To study this we used a heterotopic cardiac xenotransplant model (Syrian hamster to Lewis rat). Controls had no immunotherapy. Xenorecipients received cyclosporine to restrict T cellular response/development or cyclophosphamide, an antiproliferative, to reduce xenoreactive clones and antibody/complement injury, or anti-TNF antibody to alter cytokine cascades and endothelial activation/inflammation. Further xenorecipients received combinations. While single modalities alone did not enhance survival, combinations appeared to be at least additivein vivo,suggesting that therapy directed at specific phases of xenorejection may prove useful.

Improved Xenograft Survival with Continuous Infusion Deoxyspergualin and RATG

The critical shortage of available donor organs is the major limit to current allogeneic transplantation. Xenografting has the potential to overcome this difficult problem. Suppressing the vigorous rejection response remains a major obstacle to the clinical application of xenografting, 15-Deoxyspergualin (DOSP), a potent new immunosuppressive agent has been shown to be effective in allogeneic and xenogeneic experimental models. This study tests DOSP in combination with rabbit antithymocyte globulin (RATG) in the hamster-to-rat cardiac xenograft. Results show that combination therapy with DOSP/RATG was superior to treatment with either agent alone (p less than .05). Optimal graft prolongation (20.9 days versus control of 3.1 days, p less than .05) was achieved with combination therapy of RATG and DOSP 2.5 mg/kg day-1 by continuous infusion.

Improved xenograft survival with continuous infusion deoxyspergaulin and RATG

Improved xenograft survival with continuous infusion deoxyspergaulin and RATG