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C: Preclinical and clinical xenotransplantation

Research Area C will continue to perform preclinical xenotransplantation experiments and to plan a clinical study with macroencapsulated islets. C1 will further refine the optimal age and maturation stage of NPIs and assess their functionality after transplantation into hyperglycaemic mice; under the kidney capsule for control of glucose homeostasis, and into the anterior ocular chambers for longitudinal in vivo imaging. C3 will test NPIs with different combinations of genetic modifications developed by Project Group B in diabetic NHPs (Macaca fascicularis), initially with an anti-CD154ab based immunosuppressive regimen. In parallel novel immunosuppressive strategies will be tested, first in immune-competent mouse models and then translated to NHPs. A pipeline for isolating NPIs that conforms to GMP standards will be established and a protocol for the first clinical study of non-encapsulated NPI xenotransplantation finalised. C4 has already submitted a clinical study protocol for porcine islets macroencapsulated in the BetaAir® device. As mentioned above, this approach was successful and safe in a diabetic NHP model, but had limited metabolic efficacy. The islet product and the device will therefore both be optimised employing cutting-edge bio-polymer technologies. C7 will extend its successful efforts to generate haemodynamically effective, immunocompatible xenogeneic pulmonary heart valves from GGTA1/CMAH/B4GALNT2-3´ko pigs using advanced decellularisation and deglycosylation techniques. These valves will be systematically investigated for remnant glycan residues and human patients screened for antibodies against such epitopes, providing the basis for (pre)clinical implantation. C8 achieved the world’s first consistent long-term success in orthotopic cardiac xenotransplantation. In six of eight experiments, recipient baboons survived for at least three postoperative months, and two for over six months. GGTA1-ko, hCD46/hTBM-2´tg hearts were perfused with cold, hyperoncotic, oxygenated preservation solution to prevent early systolic graft failure. The recipients were immunosuppressed, with chimeric anti-CD40 ab providing the mainstay, and also medicated to prevent overgrowth of the xeno-hearts. In future experiments overgrowth will be prevented by growth hormone receptor (GHR) gene knockout or the use of minipigs. Hearts from the multi-ko, multi-tg pigs developed by B1&2 will be tested in the orthotopic porcine heart-to-baboon transplantation model with improved immunosuppressive regimens, e.g. humanised co-stimulation blockade. In the last year of the coming funding period, porcine hearts should be available for clinical application.

Although treatment with pig insulin is known to be safe in human patients with type 1 diabetes, there has been no comprehensive study of porcine islet biology and important functional characteristics such as glucose-sensing and insulin secretion kinetics. E. Bonifacio, M. Solimena and S. Speier (C1), who are leading researchers in beta-cell biology, will therefore carry out comparative studies of porcine and human islet function using unique experimental platforms for long-term in vivo monitoring and imaging of beta-cells. This approach may also be exploited within projects C3 and C4 to support the development of immunomodulatory and anti-inflammatory strategies.

J. Seissler (C3) has recently shown that LEA29Y expressed in porcine islet beta-cells can prevent xenogeneic graft rejection in humanised NSG mice without systemic immunosuppression and its side effects. The goal of this project is to analyse LEA29Y transgenic islets and further immunomodulatory transgenes to combat innate immunity and autoimmunity, as well as treatment with Tregs in a long-term humanised mouse model. Data from these studies will enable the design and generation of novel pigs expressing several transgenes in porcine beta-cells for preclinical studies in non-human primates.

B. Ludwig, M. Brendel and S. Bornstein have recently demonstrated that macro-encapsulation of porcine islets with a novel oxygenated device can restore hyperglycaemia in diabetic rats and pigs. Aims of project C4 include identifying the optimal pig source for large scale isolation of high quality pig islets (collaboration with C1-C4, B3), and improving the device by investigating its application in large animal models, such as the diabetic pigs developed by E. Wolf, and non-human primate models.

They will develop in vitro pretreatment of pig islets to reduce apoptosis and the instant blood mediated inflammatory reaction after transplantation, using immunomodulatory molecules and mesenchymal stromal cells. The general concept of graft encapsulation as described in C4 closes the gap between experimentally developed manipulative strategies and safe and effective clinical application.

As outlined in the background section, preliminary clinical results indicate that pig islet xenotransplantation is feasible, but the immunological barrier and other obstacles remain. Projects C1-C4 represent a combined effort by recognised experts with complementary expertise with the overall aim of translating the findings of animal studies to the clinic.

Project C6 (M. Winkler/A. Tiede/K. Johanning) will focus on xenogeneic kidney transplantation. Current maximum survival times in life supporting preclinical kidney xenotransplantation using multi-transgenic and Gal knockout donor pigs and recipient immunosuppression are 80 to 100 days. Recipient survival remains limited by acute vascular rejection, with aberrant activation of coagulation (AAC) resulting in thrombotic microangiopathy and finally organ failure. This still occurs after elimination of elicited antibodies. Prof. Winkler and colleagues have worked with activated Protein C (APC), an important natural anticoagulant that is reduced during AAC. Provision of supplemental APC can reduce AAC as indicated by the lack of consumption of coagulation factors and absence of microthrombi. Based on these data, transgenic pigs expressing human thrombomodulin have been generated within our consortium, and these are expected to have normal APC plasma levels. There is also a need for additional molecules to protect and maintain endothelial integrity in the xenograft. Among these heme oxygenase 1 (HO-1) is one of the best characterised. Ex vivo perfusion of hHO-1 transgenic porcine kidneys with human blood showed prolonged perfusion times and reduced signs of coagulopathy compared with porcine wild-type kidneys.

The aim of project C7 (A. Hilfiker/A. Haverich/U. Martin) is to develop a porcine derived decellularised matrix for heart valves that does not suffer degradation in human patients. To reduce the immunogenicity of matrix derived from wild-type pigs, decellularisation will be optimised with regard to xenoantigen removal and combined with enzymatic treatments to specifically remove known xenoantigens such as the 1,3-Gal epitope, but also unidentified xenoantigens that react with human antibodies. Since decellularisation and enzymatic digestions will affect the integrity of the matrices, mechanical properties and biocompatibility will be tested. Tests for antigenicity will be performed in humanised mouse models and compared with matrices derived from α Gal deficient and CMAH deficient pigs as they become available. Finally, successful antigen-reduced decellularised pulmonary heart valve matrices will be tested for functionality in the sheep model. Having both in vitro and in vivo systems to quantify the immunogenicity of decellularised matrices, a long term goal will be to focus on the identification of specific antigens that might be future targets for genetic modification in pigs.

The primary aim of project C8 (P. Brenner/ J. Abicht) is to investigate hyperacute, delayed and cellular rejection after xenotransplantation of transgenic pig hearts into baboons using different immunomodulatory and immunosuppressive strategies. Porcine multi-transgenic cardiac xenografts, including novel transgene combinations, will be transplanted in a life-supporting heterotopically thoracic position into baboons. Thereby strategies such as lymph node irradiation, co-stimulatory blockade and tolerance induction will be evaluated. The best result of heterotopic thoracic transplantation model will be tested in an orthotopic model (proof of principle). The goal is to achieve consistent long-term survival of cardiac xenografts for at least 90 days in a life-supporting position with tolerable immunosuppression and good quality of life of the recipient animal. Cardiac xenotransplantation will then commence in clinical patients with end-stage heart failure, either for bridging-to-transplantation, or as a destination therapy.

In summary, the comprehensive orchestration of project groups Z, A, B, and C generates a unique network of expertise and infrastructures to realise the central goal of the Transregio-CRC: moving pig-to-primate xenotransplantation from experimental studies to clinical application.