Research Areas

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. Xenotransplantation was among the ten scientific areas predicted to make headlines in 2020.

The Transregional Collaborative Research Centre (TRR) 127 aims to dissect the biology of xenogeneic cell, tissue and organ transplantation and develop evidence-based concepts that bring xenotransplantation of porcine pancreatic islets, heart valves, and hearts from bench to bedside. Breakthroughs in macroencapsulated porcine islet transplantation into diabetic macaques (C4) and life-supporting orthotopic porcine heart transplantation into baboons (C8) have been published in leading journals, demonstrating consistent positive results in the most stringent pre-clinical models. Furthermore, ongoing experiments to transplant genetically-modified neonatal islets into macaques (C3) and decellularised porcine heart valves into baboons (C7) are also very promising.

The overarching goals of the TRR 127 during the third funding period (2020 - 2024) are to overcome the remaining obstacles and to generate the safety and efficacy data required for submission of clinical study protocols in Europe and elsewhere. This has already been accomplished for the macroencapsulated islet transplantation programme (C4) and approval is currently awaited.

The Consortium is structured as three closely interacting Project Groups A, B and C, all supported by the Central Project Group Z that focuses on ethical, legal and societal issues (Z1), microbiological safety (Z2), provision of safe donor pigs with effective and stable genetic modifications (Z3), and non-human primate (NHP) studies (Z4).

A: Immunity/Tolerance

Research Area A is developing concepts to modulate immune mechanisms at the xenograft-host interface. A1 will test the hypotheses that: i) SLA class-I deficiency with additional downregulation of SLA class-II and/or transgenic expression of hPD-L1 will result in tissues with particularly low immunogenicity; and ii) clinically suitable donor-recipient combinations can be defined by SLA-HLA matching. A2 will develop strategies of local immunomodulation delivered either by recombinant adeno-associated viral vectors (rAAV), micro-RNAs, small hairpin (sh) RNAs, or by genetic modification of the donor animal to support long-term survival of cardiac xenografts. A3 and A5 will synergistically address mechanisms of the instant blood-mediated inflammatory reaction (IBMIR) induced by intraportal islet cell transplantation and test strategies to overcome them in unique ‘humanised’ mouse models. A4 will characterise the functional peculiarities of xenoreactive regulatory T cells (Treg) and establish protocols for Treg-mediated tolerance induction, e.g. by expression of xenospecific chimeric antigen receptors (CARs).

B: Novel transgenic strategies

Research Area B is developing and characterising genetically multi-modified donor pigs to overcome rejection mechanisms, functional incompatibilities, and zoonotic risks. B1 and B2 has employed gene stacking and combineering technology to generate 5´tg pigs carrying hCD46, hCD55, hCD59, hHO1 and hA20. A strategy for dynamic LEA29Y expression in response to rejection has been devised. Moreover, 4´ko pigs that lack SLA class I, GGTA1, CMAH and B4GALNT2 have also been produced. Combination of these genotypes will provide multi-modified pigs for transplantation of xeno-hearts and heart valves. B3 focuses on optimised donor pigs for xenogeneic islet transplantation. Pigs that lack GGTA1 and CCL2 and express LEA29Y and hPD-L1 (2´ko, 2´tg) have been produced, and a hCD47 transgene will be added to overcome cellular rejection by macrophages. Several pig lines with reporter genes are available for studies of beta-cell maturation, function and survival after transplantation. Single-cell RNA sequencing will be employed to inform and improve methods of maturing neonatal porcine islets (NPIs). Both B projects use donor animals with a PERV-C free genetic background.

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.

Z: Central Projects

The TRR 127 has established four central projects, which cover ethical and legal aspects (Z1) and microbiological/virological safety (Z2) of xenotransplantation or provide the infrastructure for the routine supply of genetically multi-modified pigs (Z3) and for experiments with non-human primates (Z4).