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.

One remaining difficulty with livestock transgenesis is how best to achieve predictable, ubiquitous and high expression of transgenes. To address this, complementary approaches will be pursued in projects B1 and B2 focussing in the first instance on two xenoprotective transgenes: the human complement regulatory gene CD55 (for which there is as yet no high expressing pig available worldwide); and human heme oxygenase-1 (hHO-1) which protects endothelial tissue from TNF- induced apoptosis.

H. Niemann and W. Kues (B1) will produce lines of transgenic pigs using predetermined genomic loci defined by sleeping beauty transposon vectors, which in previous experiments showed preferential integration at transcriptionally permissive loci. In addition, the group will use ZFN technology for knocking out the porcine GGTA1 gene. One line will be built on the existing human heme oxygenase-1(hHO-1) transgenic pig line and will also carry a CD55 transgene and be αGal deficient. hA20 and hTFPI transgenes also have promise to combat AVR and will be expressed in triple-transgenic pigs. Strategies will be developed to enable propagation of triple-transgenic pigs by conventional breeding. Recombination-mediated cassette exchange will also be used to place the transgenes at a predetermined permissive locus. Inactivation of the GGTA1 gene in pigs has been the single most important advance in xenotransplantation, but barriers due to non-Gal surface antigens still exist resulting in endothelial cell activation which is characterised by expression of adhesion molecules and presentation of tissue factor which initiates the coagulation cascade. To counteract this, research within project B1 will investigate the endothelium protective effects of hHO-1 and the anti-thrombotic effects of tissue factor down-regulation in the porcine organ.

A. Schnieke and T. Flisikowska (B2) will address the issue of transgene expression by investigating the porcine ROSA26 locus as a site for transgene placement, and the use of novel genomic constructs under the control of endogenous or exogenous promoters to express complement regulatory genes (CD46, CD55, CD59). To date nearly all xenotransgenes have been based on cDNA constructs, which are convenient but known to be less efficiently expressed. Project B2 will also investigate the use of high capacity bacterial artificial chromosomes for the production of multi-transgenic animals. Project B2 will also generate pigs lacking the major non-Gal xenoreactive antigen N-glycolylneuraminic acid (Neu5GC or H-D antigen) by targeted inactivation of the CMP-N-acetylneuraminic acid hydroxylase gene (CMAH) using TALENs. Removal of Neu5GC is a unique requirement for human recipients of porcine grafts because humans are the only mammals deficient in CMAH activity and carry anti-Neu5GC antibodies. This is viewed as essential for clinical xenotransplantation.

The focus of project B3 (N. Klymiuk/A. Wünsch/E. Wolf) is the production of transgenic animals specifically designed for improved xeno-islet transplantation. Animals with beta-cell specific expression of LEA29Y to avoid immune rejection have already been generated. To block pro-inflammatory cytokine induced islet impairment an IL-1 receptor antagonist will also be expressed in the porcine islet cells. To improve islet viability, transgenic pigs with islet-specific expression of the X-linked inhibitor of apoptosis (XIAP) will be produced. To support vascularisation of transplanted islets an inducible islet specific VEGF-A (vascular endothelial growth factor A) expression system will be established. Principally this will be based on successful experiments using the tetracycline-inducible (Tet-on) system in transgenic pigs. There is a difference between human and pig islet architecture. Porcine islets are smaller and less compact, which makes handling of porcine islets difficult without encapsulation. In silico comparison of extracellular matrix components may provide insights into the cause of fragility and inform future genetic engineering to improve islet structure.