Work package 1

In Work Package (WP) 1 primary porcine kidney cells (pKC) will be genetically modified by the Munich group using CRISPR-Cas9 technology. Three genes (GGTA1, CMAH, B4GALNT2), which encode the enzymes synthetizing the major carbohydrate xenoantigens recognized by pre-formed human antibodies, will be knocked out and several transgenes with complement inhibiting, anticoagulant and anti-inflammatory functions stacked into the GGTA1 locus. This targeted approach will prevent random segregation of transgenes and facilitate the propagation of genetically multi-modified pigs by breeding. The Bern group will test the genetically modified pKC in vitro, mainly in microfluidic assays. The aim of these tests is to assess the effect of the genetic modifications on prevention of activation of the humoral arm of innate immunity. The pKC will be exposed to human plasma and binding of human antibodies as well as activation of the plasma cascade systems will be quantified. The Geneva group will use in vitro assays to assess the effect of the genetic modifications on the cellular arm of innate immunity, in particular NK cells, macrophages and neutrophil granulocytes, as well as T cells. In a first round, we will create a basic set of genetic modifications, i.e. knockouts of the GGTA1, CMAH, and B4GALNT2 genes, combined with transgenic expression of the human complement regulator hCD46, as well as human thrombomodulin (hTBM). In a second round, advanced sets of genetic modifications will be created by additional gene knockouts and/or integration of additional expression vectors to compile an evidence-based set of genetic modifications for xenotransplantation. Candidates for additional human transgenes to be tested are HLA-E, hCD47, hCD31, hCD274, and hTNFAIP3 (A20), but the final selection of genes may change as the field develops and new insights into xenorejection mechanisms are available. Criteria for selection will be minimal antibody binding, optimal prevention of plasma cascade activation – in particular complement – and minimal activation of innate immune cells and platelets, as well as minimal activation of human T cells.