Purpose: Significant progress in genetic engineering has enabled the generation of knock-out pigs that additionally express multiple human “transgenes” chosen to address various known xenogeneic rejection pathways. We evaluated which transgene combinations are associated with prevention of lung xenograft injury in a rigorous life-supporting pig-to-baboon lung xenograft model.
Methods: GTKO.hCD46 porcine donor organs with up to 4 additional genetic modifications, including hCD55, hTBM, hEPCR, hTFPI, hCD47, hCD39, hHO-1, HLA-E, A20 and humanized vWF were used in 48 left single lung transplants into baboons. A rationally designed “platform drug regimen” consisted of steroids, sC1Inh, thromboxane synthase inhibitor, histamine receptor blockers, and anti-GPIb Fab. Desmopressin (DDAVP) was given to the donor animals prior to lung procurement, to deplete pig endothelial VWF. Immunosuppression consisted of aCD20, ATG, MMF, aCD40, coupled with anti-IL6R moAb and/or Alpha-1 Antitrypsin. Xenograft function was assessed intermittently by transplant blood flow measurements and radiographs.
Results: Xenogeneic lungs with 6 genetic modifications (6-GE) transplanted into baboons survived for up to 8d (hCD55.hEPCR.hTBM.hCD39) and 9d (hEPCR.hTBM.hCD47.HO-1), vs <12hrs for most 3- or 4-GE lungs. Several other donor transgene-combinations resulted in 47d survival. Lungs of GTKO.hCD46.hCD55.hEPCR.hCD47.hTFPI and a few 3- or 4-GE phenotypes (eg including HLA-E and hvWF) consistently exhibited life-supporting (LS) lung function for 24-30h, with relatively normal macro- and microscopic lung appearance until 2-3 d. Lung xenograft failure was usually associated with rebounding anti-pig antibody titer and loss of lung vascular barrier function leading to alveolar flooding and consolidation, consistent with “delayed xenograft rejection”. Accumulated evidence suggests that, in addition to anti-non-Gal antibody, recipient NK cells (HLA-E) and donor macrophage activation (thromboxane) each play important roles to drive the residual inflammation.
Conclusion: Combining multi-transgenic donor organs with mechanism-directed drug treatments significantly prolongs life-supportive lung xenograft function and recipient survival. Aggregation of existing targeted genetic modifications, along with antibody depletion and mechanism-based drug additions appear likely to successfully control known pathogenic pathways, and further advance lung xenotransplantation towards clinical application.