*Purpose: CRISPR/Cas9 gene editing techniques facilitate generation of pigs with multiple genetic modifications. We report results associated with multiple mechanistically targeted gene knock-outs and transgenes, evaluated in an established in vivo pig-to-baboon lung xenograft model.

*Methods: GalTKO.hCD46 porcine lungs with up to 5 additional genetic modifications, including hCD55, hTBM, hEPCR, hTFPI, hCD47, hCD39, hHO-1, HLA-E, A20 and humanized vWF were used in 49 single lung transplants into baboons; 6 also included β4GalKO. An evidence-based “platform drug regimen” consisted of steroids, sC1Inh, thromboxane synthase inhibition, anti-histamine, and αGPIb Fab. Donors were treated with Desmopressin (DDAVP) to deplete pig endothelial vWF. Immunosuppression consisted of αCD20, ATG, MMF, αCD40, usually with αIL6R moAb and/or Alpha-1 Antitrypsin. Xenograft function was assessed intra-operatively by transplant blood flow measurements and life-supporting lung capacity, and postoperatively by radiographs.

*Results: Few GalTKO.hCD46 pig lungs with 1, 2, or 3 additional genetic modifications (3/4/5-GE) exhibited durable life-supporting xeno lung function, and baboon recipients rarely survived for >12h due to systemic inflammation and/or lung xenograft failure. Multiple 6-GE lungs and a few 2-, 3- or 4-GE lungs (e.g. β4GalKO, hvWF) usually fully supported recipient gas transport and hemodynamics; overall, 9/49 exhibited recipient survival >2d; occasional recipient baboons survived for up to 8d (hCD55.hEPCR.hTBM.hCD39) and 9d (hEPCR.hTBM.hCD47.HO-1), and one 7-GE (hEPCR.hTBM.hCD47.HO-1 with β4GalKO) lung recipient survived for 31d. Lung xenograft failure beyond 4d was usually associated with rebounding anti-pig antibody titer and loss of lung vascular barrier function.

*Conclusions: Multi-transgenic pig lungs designed to modulate anti-pig antibody binding and other previously identified pro-inflammatory mechanisms, combined with evidence-based, mechanism-directed drug treatments, significantly prolong life-supportive xenolung function and recipient baboon survival. Accumulated evidence suggests that, in addition to anti-non-Gal antibody, complement, and coagulation pathway dysregulation, recipient NK cells (HLA-E) and donor macrophage activation (thromboxane, histamine) play important roles in driving residual inflammation. Controlling these pathways with additional targeted lung donor gene modifications (or drug treatments) appear likely to successfully protect lung xenografts, and further advance lung xenotransplantation towards clinical application.

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