*Purpose: Physiologic behavior of porcine lungs with multiple xeno-targeted genetic modifications, Pig 2.0, have not previously been evaluated.

*Methods: Eight pairs of lungs from Pig 2.0 with combined Gal1,3αGal, β4Gal, and Neu5Gc triple-knockouts (TKO) and containing additional human transgenes addressing molecular incompatibilities in complement activation (hCD46, hCD55, hCD59) and innate immune and coagulation-related cell function (CD47, HLA-E/b2M, hCD39) were perfused for up to 8 hours with human blood using an ex vivo perfusion circuit. GTKO.hCD55 lungs served as a reference group. In each pair of lungs, blood was left ‘untreated’ on one side (n=5 Pig 2.0, n=3 reference), and the other side was ‘treated’ with 1-BIA, a thromboxane synthase inhibitor, and histamine (H) receptor blockade (n=8 Pig 2.0, n=4 reference).

*Results: Median survival time was 450 min (range 300-480 min) for Pig 2.0 lungs vs 30 min (range 20-300 min) for reference lungs (p=0.04) in the untreated groups and 440 min (range 290-480 min) vs 300min (range 145-360 min) in the treated cohorts (p=0.018) (Fig.1A). Pulmonary vascular resistance (PVR) rise was significantly attenuated and delayed in untreated Pig 2.0 lungs relative to GTKO.hCD55 lungs, simulating additional blood treatment with 1-BIA and H-blockade in the reference group (Fig.1B). Platelet and granulocyte sequestration occurred within 5-15 min of reference lung perfusion and were not attenuated in association with Pig 2.0 lungs.

*Conclusions: TKO combined with human complement-, coagulation-, and immune-related regulatory genes protect lungs from PVR increase and improve lung survival during human blood perfusion similar to anti-inflammatory drug treatment in reference lungs. Platelet and granulocyte sequestration were not prevented, as previously described with other lung genetics. The transgene combination expressed by Pig 2.0 may be helpful to achieve successful xenotransplantation of the lung and other organs, but additional regulation of coagulation and inhibition of adhesion pathways may be required for further improvement.

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