Background: Xenotransplantation has made significant advances recently using pigs genetically engineered to remove carbohydrate antigens, alone or with addition of various human complement, coagulation, and anti-inflammatory ‘transgenes’.  Here we evaluated results associated with 3-, 9-, or 10-GE pig hearts treated using an established costimulation-based immunosuppressive regimen and a cold-perfused storage technique designed to minimize graft ischemia, as used for the recent clinical heart xenograft case.

Methods: Eight baboons received heterotopic abdominal heart transplants from GE pigs using Steen’s cold-perfusion ischemia minimization. Three reference hearts were from 3-GE pigs (GalKO.β4GalNT2KO.CD55) [NSRRC, Columbia, MO].  Three pigs were 9-GE (GalKO.β4GalNT2KO. GHRKO. CD46. CD55.TBM.EPCR.CD47.HO-1) and 2 were 10G (9-GE+CMAHKO) [Revivicor, Blacksburg, VA]. Induction with ATG (5mg/kg on d-3 and -1) and aCD20 (20mg/kg on d-1, d0) was followed by aCD154 [Tonix Pharmaceuticals, Chatham, NJ] or primatized hu5c8 [NHPRRC, Boston, MA]; 20mg/kg/wk x6 mo, then 10 mg/kg/wk), MMF (40mg/kg/d), and tapered corticosteroid.  Periop aIL6R Ab and C1 esterase inhibitor were given.  IV heparin was given for the first 8-10 days.

Results: Two of the 3-GE grafts functioned well initially but failed within 5 days, with graft necrosis and rupture associated with complement deposition, intravascular thrombi, and myocardial infarction, while the third failed intraoperatively with refractory ventricular fibrillation.  One 9-GE graft was lost intraoperatively due to a technical issue. One 9-GE heart in a baboon with a strongly positive preop crossmatch failed with antibody mediated rejection at d13.  One 10-GE heart failed at d113 with combined cellular and antibody mediated rejection. One 10-GE and 1 9-GE heart have preserved graft function, normal myocardium on protocol biopsies, and slowly progressive graft hypertrophy after 210 and 350 days, respectively (both ongoing).  Transient intraventricular clot in some 9- and 10-GE grafts peri-transplant resolved with heparin; no treatment-related complications were seen.

Conclusion: Relative to reference genetics without thrombo-regulatory and anti-inflammatory gene expression, 9- or 10-GE pig hearts exhibit promising performance in the context of a clinically applicable regimen including ischemia minimization and aCD154-base IS, justifying further evaluation in an orthotopic model.