Introduction: Significant advances have been made recently in xenotransplantation using gene-edited (GE) pigs with up to 3 carbohydrate gene knockouts in various combinations, alone or with additional human complement (CD46, CD55, CD59), coagulation (TBM, EPCR, TFPI), and anti-inflammatory (CD47, CD39, HLAE/β2M, A20, PD-1, HO-1) ‘transgenes’. In this pilot study we evaluated hearts from 3-GE and multi-GE pigs treated with an established costimulation-based immunosuppressive (IS) regimen and cold-perfused ‘ischemia minimization’ (IM).

Methods: Five baboons (7.8 to 14.8 kg) received heterotopic abdominal heart transplants from GE pigs. Results with 3 reference GTKO.B4GALNT2KO.CD55 pig hearts (3-GE; NSRRC, MO) were compared with 2 hearts that share eight GE’s (GTKO.CMAHKO.B4GALNT2KO.hCD46.CD55.TBM.TFPI.HLAE/β2M. CD47) but differed by added GE modifications (CD59.CD39.A20.HO1.PD-1: EGEN-4737; or EPCR: EGEN-4417) (eGenesis, MA). Hearts were preserved with Steen’s cold-perfused IM method. Induction with ATG (5 mg/kg, d-3 and -1), αCD20 (20 mg/kg on d-1, d0) was followed by αCD154 (20 mg/kg/wk x6 mo, then 10 mg/kg/wk), MMF (40 mg/kg/d), and tapered-dose corticosteroid; αIL6R Ab and C1 esterase inhibitor were administered as single doses around the time of surgery. Intravenous heparin was given for the first 10 days in the reference group only.

Results: All three 3GE grafts functioned well initially but failed within 5 days, with graft necrosis and rupture associated with compliment deposition, intravascular thrombi, and myocardial infarction.  The EGEN-4737 heart recipient was euthanized on POD20 with a beating graft due to abdominal wound dehiscence two days after protocol biopsy, with edema and C4d deposition but otherwise preserved myocardial histology. The EGEN-4417 heart recipient exhibited good function until around POD284; IS was reduced around POD200, and the graft stopped functioning at POD300. No treatment-related complications were seen. Intra-graft thrombi were seen in all three 3GE hearts but not in the multi-GE hearts.

Conclusion: Relative to reference genetics without thrombo-regulatory and anti-inflammatory gene expression, while limited in numbers, this pilot experience with multi-GE porcine hearts in the context of a clinically applicable IM and IS regimen appears to support our working hypothesis that the additional GE’s tested in these 2 multi-GE constructs will prove effective to prolong heart xenograft survival in an orthotopic model.