To evaluate the optimal strategy of gene editing type and immunosuppressive regimen by applying a novel anti-CD40 monoclonal antibody (anti-CD40) in combination with tacrolimus-based immunosuppressive regimen in a humanized genome engineering porcine-non-human primates (NHPs) allogeneic cardiac ventral allograft experiment.

The heart of a humanized genome engineering (GTKO/hCD39/hCD55/hTBM) male Bama miniature pig was transplanted into the peritoneal cavity of a male cynomolgus to establish a cardiac xenotransplantation model. During the perioperative period, a new immunosuppressive regimens consisting of domestic anti-CD40 in combination with mycophenolate mofetil, tacrolimus, methylprednisolone, anti-CD20 monoclonal antibody and anti-thymocyte globulin were administered, and the electrical activity of the transplanted heart was monitored in real-time using an extracorporeal telemetry monitoring device. The main outcomes were coronary artery blood flow and function after revascularization, electrocardiographic signal changes and myocardial enzyme changes in the transplanted heart, and immunosuppression status and survival time of the recipient. The secondary outcomes were the physiological parameters of the recipient, including routine blood tests, liver and kidney function, serum protein and electrolytes, and life support therapy based on the results. When the xenograft suffered failure of function, the cardiac xenograft was removed and sent for pathological examination using HE staining and electron microscopy.

The transplanted heart showed a ruddy color and had a soft texture, autonomous heartbeat and strong myocardial contraction after revascularization. One week after transplantation, the transplanted heart had good coronary artery perfusion. The cardiac ultrasound indicated normal myocardial systolic function, and the condition of the recipient was normal. The levels of serum creatine kinase and lactic dehydrogenase in the recipient transiently increased after transplantation and decreased to normal levels at postoperative day 6. The hemoglobin, electrolytes and liver and kidney function of the recipient significantly improved at 1 week after transplantation. Cardiac ultrasound indicated significant myocardial hypertrophy of the cardiac xenograft at 2 weeks after transplantation. At 20 days after transplantation, the heartbeat of the xenograft decreased from 120-140 beats per minute to 50-80 beats per minute, the blood flow of the anastomotic site was normal, and the coronary artery was well-perfused, but the myocardium was debilitated in the systolic phase. The experiment was terminated at 20 days after transplantation because the function of the transplanted heart was significantly weakened. HE staining showed myocardial fiber hyperplasia, local myocardial fibrosis, interstitial edema and monocyte infiltration. Myofibril distortion, intermuscular space broadening, and structural damage to the rough endoplasmic reticulum, mitochondria and myocardial myofilament were observed under an electron microscope.

A humanized genome engineering porcine-NHPs model of intra-abdominal heterotopic cardiac xenotransplantation was successfully established. The application of a novel anti-CD40 monoclonal antibody with tacrolimus-based combination immunosuppressive regimens effectively inhibited hyperacute rejection, delayed the occurrence of acute rejection, and achieved longer survival of the xenograft organ.