Clinical treatment procedure and experience of six gene-edited pig-rhesus monkey heterotopic heart xenotransplantation

Heart transplantation is an effective treatment for end-stage heart failure; however, problems such as the severe shortage of donor hearts, rejection, the toxic effects of immunosuppressants, and serious postoperative complications remain.[1] Heart xenotransplantation is one of the most promising alternatives to address the shortage of allogeneic donor hearts. However, the recipient survival rate and quality of life are affected by major obstacles, including immune rejection, complement and coagulation dysfunction, and infection.[2,3] Owing to the continuous improvement of gene editing strategies for donor pigs and anti-immune rejection strategies for non-human primate recipients, heart xenotransplantation research and clinical applications have made important breakthroughs. However, using multiple types and high doses of immunosuppressive drugs increases the incidence of postoperative infections in the recipient, also affecting the survival rate.[4] On January 7, 2022, surgeons at the University of Maryland Medical Center transplanted a genetically modified pig heart into a human body. The recipient survived only for 60 days, demonstrating that more preclinical studies are still needed to address the issue of long-term graft survival.[5] In this study, we performed six-gene edited pig-rhesus heterotopic heart transplantations. The recipient survival times were significantly extended, and the comprehensive treatment plan after xenograft transplantation, including anti-inflammation, anticoagulation, anti-immune rejection, and anti-infection therapies, was further improved. Here, we summarized our experience and experimental results in detail to guide further study on heart xenotransplantation. The experiments were approved by the Laboratory Animal Ethics Committee of the Air Force Medical University (No. IACUC2022001). The pig-rhesus monkey heterotopic heart xenotransplantation surgery was performed on October 16, 2022. Basic information about the animals, surgical methods and procedures, immunosuppressive protocols, postoperative monitoring and examination methods are given in Supplementary Methods, https://links.lww.com/CM9/B914. Graft gradually dwindled, and its contractility weakened. After surgery, the graft successfully regained its beating and pulse, as indicated via intraoperative imaging and ultrasonography performed the day after surgery [Figure 1A]. Due to factors such as abdominal organ compression and intraoperative injury, the grafted heart had an ejection fraction (EF) of 46% the day after transplantation. Postoperative measurement of the EF decreased slowly over time. The EF was equal to 32% on postoperative day 8, indicating dysfunction of the grafted heart.Figure 1: (A) Representative echocardiograms showing reduced donor heart systolic function; (B) Representative echocardiograms showing thickened and echogenic mitral and aortic valves in donor heart; (C) Line plots reflecting postoperative changes in serum complement series (C3 and C4) concentrations in recipient rhesus monkey; (D) Line chart reflecting changes in serum IL-6 level in rhesus monkey after transplantation; (E) General morphology of donor heart and left ventricle at death of recipient rhesus monkey. (F) Representative images of donor heart stained with HE and Masson's trichome. (G) Representative immunofluorescence image of donor heart section stained with IgG (up) and IgM (down); (H) Echocardiography indicated a thrombus in left atrium of donor heart on postoperative day 2; (I) General morphology (left) and HE staining (right) of thrombus in left atrium; (J) Line chart reflecting changes in serum platelet level in rhesus monkey after transplantation. C3: Complement 3; C4: Complement 4; HE: Hematoxylin and eosin; IgG: Immunoglobulin G; IgM: Immunoglobulin M; IL-6: Interleukin 6; LV: Left ventricle.Graft suffered from immune rejection injury. The pathological results of the graft still showed signs of immune rejection. Ultrasonography revealed that the graft valves, especially the aortic valve, began to show an enhanced echo on the third day after surgery [Figure 1B]. The postoperative complement 3 (C3) and complement 4 (C4) were all at very low levels. However, the C3 level gradually recovered 12 days after surgery [Figure 1C]. The C-reactive protein and procalcitonin levels remained normal after the transplantation. However, the Interleukin 6 (IL-6) concentration started to gradually rise on the 7th day, decreased after the 10th day, exhibited a slight increase on the 11th day, and peaked on the 14th day [Figure 1D]. The overall size of the heart had decreased, the ventricular walls had stiffened, and the heart surface was dark red [Figure 1E]. Hematoxylin and eosin (HE) staining suggested abnormal arrangement of cardiomyocytes, and diffuse interstitial hyperplasia. Masson's trichrome staining revealed severe fibrosis of the ventricular wall [Figure 1F]. Furthermore, we detected the presence of the receptors immunoglobulin G (IgG) and immunoglobulin M (IgM) in the graft tissue [Figure 1G]. Recipient developed significant coagulation dysfunction in early stage and continued until death. Cardiac ultrasonography performed the day after surgery revealed a thrombus in the left atrium, which moved into the atrium with blood flow and was approximately 11 × 8 mm in size [Figures 1H–I]. The platelet count suddenly decreased to 20 × 109/L on postoperative day 10 and showed an overall downward trend, with a platelet count of 2 × 109/L on the day the recipient died [Figure 1J]. Ultimately, the recipient died from coagulation disorders, multiple blood clots, and bleeding from the abdominal cavity and digestive tract. We modified the anti-immune-rejection treatment regimen before surgery based on the latest heart transplant research and progress in anti-immune rejection therapy.[6,7] In this study, the recipient survived for 20 days. The gene-edited donor was successfully constructed and verified; however, immune rejection still occurred in the recipient rhesus monkey. An intraperitoneal heterotopic xenotransplantation was performed to determine further the feasibility of immunosuppression and postoperative management. The heterotopic transplant model is useful for studying the immunological barriers to xenotransplantation. Multiple immunosuppressive and immunomodulatory agents and approaches and various genetic modifications to donor pigs were tested and refined in this model. The success obtained in a heterotopic model is a foundation for further experiments with the orthotopic model.[8] This model enables the assessment of heart transplant rejection without cardiopulmonary bypass support and life-threatening consequences for the recipient.[9] Damage to the endothelial cells (heart valves), activation of inflammatory mediators and complement system, the main pathological manifestations of cardiomyocyte apoptosis and myocardial fibrosis suggested the immune rejection injury. Chronic allogeneic graft rejection is characterized by arterial lesions with concordial endocardial thickening, and the basic pathology involves smooth muscle cell proliferation, monocyte infiltration, and fibrosis. Xenograft-specific manifestations include endocardial edema, stromal deposition, and fibrinoid necrosis.[10] Coagulopathy is another major barrier to xenografting. Both acute humoral and cellular xenograft rejections are accompanied by coagulation dysregulation, leading to thrombotic microangiopathy in the graft.[11] Based on the experience of this study, we believe that in future studies, the risk assessment of thrombosis, monitoring of gene loci responsible for the action of heparinase, aspirin, and warfarin before surgery, and individualization of the postoperative anticoagulation strategy should be improved. In addition, during transplantation in the left ventricle and left atrium of the heart and tube insertion, hemodynamic parameters should be monitored. Also, timely and targeted anticoagulation and thrombolytic drugs should be administered. After surgery, the types of anticoagulant drugs (heparin, low-molecular-weight heparin, warfarin, aspirin, etc.), mode of administration (intravenous, subcutaneous, oral, gastric tube, and injection), and the interval between administration (duration and interval duration) should be adjusted promptly according to the test indicators and severity of thrombosis. Finally, the coagulation status, such as thromboelastography parameters and coagulation factors of the recipient, should be fully understood in the early postoperative period to avoid hemorrhage. In conclusion, the survival time of the recipient in this study is relatively advanced in China; however, the cause of death was still coagulation disorder. In our future study, we intend to adjust the gene-editing scheme to modify the key coagulation mechanism of regulatory target genes such as vWF and TFPI1. Further, the postoperative use of anti-immune-rejection, anticoagulation, and anti-infection drugs has been refined. Thus, overcoming the coagulation disorders of xenografts, maintaining the donor heart function, prolonging the survival time of the recipient as much as possible, and accumulating a research basis for the successful clinical application of gene-edited pig-human heart xenografts will be possible. Funding This work was supported by the Xijing Hospital Clinical New Technology Project (grant Nos. XJSZ03 and XJSM02) Conflicts of interest None.