Until recently, clinical translation of xenotransplantation was impeded by vigorous innate and adaptive immune responses to the transplanted organ.¹ On January 7, 2022, the first clinical xenotransplant of a heart from a genetically engineered pig (with ten genetic modifications) was conducted at the University of Maryland in Baltimore.² The patient, Mr. David Bennett, Sr., lived for 60 days before dying from a complex, but uncertain, interaction of co-morbidities.

What Advances Have Resulted from Preclinical Studies?

This partial success was a result of decades of research by scientists around the world, which can be summarized by four achievements:³

(i) Advances in gene-editing techniques, the most recent of which is CRISPR-Cas9, have enabled deletion of the three known pig carbohydrate xenoantigens (galactose-α1,3-galactose, N-glycolylneuraminic acid, and Sda) against which humans have performed antibodies, thus minimizing activation of the complement cascade.

(ii) A better understanding of incompatibilities between pigs and primates with regard to the complement and coagulation systems and 'self-identification' mechanisms has allowed the transgenic introduction into the pigs of human complement-regulatory and other proteins that provide added protection from the primate innate immune response.⁴ Furthermore, the rapid growth of a pig heart after transplantation into a nonhuman primate (NHP), which can result in compression of the heart, has been reduced by deleting growth hormone receptors in the pig.⁵

(iii) Novel immunosuppressive regimens, based on blockade of the CD40/CD154 co-stimulation pathway, prevent an adaptive immune response to a pig xenograft more efficiently than do conventional immunosuppressive agents.^6,7

(iv) A better understanding of the susceptibility of the pig heart to ischemic injury led to the introduction of enhanced myocardial preservation strategies.^8-10

These advances have extended survival of NHPs with orthotopic pig heart transplants to approximately 8-9 months.^8,11,12

The First Clinical Gene-Edited Pig Cardiac Transplant

The exact cause of Mr. Bennett's demise remains uncertain, but certain factors are suspected of playing roles. He was in a debilitated state of health at the time of the transplant, and possibly did not have sufficient energy reserves to enable him to fully recover from the operation. The surgical procedure was complicated by a dissection of the thoracic aorta which, though successfully replaced, contributed to acute renal failure requiring dialysis throughout his postoperative life [M. Mohiuddin personal communication]. Furthermore, porcine cytomegalovirus was inadvertently transferred with the pig heart;¹³ subsequent viral activation may have been detrimental to myocardial function. In allotransplantation, human cytomegalovirus is more likely to cause a systemic infection, whereas in xenotransplantation porcine cytomegalovirus is believed to have only a local effect within the pig graft.

Are We Ready for a Formal Clinical Trial?

To date, no NHP supported by a pig heart has survived for >9 months,^8,10-12 and so further advances will likely be required before cardiac xenotransplantation can be offered even in a formal clinical trial, although further transplants may be approved on 'compassionate' grounds. Relatively consistent survival of NHPs supported by pig hearts for at least 6 months, and in selected cases for 12 months, would encourage further clinical transplants. Although there is a specific problem with NHP models,^14,15 extended graft survival might be achieved by (i) further gene-editing of the pig, (ii) the replacement in the immunosuppressive regimen of an anti-CD40 agent by an anti-CD154 agent,¹⁶ and (iii) improved monitoring of the microbial status of the organ-source pig.

Which Patients Might Benefit?

It will be critical to select the first clinical trial patients to ensure they have a realistic possibility of benefitting from the procedure.^3,17 The following patients might be considered:

(i) Patients eligible for cardiac allotransplantation, but in whom mechanical circulatory support is contraindicated or associated with an elevated risk. Those with a restrictive or hypertrophic cardiomyopathy, refractory ventricular arrhythmias, or with a mechanical valve prosthesis, are at substantial risk of dying before obtaining a suitable allograft. Bridging with a pig xenograft may be lifesaving.

(ii) Patients with high titers of anti-HLA antibodies experience long wait times and are at risk of sudden death. However, there is a small risk that their anti-HLA antibodies may cross-react with pig antigens, e.g., swine leukocyte antigens (SLA), increasing the risk of early xenograft failure.¹⁸ If cross-reacting antibodies are excluded, cardiac xenotransplantation might be preferable to methods of desensitization to enable cardiac allotransplantation.

(iii) Cardiac allograft recipients with extensive graft vasculopathy also have a high risk of sudden death, and experience suboptimal support by a mechanical device.

(iv) Infants with complex congenital heart disease have limited access to allotransplantation due to the scarcity of size-matched donor organs. In these infants, the results of mechanical support are poor, and both survival and quality of life after multiple staged surgical reconstructive procedures remain limited, particularly for those relying on univentricular 'Fontan' physiology. A gene-edited pig heart could provide a bridge until a heart from a size-appropriate deceased human donor becomes available.^11,19

Not committing the patient to life-long dependency on a pig heart (by bridging) would be advantageous until more experience of the potential of pig organ transplantation is known. Importantly, the limited data available suggest that sensitization to a pig organ would not appear to be detrimental to the outcome of a subsequent allotransplant.²⁰

Which Patients Should be Excluded?

Patients whose prognosis is poor based on risk factors not directly related to their heart pathology, such as frailty or malignancy, that would disqualify them as candidates for cardiac allotransplantation, should be excluded from the first clinical trials. Poor outcomes would not reflect the potential of xenotransplantation. Adult patients presenting in extremis and unable to participate fully in a robust informed consent process would need to be assessed particularly carefully on ethical grounds.

Conclusion

Xenotransplantation offers a promising solution by potentially providing a timely source of organs for transplantation. If mechanical circulatory support of the patient is contraindicated, the ready availability of hearts from genetically engineered pigs would reduce the need for a patient's prolonged and costly pre-transplant stay in an intensive care unit. We predict that, when the success of cardiac xenotransplantation is proven, far more patients will be referred for heart transplantation than at present. Based on the initial limited success achieved by the Maryland team, our vision for cardiac xenotransplantation now seems within reach.

References

1. Cooper DKC, Ezzelarab MB, Hara H, et al. The pathobiology of pig-to-primate xenotransplantation: a historical review. Xenotransplantation 2016;23:83-105.
2. Rothblatt M. Commentary on achievement of first lifesaving xenoheart transplant. Xenotransplantation 2022;29:e12746.
3. Chaban R, Cooper DKC, Pierson RN 3rd. Pig heart and lung xenotransplantation: present status. J Heart Lung Transplant 2022;41:1014-22.
4. Cooper DKC, Hara H, Iwase H, et al. Justification of specific genetic modifications in pigs for clinical kidney or heart xenotransplantation. Xenotransplantation 2019; 26:e12516.
5. Hinrichs A, Kessler B, Kurome M, et al. Growth hormone receptor-deficient pigs resemble the pathophysiology of human Laron syndrome and reveal altered activation of signaling cascades in the liver. Mol Metab 2018;11:113-28.
6. Bühler L, Awwad M, Baker M, et al. High-dose porcine hematopoietic cell transplantation combined with CD40 ligand blockade in baboons prevents an induced anti-pig humoral response. Transplantation 2000;69:2296-2304.
7. Yamamoto T, Hara H, Foote J, et al. Life-supporting kidney xenotransplantation from genetically engineered pigs in baboons: a comparison of two immunosuppressive regimens. Transplantation 2019;103:2090-2104.
8. Längin M, Mayr T, Reichart B, et al. Consistent success in life-supporting porcinecardiac xenotransplantation. Nature 2018;564:430-33.
9. Längin M, Reichart B, Steen S, et al. Cold non-ischemic heart preservation with continuous perfusion prevents early graft failure in orthotopic pig-to-baboon xenotransplantation. Xenotransplantation 2021;28:e12636.
10. Goerlich CE, Griffith B, Singh AK, et al. Blood cardioplegia induction, perfusion storage and graft dysfunction in cardiac xenotransplantation. Front Immunol 2021;12:667093.
11. Cleveland DC, Jagdale A, Carlo WF, et al. The genetically engineered heart as a bridge to allotransplantation in infants just around the corner? Ann Thorac Surg 2022114:536-44.
12. Mohiuddin MM, Goerlich CE, Singh AK, et al. Progressive genetic modifications of porcine cardiac xenografts extend survival to 9 months. Xenotransplantation 2022;29:e12744.
13. Regalado A. The gene-edited pig heart given to a dying patient was infected with a pig virus (MIT Technology Review). 2022. Available at: https://www.technologyreview.com/2022/05/04/1051725/xenotransplant-patient-died-received-heart-infected-with-pig-virus/. Accessed 06/01/2022.
14. Yamamoto T, Hara H, Iwase H, et al. The final obstacle to successful preclinical xenotransplantation? Xenotransplantation 2020;25:e12596.
15. Iwase H, Jagdale A, Yamamoto T, et al. Evidence suggesting that deletion of expression of N-glycolylneuraminic acid (Neu5Gc) in the organ-source pig is associated with increased antibody-mediated rejection of kidney transplants in baboons. Xenotransplantation 2021;28:E12700.
16. Perrin S, Magill M. The inhibition of CD40/CD154 costimulatory signaling in the prevention of renal transplant rejection in nonhuman primates: a systematic review and meta analysis. Front Immunol 2022;13:861471.
17. Pierson RN 3rd, Burdorf L, Madsen JC, Lewis GD, d'Alessandro A. Pig-to-human heart transplantation: who goes first? Am J Transplant 2020;20:2669-74.
18. Martens GR, Reyes LM, Li P, et al. Humoral reactivity of renal transplant-waitlisted patients to cells from GGTA1/CMAH/B4GalNT2, and SLA class I knockout pigs. Transplantation 2017;101:e86-92.
19. Raza SS, Hara H, Cleveland DC, Cooper DKC. The potential of genetically engineered pig heart transplantation in infants with complex congenital heart disease. Pediatr Transplant 2022;26:e14260.
20. Li Q, Hara H, Zhang Z, Breimer ME, Wang Y, Cooper DKC. Is sensitization to pig antigens detrimental to subsequent allotransplantation? Xenotransplantation 2018; 25:e12393.

Clinical Topics: Arrhythmias and Clinical EP, Cardiac Surgery, Cardiovascular Care Team, Congenital Heart Disease and Pediatric Cardiology, Heart Failure and Cardiomyopathies, Invasive Cardiovascular Angiography and Intervention, Implantable Devices, SCD/Ventricular Arrhythmias, Atrial Fibrillation/Supraventricular Arrhythmias, Aortic Surgery, Cardiac Surgery and Arrhythmias, Cardiac Surgery and CHD and Pediatrics, Cardiac Surgery and Heart Failure, Congenital Heart Disease, CHD and Pediatrics and Arrhythmias, CHD and Pediatrics and Interventions, CHD and Pediatrics and Prevention, CHD and Pediatrics and Quality Improvement, Heart Transplant, Interventions and Structural Heart Disease

Keywords: Transplantation, Heterologous, Cytomegalovirus, Galactose, Quality of Life, Receptors, Somatotropin, Graft Survival, Heterografts, Aorta, Thoracic, Baltimore, CRISPR-Cas Systems, Frailty, Gene Editing, Virus Activation, Waiting Lists, Heart Transplantation, Antigens, Heterophile, HLA Antigens, Immunosuppressive Agents, Risk Factors, Prognosis, Immunity, Innate, Cardiomyopathy, Hypertrophic, Heart Defects, Congenital, Adaptive Immunity, Acute Kidney Injury, Intensive Care Units, Death, Sudden, Morbidity, Morbidity, Allografts, Arrhythmias, Cardiac, Prostheses and Implants, Neoplasms

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