Background: Many genetically multi‐modified donor lines for xenotransplantation have a background of domestic pigs with rapid body and organ growth. The intrinsic growth potential of porcine xeno‐organs may impair their long‐term function after orthotopic transplantation in non‐human primate models. Since growth hormone is a major stimulator of postnatal growth, we deleted its receptor (GHR‐KO) to reduce the size of donor pigs in one step.Methods: Heart weight and proteome profile of myocardium were investigated in GHR‐KO and control pigs. GHR‐KO mutations were introduced using CRISPR/Cas9 in an α1,3‐galactosyltransferase (GGTA1)‐deficient background expressing the human cluster of differentiation (hCD46) and human thrombomodulin (hTHBD) to generate quadruple‐modified (4GM) pigs.Results: At age 6 months, GHR‐KO pigs had a 61% reduced body weight and a 63% reduced heart weight compared with controls. The mean minimal diameter of cardiomyocytes was 28% reduced. A holistic proteome study of myocardium samples from the two groups did not reveal prominent differences. Two 4GM founder sows had low serum insulin‐like growth factor 1 (IGF1) levels (24 ± 1 ng/mL) and reached body weights of 70.3 and 73.4 kg at 9 months. Control pigs with IGF1 levels of 228 ± 24 ng/mL reached this weight range three months earlier. The 4GM sows showed normal sexual development and were mated with genetically multi‐modified boars. Offspring revealed the expected Mendelian transmission of the genetic modifications and consistent expression of the transgenes.Conclusion: GHR‐KO donor pigs can be used at an age beyond the steepest phase of their growth curve, potentially reducing the problem of xeno‐organ overgrowth in preclinical studies.

For many patients with terminal/advanced cardiac failure, heart transplantation is the most effective, durable treatment option, and offers the best prospects for a high quality of life. The number of potentially life-saving donated human organs is far fewer than the population who could benefit from a new heart, resulting in increasing numbers of patients awaiting replacement of their failing heart, high waitlist mortality, and frequent reliance on interim mechanical support for many of those deemed among the best candidates but who are deteriorating as they wait. Currently, mechanical assist devices supporting left ventricular or biventricular heart function are the only alternative to heart transplant that is in clinical use. Unfortunately, the complication rate with mechanical assistance remains high despite advances in device design and patient selection and management, and the quality of life of the patients even with good outcomes is only moderately improved. Cardiac xenotransplantation from genetically multi-modified (GM) organ-source pigs is an emerging new option as demonstrated by the consistent long-term success of heterotopic (non-life-supporting) abdominal and life-supporting orthotopic porcine heart transplantation in baboons, and by a recent ‘compassionate use’ transplant of the heart from a GM pig with 10 modifications into a terminally ill patient who survived for 2 months. In this review, we discuss pig heart xenotransplantation as a concept, including pathobiological aspects related to immune rejection, coagulation dysregulation, and detrimental overgrowth of the heart, as well as GM strategies in pigs to prevent or minimize these problems. Additional topics discussed include relevant results of heterotopic and orthotopic heart transplantation experiments in the pig-to-baboon model, microbiological and virologic safety concepts, and efficacy requirements for initiating formal clinical trials. An adequate regulatory and ethical framework as well as stringent criteria for the selection of patients will be critical for the safe clinical development of cardiac xenotransplantation, which we expect will be clinically tested during the next few years.

Porcine circovirus 3 (PCV3) is a newly described member of the virus family Circoviridae. PCV3 is highly distributed among pigs and wild boars worldwide. A sudden introduction of PCV3 was recently observed in a herd of triple genetically modified pigs generated for xenotransplantation. These animals were used as donor pigs for orthotopic heart transplantation into baboons. In four cases, PCV3-positive hearts were transplanted, and transmission of PCV3 to the recipient was observed. PCV3 was found in all organs of the recipient baboons and a higher virus load was found in animals with a longer survival time of the transplant, indicating replication of the virus. This is the first report showing trans-species transmission of PCV3 to baboons by transplantation of a heart from a PCV3-positive donor pig. Sequence analysis showed that PCV3a and PCV3b were present in the infected pigs and were transmitted. Experiments to infect human 293 cells with PCV3 failed.

Heart transplantation is the only long-lasting lifesaving option for patients with terminal cardiac failure. The number of available human organs is however far below the actual need, resulting in substantial mortality of patients while waiting for a human heart. Mechanical assist devices are used to support cardiac function but are associated with a high risk of severe complications and poor quality of life for the patients. Consistent success in orthotopic transplantation of genetically modified pig hearts into baboons indicates that cardiac xenotransplantation may become a clinically applicable option for heart failure patients who cannot get a human heart transplant. In this overview, we project potential paths to clinical cardiac xenotransplantation, including the choice of genetically modified source pigs; associated requirements of microbiological, including virological, safety; optimized matching of source pig and recipient; and specific treatments of the donor heart after explantation and of the recipients. Moreover, selection of patients and the regulatory framework will be discussed.