ABSTRACT:Starting from early 2000, with the introduction of Gal-knockout pigs, prolonged survival especially in heart and kidney xenotransplantation was recorded. However, remaining antibody barriers to nonGal antigens continue to be the hurdle to overcome. The production of genetically-engineered pigs was difficult requiring prolonged time. However, advances in gene editing, such as zinc finger nucleases, transcription activator-like effector nucleases, and most recently CRISPR technology made the production of genetically-engineered pigs easier and available to more researchers. Today, the survival of pig-to-nonhuman primate heterotopic heart, kidney, and islet xenotransplantation reached >900 days, >400 days, and >600 day, respectively. The availability of multiple-gene pigs (5 or 6 genetic modifications) and/or newer costimulation blockade agents significantly contributed to this success. Now, the field is getting ready for clinical trials with an international consensus. Clinical trials in cellular or solid organ xenotransplantation are getting closer with convincing preclinical data from many centers. The next decade will show us new achievements and additional barriers in clinical xenotransplantation.

INTRODUCTION

Outcomes of organ and cell allotransplantation continue to improve. However, the shortage of transplantable organs remains as the major hurdle in the field of transplantation despite the use of marginal deceased donors and living donors. Xenotransplantation (i.e., cross- species transplantation between pig and humans) could offer an unlimited and prompt supply of transplantable organs, when needed. In addition to organ transplantation, many disorders could be treated by xenotransplantation.

PAST

The concept of xenotransplantation is not new, and there have been numerous clinical attempts during the past 300 years or more. Clinical blood xenotransfusion was attempted in the 17th century by Jean Baptiste Denis, corneal xenotransplantation from pigto-human followed in the early 19thcentury, and attempts were made at nonhuman primate (NHP) kidney xenotransplantation in the 1960s by Reemtsma.

PRESENT

Xenotransplantation research was stimulated by the production of pigs in which the important antigen, galactose-α1,3-galactose (Gal), had been deleted by gene-knockout (GTKO pigs) in 2003. More recently, the identification of other xenoantigens has also been important.

Techniques for making genetically-engineered pigs have become easier and faster. Rapid improvement in the results of preclinical studies has made the field more hopeful of the initiation of clinical trials.

1. Heart xenotransplantation—Mohiuddin demonstrated that long-term survival of genetically-engineered pig heterotopic heart grafts could be achieved in NHPs. Genetic modifications in the pig (GTKO.hCD46.hThrombomodulin) combined with a successful treatment regimen based on a chimeric anti-CD40 monoclonal antibody (mAb), consistently prevented humoral rejection and systemic coagulation pathway dysregulation, sustaining cardiac xenograft survival in one case beyond 900 days. Iwase tested three different costimulation blockade-based immunosuppressive regimens in the pig-to-baboon heterotopic heart xenotransplantation model, and demonstrated that the combination of anti-CD40mAb+belatacept proved effective in preventing a T cell response

2. Kidney xenotransplantation—The Emory group performed pre-transplant antibody screening in recipient monkeys and showed that the combination of low titer antibody and anti-CD154mAb costimulation blockade promoted long-term renal xenograft survival [16]. The Pittsburgh group showed that specific genetic modifications of the pig are important in achieving prolonged survival . Most recently, Kim et al reported the longest survival (405 days) of a life-supporting pig kidney xenograft in a preclinical model, emphasizing the importance of CD4+ T cell depletion.

3. Lung xenotransplantation—Burdorf showed that platelet sequestration and activation during GTKO.hCD46 pig lung perfusion by human blood was primarily mediated by GPIb, GPIIb/IIIa, and von Willebrand Factor. Laird showed that transgenic expression of human leukocyte antigen (HLA)-E attenuates GTKO.hCD46 pig lung xenograft injury. A recent review from the same group concluded that genetic modification of pigs coupled with drugs targeting complement activation, coagulation, and inflammation have significantly increased duration of pig lung function in ex vivo human blood perfusion models, and life-supporting lung xenograft survival in vivo.

4. Liver xenotransplantation—Although limited, fairly consistent 7–9 days’ survival has been reported by different groups using GTKO and GTKO.hCD46 pig liver xenografts in NHPs after orthotopic pig liver xenotransplantation . The Boston group increased survival to 29 days by the exogenous administration of human coagulation factors using the same model .

5. Islet xenotransplantation—

6. Matsumoto published a clinical trial using encapsulated neonatal wild-type pig islets in patients with type 1 diabetes. Their study showed that there was a clinical benefit of islet xenotransplantation with improved HbA1c, especially when a greater number of islets was transplanted. Arefanian showed that porcine islet-specific tolerance induced by the combination of antilymphocyte function-associated antigen-1 and anti-CD154mAb is dependent on PD-1 (programmed cell death protein-1).

7. Tissue (cornea, heart valve, skin) xenotransplantation—Porcine corneal xenotransplantation shows promising application in the clinic. Lee studied the impact of the expression of N-glycolylneuraminic acid on pig corneas, concluding that the absence of N-glycolylneuraminic acid expression on GTKO pig corneas may not prove an advantage over GTKO pig corneas. They studied the impact of N-glycolylneuraminic acid expression in bioprosthetic pig heart valves on human antibody recognition and structural deterioration. Tena demonstrated that pig cells expressing human CD47 are associated with an immune-modulating effect, which leads to markedly-prolonged survival of pig skin grafts in NHPs.

8. Cellular (hepatocyte, neuronal cell) xenotransplantation—Machaidze tested porcine hepatocytes in alginate-poly-l-lysine microspheres transplanted intraperitoneally immediately after hepatectomy in a model of fulminant liver failure in baboons . The microencapsulated porcine hepatocytes provided temporary functional support. Parkinsonian NHPs received wild-type or CTLA4-Ig-transgenic porcine xenografts and different durations of exogenous immunosuppressive therapy to test whether systemic plus graft-mediated local immunosuppression might avoid rejection.

9. Inflammation and coagulation—Further attention was directed to inflammation in xenotransplantation. Ezzelarab showed that systemic inflammation in xenograft recipients precedes activation of coagulation. Iwase measured serum free triiodothyronine (thyroid hormone) as a marker of inflammation in healthy naïve baboons, healthy naïve monkeys, and after pig-to-baboon heterotopic heart xenotransplantation, orthotopic liver xenotransplantation, artery patch xenotransplantation, and in monkey heterotopic heart allotransplantation.

10. Zoonosis—The potential for the transmission of infection from animal-to-human has always been of concern. Therefore, several porcine viruses have been studied in regard to xenotransplantation. Denner published seminal reviews on virological safety in xenotransplantation. Particular attention has been directed to porcine endogenous retroviruses (PERV) , their susceptibility to retroviral inhibitors , and their genomewide inactivation by genetic technology

11. Genetic engineering—The introduction of CRISPR (clustered regularly interspaced short palindromic repeats) technology in xenotransplantation has increased the speed in which genetic manipulations can be achieved in pigs. In the early years, genetic engineering of pigs was performed by homologous recombination, which might take longer than 2 years from cell work to pregnancy. Today, research groups can produce multiple gene knock-out or knock-in pigs using CRISPR technology , which can also be used to delete PERV expression. Genetically-modified pigs using CRIPSR technology have been used in several important studies relating to antibody binding and coagulation dysfunction.

CONCLUSION

FUTURE

With our accumulated experience and recent achievements [in xenotransplantation, the stage may now be set for the first-in-human exploration. The future is set for well-controlled trials of genetically engineered pig islet xenotransplantation. The xenotransplantation research community needs to decide (i) whether successful orthotopic heart transplantation in the pig-to-NHP model is required before proceeding to a clinical trial [104], and (ii) whether the preclinical threshold for a clinical renal xenotransplantation trial can be reduced .

The resurgence of xenotransplantation is now obvious, with prolonged survival of cellular and solid organ xenografts associated with the administration of newer costimulation blockade agents and access to genetically-engineered pigs. Our increasing knowledge of the pig genome will almost certainly lead to further genetic manipulations. The future of xenotransplantation is vibrant.

 

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