Control of xenograft rejection in experimental models has been a challenge. The direct targeting of T cells enabled allotransplantation, but this approach has been less effective in xenotransplantation. Various genetic modifications like α-1,3 galactosyltransferase gene-knockout, transgenic expression of human complement regulatory proteins (CD46), and coagulation-regulatory protein thrombomodulin have been the key to avoiding early cardiac xenograft rejection. The potential of the CRISPR technique1,2 in addition to an improved immunosuppression regimen including costimulation blockade, for example, anti-CD154mAb and anti-CD40mAb, plus elimination of the B-cell response have advanced the field.3,4 With these manipulations, nonlife supporting, heterotopic cardiac xenograft survival has been extended to 945 days.5,6
In their recent letter to Nature, Längin et al7 report early, consistent survival in an experimental orthotopic cardiac xenotransplantation model. Those results demonstrate yet another milestone in moving the xenotransplant closer to a clinical application. The data by the Munich group can be considered a game changer as the authors have successfully translated the recently reported significant cardiac xenograft survival in a heterotopic position5 to an orthotopic transplant model and have demonstrated the required life-sustaining capability of a genetically engineered pig hearts transplanted into baboon.
It is not clear what regulatory agencies will consider sufficient to move forward with clinical xenotransplantation. The International Society for Heart and Lung Transplantation suggests that success in a large animal model will include 60% graft survival for a follow-up of 3 months and 50% survival in a 6 months’ observation.8 The study by Längin et al7 included 12 transplants, with 2 baboons surviving 90 days and 2 animals surviving beyond 6 months. The first 2 transplants were electively terminated due to restriction of only 90-day survival by their study protocol. The authors then received permission to extend the survival for 6 months, and the latter 2 transplants were therefore electively terminated after crossing 6-month survival (182 and 195 days, respectively).7
A few key findings of this study should be emphasized. According to the authors, the use of cold (−8°C) nonischemic continuous perfusion of the donor’s heart in a special chamber with Steen solution9 containing red blood cells (15% hematocrit) for 3 hours before implant appeared beneficial. Maintenance immunosuppression included anti-inflammatory drugs targeting interleukin (IL)-6, IL-1, and tumor necrosis factor (TNF)-α. Treatment also included temsirolimus, a prodrug of rapamycin and a specific inhibitor of the phosphatidylinositol 3-protein kinase (mTOR) family that inhibits cell cycle progression. The authors demonstrated in the 2 longest-term survivors that the withdrawal of temsirolimus led to hypertrophy and physiological overgrowth of the naturally aging porcine heart in the baboon. It is unclear if temsirolimus application had been essential as directly inhibiting xenograft hypertrophy or as an immunosuppressant. Potential side effects of extended use of temsirolimus are not discussed, and it is not known whether the withdrawal of this drug was elective or due to complications. A continuous intravenous infusion of antihypertensive drugs, enalapril and metoprolol, was implemented to achieve “porcine-like” hemodynamics, with a target systolic pressure of 80 mm Hg and heart rate of 100, in an effort to further mitigate xenograft overgrowth. Controlling hemodynamic changes alone appeared insufficient to prevent xenograft overgrowth, as the addition of temsirolimus seemed necessary, suggesting that intrinsic factors beyond hemodynamic control contribute to the porcine heart.
While the overall clinical success is significant, it remains difficult to tease out which of the individual therapeutics have been necessary. The mechanism by which nonischemic continuous organ perfusion is superior compared to conventional cold preservation techniques for myocardial preservation is unclear. The authors used the Steen solution9 that has traditionally been used for lung preservation with a composition that differs from conventional crystalloid cardioplegia. Moreover, detailed mechanisms on how appropriate xenograft size is maintained remain unclear. If growth restriction is necessary, use of minipigs or growth hormone receptor-deficient pigs could help avoiding additional drugs.
While the use of costimulation blockade with anti-CD40 antibody is now accepted as a mainstay of immunosuppression in avoiding cardiac xenograft rejection, the approach poses an additional hurdle to clinical translation, as it is not currently approved for use in human recipients. Another threat to xenotransplantation and a major concern by the regulatory agencies is the possibility of the transmission of a porcine virus to humans. To address this, Yang’s team used CRISPR technology snipping out 62 copies of the PERV gene in pig-kidney cells.10 However, to date, there is no evidence of active transmission of PERV to humans, and these modifications may not be necessary.
Survival of cardiac xenografts is improving with the availability of genetically engineered organs and modified immunosuppression. With this, a structured approach to elucidating what genetic modifications and immunosuppressive agents are key to clinical success will be necessary.
At the same time, it will be important to engage regulatory agencies into a continuous dialogue to define next steps and critical components for clinical xenotransplantation trials. A critical next step will be to define the ideal group of patients that will benefit from xenotransplantation. In our opinion, pediatric patients seem a good first choice as size-matched organs will be from pigs that are only a few months old. Other targeted groups may include patients with restrictive cardiomyopathy or those with complex congenital malformations, HLA-sensitized patients, those waiting for a retransplant, patients with complex pathological considerations preventing VAD implantation, or older patients with a less robust immune response.
Building xenotransplant consortia working on similar approaches in parallel yielding comparable results may represent an additional and essential step toward a clinical application.
Xenotransplantation as a field has made great strides toward clinical application. This progress has brought the field back into a constructive public discussion. The report by Längin et al7 presents another milestone in moving cardiac xenotransplantation forward.
1. Hsu PD, Lander ES, Zhang F. Development and applications of CRISPR-Cas9 for genome engineering. Cell. 2014;157:1262–1278.
2. Mali P, Yang L, Esvelt KM, et al. RNA-guided human genome engineering via Cas9. Science. 2013;339:823–826.
3. Mohiuddin MM, Singh AK, Corcoran PC, et al. Role of anti-CD40 antibody-mediated costimulation blockade on non-gal antibody production and heterotopic cardiac xenograft survival in a GTKO.hcd46tg pig-to-baboon model. Xenotransplantation. 2014;21:35–45.
4. Mohiuddin MM, Corcoran PC, Singh AK, et al. B-cell depletion extends the survival of GTKO.hcd46tg pig heart xenografts in baboons for up to 8 months. Am J Transplant. 2012;12:763–771.
5. Mohiuddin MM, Singh AK, Corcoran PC, et al. Chimeric 2C10R4 anti-CD40 antibody therapy is critical for long-term survival of GTKO.hcd46.htbm pig-to-primate cardiac xenograft. Nat Commun. 2016;7:11138.
6. Singh AK, Chan JL, DiChiacchio L, et al. Cardiac xenografts show reduced survival in the absence of transgenic human thrombomodulin expression in donor pigs. Xenotransplantation. [Epub ahead of print. Oct 5, 2018]. doi: 10.1111/xen.12465.
7. Längin M, Mayr T, Reichart B, et al. Consistent success in life-supporting porcine cardiac xenotransplantation. Nature. 2018;564:430–433.
8. Cooper DK, Keogh AM, Brink J, et al. Xenotransplantation Advisory Committee of the International Society for Heart and Lung Transplantation. Report of the Xenotransplantation Advisory Committee of the International Society for Heart and Lung Transplantation: the present status of xenotransplantation and its potential role in the treatment of end-stage cardiac and pulmonary diseases. J Heart Lung Transplant. 2000;19:1125–1165.
9. Steen S, Paskevicius A, Liao Q, et al. Safe orthotopic transplantation of hearts harvested 24 hours after brain death and preserved for 24 hours. Scand Cardiovasc J. 2016;50:193–200.
10. Yang L, Güell M, Niu D, et al. Genome-wide inactivation of porcine endogenous retroviruses (PERVs). Science. 2015;350:1101–1104.
The authors have successfully translated the recently reported significant cardiac xenograft survival in a heterotopic position to an orthotopic transplant model and have demonstrated the required life-sustaining capability of a genetically engineered pig hearts transplanted into baboon.