David K. C. Cooper

Production of α1,3-galactosyltransferase and cytidine monophosphate-N-acetylneuraminic acid hydroxylase gene double-deficient pigs by CRISPR/Cas9 and handmade cloning

Gene-knockout pigs hold great promise as a solution to the shortage of organs from donor animals for xenotransplantation. Several groups have generated gene-knockout pigs via clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) and somatic cell nuclear transfer (SCNT). Herein, we adopted a simple and micromanipulator-free method, handmade cloning (HMC) instead of SCNT, to generate double gene-knockout pigs. First, we applied the CRISPR/Cas9 system to target α1,3-galactosyltransferase (GGTA1) and cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH) genes simultaneously in porcine fetal fibroblast cells (PFFs), which were derived from wild-type Chinese domestic miniature Wuzhishan pigs. Cell colonies were obtained by screening and were identified by Surveyor assay and sequencing. Next, we chose the GGTA1/CMAH double-knockout (DKO) cells for HMC to produce piglets. As a result, we obtained 11 live bi-allelic GGTA1/CMAH DKO piglets with the identical phenotype. Compared to cells from GGTA1-knockout pigs, human antibody binding and antibody-mediated complement-dependent cytotoxicity were significantly reduced in cells from GGTA1/CMAH DKO pigs, which demonstrated that our pigs would exhibit reduced humoral rejection in xenotransplantation. These data suggested that the combination of CRISPR/Cas9 and HMC technology provided an efficient and new strategy for producing pigs with multiple genetic modifications.

Pig-to-Primate Islet Xenotransplantation: Past, Present, and Future:

Islet allotransplantation results in increasing success in treating type 1 diabetes, but the shortage of deceased human donor pancreata limits progress. Islet xenotransplantation, using pigs as a source of islets, is a promising approach to overcome this limitation. The greatest obstacle is the primate immune/inflammatory response to the porcine (pig) islets, which may take the form of rapid early graft rejection (the instant blood-mediated inflammatory reaction) or T-cell-mediated rejection. These problems are being resolved by the genetic engineering of the source pigs combined with improved immunosuppressive therapy. The results of pig-to-diabetic nonhuman primate islet xenotransplantation are steadily improving, with insulin independence being achieved for periods >1 year. An alternative approach is to isolate islets within a micro- or macroencapsulation device aimed at protecting them from the human recipients immune response. Clinical trials using this approach are currently underway. This review focuses on the major aspects of pig-to-primate islet xenotransplantation and its potential for treatment of type 1 diabetes.

Human IL‐6, IL‐17, IL‐1β, and TNF‐α differently regulate the expression of pro‐inflammatory related genes, tissue factor, and swine leukocyte antigen class I in porcine aortic endothelial cells

Pro‐inflammatory cytokines play important pathological effects in various diseases and allotransplantation; however, their pathological role in xenotransplantation remains elusive. In pig‐to‐human cell or organ transplantation, whether porcine cells or organs are activated by human cytokines or not as an important question needs to be investigated.

Xenotransplantation—the current status and prospects

IntroductionnThere is a continuing worldwide shortage of organs from deceased human donors for transplantation into patients with end-stage organ failure. Genetically engineered pigs could resolve this problem, and could also provide tissues and cells for the treatment of conditions such as diabetes, Parkinsons disease and corneal blindness.nnnSources of datanThe current literature has been reviewed.nnnAreas of agreementnThe pathobiologic barriers are now largely defined. Research progress has advanced through the increasing availability of genetically engineered pigs and novel immunosuppressive agents. Life-supporting pig kidneys and islets have functioned for months or years in nonhuman primates.nnnAreas of controversynThe potential risk of transfer of a pig infectious microorganism to the recipient continues to be debated.nnnGrowing pointsnIncreased attention is being paid to selection of patients for initial clinical trials.nnnAreas timely for developing researchnMost of the advances required to justify a clinical trial have now been met.

Reducing immunoreactivity of porcine bioprosthetic heart valves by genetically-deleting three major glycan antigens, GGTA1/β4GalNT2/CMAH

Bioprosthetic heart valves (BHVs) originating from pigs are extensively used for heart valve replacement in clinics. However, recipient immune responses associated with chronic calcification lead to structural valve deterioration (SVD) of BHVs. Two well-characterized epitopes on porcine BHVs have been implicated in SVD, including galactose-α1,3-galactose (αGal) and N-glycolylneuraminic acid (Neu5Gc) whose synthesis are catalyzed by α(1,3) galactosyltransferase (encoded by the GGTA1 gene) and CMP-Neu5Ac hydroxylase (encoded by the CMAH gene), respectively. It has been reported that BHV from αGal-knockout pigs are associated with a significantly reduced immune response by human serum. Moreover, valves from αGal/Neu5Gc-deficient pigs could further reduce human IgM/IgG binding when compared to BHV from αGal-knockout pigs. Recently, another swine xenoantigen, Sd(a), produced by β-1,4-N-acetyl-galactosaminyl transferase 2 (β4GalNT2), has been identified. To explore whether tissue from GGTA1, CMAH, and β4GalNT2 triple gene-knockout (TKO) pigs would further minimize human antibody binding to porcine pericardium, TKO pigs were successfully produced by CRISPR/Cas9 mediated gene targeting. Our results showed that the expression of αGal, Neu5G and Sd(a) on TKO pigs was negative, and that human IgG/IgM binding to pericardium was minimal. Moreover, the analysis of collagen composition and physical characteristics of porcine pericardium from the TKO pigs indicated that elimination of the three xenoantigens had no significant impact on the physical proprieties of porcine pericardium. Our results demonstrated that TKO pigs would be an ideal source of BHVs.nnnSTATEMENT OF SIGNIFICANCEnSurgical heart valve replacement is an established lifesaving treatment for diseased heart valve. Bioprosthetic heart valves (BHVs) made from glutaraldehyde-fixed porcine or bovine tissues are widely used in clinics but exhibit age-dependent structural valve degeneration (SVD) which is associated with the immune response against BHVs. Three major xenoantigens present on commercial BHVs, Galactosea α1,3 galactose (αGal), N-glycolylneuraminic acid (Neu5Gc) and glycan products of β-1,4-N-acetyl-galactosaminyl transferase 2 (β4GalNT2) are eliminated through CRISPR/Cas9 mediated gene targeting in the present study. The genetically modified porcine pericardium showed reduced immunogenicity but comparable collagen composition and physical characteristics of the pericardium from wild-type pigs. Our data suggested that BHVs from TKO pigs is a promising alternative for currently available BHVs from wild-type pigs.

Early clinical xenotransplantation experiences—An interview with Thomas E. Starzl, MD, PhD

Dr Thomas E. Starzl, who died on March 4, 2017, was one of the great pioneers of organ transplantation. He was also a pioneer in the field of xenotransplantation. In 1964, he carried out baboon kidney transplants in six patients with terminal renal disease for whom no living or deceased donor became available; graft survival was for 19‐60 days, the grafts being lost largely through continuous complement activation. Between 1966 and 1974, he carried out one ex vivo liver perfusion and three orthotopic liver transplants using chimpanzees as sources of organs; graft survival was for <14 days. In 1992 and 1993, his team carried out baboon liver transplantation in two patients with cirrhosis from hepatitis B infection; graft survival was for 70 and 26 days, respectively. This early clinical experience is briefly discussed. Toward the end of his life, Dr Starzl was somewhat disillusioned by what he considered excessive regulation of medical research in the United States and believed that new advances were now likely to take place in countries such as China, where the regulatory framework is less developed.

Immunomodulation Strategies in Xenotransplantation

Many believe that xenotransplantation – i.e., the transplantation of tissues and/or organs between different species – holds the future of transplantation. Clinical application, however, is currently hampered by many barriers, of which rejection is the most formidable. This chapter focuses on the immunopathological background of xenograft rejection and the therapeutic modalities being explored to prevent it. The pig-to-nonhuman primate experimental model is emphasized, as the pig is the animal most likely to be the organ-source for organ xenotransplantation in humans.

Jewish, Christian and Muslim theological perspectives about xenotransplantation

This paper is based on a theological symposium presented at the International Xenotransplantation Associations 14th Congress held in Baltimore, MD, September, 2017.

Is sensitization to pig antigens detrimental to subsequent allotransplantation

An important question in xenotransplantation is whether an allotransplant can safely be carried out in a patient who has become sensitized to a pig xenograft. To answer this question, we have searched the literature. We primarily limited our review to the clinically relevant pig‐to‐non‐human primate (NHP) model and found five studies that explored this topic. No NHP that had received a pig graft developed antibodies to alloantigens, and in vitro studies indicated no increased humoral and/or cellular alloreactivity. We carried out a small in vitro study ourselves that confirmed this conclusion. There have been three experiments in which patients undergoing dialysis were exposed to wild‐type pig kidneys and three clinical studies related to bridging a patient in hepatic failure to liver allotransplantation. Despite the development of anti‐pig antibodies, all subsequent organ (kidney or liver) allografts were successful (except possibly in one case). In addition, pig fetal islets were transplanted into patients with kidney allografts; there was no increase in panel‐reactive alloantibodies and the kidney grafts continued to function satisfactorily. In conclusion, the limited data suggest that, after sensitization to pig antigens, there is no evidence of antibody‐mediated or accelerated cellular rejection of a subsequent allograft.

Data on B cell phenotypes in baboons with pig artery patch grafts receiving conventional immunosuppressive therapy

This report is related to the research article entitled “B cell phenotypes in baboons with pig artery patch grafts receiving conventional immunosuppressive therapy” (Yamamoto et al., in press). Herein we provide the data regarding pig artery patch xenotransplantation into the baboon׳s aorta, trough levels of tacrolimus and rapamycin in the blood after transplantation, analysis of B cell phenotype on the basis of IgD and CD27 expression in the blood, and analysis of T cell phenotype on the basis of CD28 and CD95 expression in the blood.