© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.
Xenotransplantation 2015: 22: 325–327 doi: 10.1111/xen.12181
XENOTRANSPLANTATION
Literature Update
Xenotransplantation literature update, May–June 2015 Burlak C, Mueller KR, Beaton BP. Xenotransplantation literature update, May–June 2015. Xenotransplantation 2015: 22: 325–327. © 2015 John Wiley & Sons A–S. Published by John Wiley & Sons Ltd.
Christopher Burlak,1 Kate R. Mueller1 and Benjamin P. Beaton2 1
Department of Surgery, Schulze Diabetes Institute, University of Minnesota School of Medicine, Minneapolis, MN, , 2Division of Animal Sciences, University of Missouri-Columbia, Columbia, MO, USA Key words: genetic engineering – pig – xenotransplantation Abbreviations: NHP, non-human primate; aGal, galactose-a1,3-galactose; ASGR1, asialoglycoprotein receptor. Address reprint requests to Christopher Burlak, Department of Surgery, Schulze Diabetes Institute, University of Minnesota School of Medicine, 420 Delaware St. SE, Minneapolis 55455, MN, USA (E-mail: [email protected]) Received 23 June 2015; Accepted 26 June 2015
Commentary and review
The use of animal models in research is essential for the development and validation of methods for the diagnosis and treatment of diseases. Specific to diabetes research, several animal models are used for the development of treatments using islet cells. Denner and Graham [1] provided commentary on the efficacy and safety of nonhuman primates (NHPs) as a model for the evaluation of porcine-to-NHP islet cell transplantation. Their commentary gave a brief overview on the use of NHPs for the evaluation of islet cell transplantation, including limitations associated with using NHPs. New techniques (macro- and micro-encapsulated islet cell transplantation) and deficiencies (differences between human and NHP metabolic differences in glucose metabolism, zoonotic pathogens) associated with NHPs have recently called into question the efficacy of NHPs. The authors do grant that the pig-to-NHP xenotransplantation has provided advancements toward clinical translation but that they may no longer be required for all preclinical data. Joachim Denner has also
reviewed the impact and biology of hepatitis E virus on pigs and humans and the various genotypes of HEV that may pose a risk to specific pathogen-free or designated pathogen-free pigs [2]. Ethics of xenotransplanation
The ethics of xenotransplantation from a theological perspective take into account the status of man, the therapeutic intent, and the treatment of animals used for therapy [3]. Citing the Prospects for Xenotransplantation by the Pontifical Academy for Life and a collaborative statement of Catholic and Protestant point of view, Sautermeister considers the aspects of medicine, social ethics, and animal ethics in relation to xenotransplantation. Within the Catholic faith and theology, there is an intrinsic ethical imperative to heal; however, given the “relatively early stage of xenotransplantation,” all medical risks and expectations, both positive and negative, need to be considered. Theological ethics do not inherently prohibit xenotransplantation as long as certain conditions of personhood and autonomy are not violated and animals are treated responsibly and not merely as instruments. 325
Burlak et al. He acknowledges how the “pluralization of lifestyles” makes a unified societal agreement unlikely and that sociocultural conditions do not afford a general rejection of xenotransplantation. Theological ethics do not focus only on the religious aspect, but offer a perspective to the ethical and social discourse on xenotransplantation that takes into account the impact of biographical, psychosocial, culture-bound, and ideological preconditions and how they relate to a personal identity. He concludes that upon consideration, xenotransplantation is a permissible form of therapy, so long as biomedical ethics are followed, including a respectful treatment of animals. In a companion piece, Sautermeister et al. [4] address the theological ethical considerations of xenotransplantation from an interdisciplinary perspective. In a summary of lectures given at the symposium “Xenotransplantation – a challenge to theological ethics”, viewpoints on xenotransplantation from Christian, Jewish, and Muslim perspectives are given. Regarding Christian ethics, from an anthropocentric perspective, xenotransplantation cannot be definitively rejected as long as man’s identity, integrity, and dignity are not compromised. The use of animals is permissible, akin to use for agriculture or consumption, so long as the moral imperative that they are not treated arbitrarily is observed. Historically, there has been a progression of compassion for fellow mankind and animals such that in the context of xenotransplantation, there should be a transparency of motives other than a therapeutic intent. The core principle of Jewish ethics that human life should be preserved supersedes most other religious obligations, and with regard to xenotransplantation, using organs from animals that are considered impure is permissible because there is no “pleasure” (Hana’a) and they are being used for the benefit of man. Islam also ascribes to the notion that man has an “obligation to preserve one’s own health”. Animals are seen as a sign of God’s mercifulness and as moral objects that do not have the same dignity as man; however, exploitation or infliction of pain should be avoided. Hence, xenotransplantation is justifiable for therapeutic use in Islam. There is also a moral obligation to the human recipients of xenotransplantation. Building on the foundation of informed consent, there needs to be a transparent public discourse that addresses fair distribution, organ shortage, and animal welfare. While there are no fundamental religious prohibitions to xenotransplantation, the authors acknowledge that patient autonomy may lead to an individual’s refusal of treatment and should be respected. 326
Genetically engineered pigs
When discussing the possibility of generating a pig-to-human xenotransplantation model, the first barriers to overcome are antigens on the surface of pig cells/tissues. Two of the more characterized xenoantigens are the carbohydrate galactose-a1, 3-galactose (aGal, synthesized by GGTA1) and N-glycolylneuraminic acid (Neu5Gc, synthesized by CMAH), but additional xenoreactive antibodies are of concern such as b1,4 N-acetylgalactosaminyl transferase (b4GalNT2). Estrada et al. [5] examined human and NHP antibody binding to peripheral blood mononuclear cells (PBMCs) derived from genetically engineered pigs devoid of these carbohydrate-modulating genes (GGTA1, GGTA1/ CMAH, and GGTA1/CMAH/b4GalNT2). The cells from GGTA1/CMAH/b4GalNT2 null pigs exhibited a reduction in human IgM and IgG binding when compared to cells lacking both GGTA1 and CMAH. However in the NHP antibody reactivity experiments, cells lacking both GGTA1 and CMAH had an increase in NHP antibodies binding when compared to cells lacking either GGTA1 alone or to those null in GGTA1/CMAH/ b4GalNT2. The authors concluded that ablation of the b4GalNT2 gene reduces antibody binding in both humans and NHPs but that immunity in NHPs highlights the complexities of xenoantigens. These results overall were useful in human sera but demonstrated a potential limitation in the use of NHP as a model for examining genetic modifications in the pig for the use of human xenotransplantation. Using pig organs for liver xenotransplantation results in lethal thrombocytopenia due to the mechanism of human platelet removal in the donor pig organ. It has been suggested that the mechanism associated with platelet removal is partially dependent upon the asialoglycoprotein receptor (ASGR) [6–8]. Using the transcription activator-like effector nuclease (TALEN) platform, Paris et al. [9] have targeted the ASGR1 gene resulting in ASGR1 knockout pigs. The authors originally mutated the ASGR1 gene in adult porcine liver-derived cells and generated six cloned fetuses using somatic cell nuclear transfer (SCNT). Fibroblast cells were harvested from the cloned fetuses and SCNT was performed using these cells to generate 22 live piglets. The live piglets appeared normal and were evaluated for xenogeneic human platelet uptake. Compared to wild-type pigs (ASGR1 +/+), livers from ASGR1 null pigs (ASGR1 / ) exhibited a decrease in human platelet uptake. The authors concluded that the disruption of the ASGR1 gene does reduce platelet destruction, but additional
Xenotransplantation literature update modifications, such as the CD18 receptor, are likely required to completely reduce thrombocytopenia. Preclinical research
Higginbotham et al. [10] sought to enhance the survival of pig-to-primate kidney xenografts by screening potential NHP for low anti-pig antibody titers prior to xenotransplantation. The authors then transplanted NHP using GGTA1 KO / CD55 transgenic pig kidneys with T-cell depletion and immune suppression. As has been seen in allotransplantation, the NHP with a high pre-transplant antibody titer rejected the pig kidney in the first week despite the use of genetically modified pigs and immune suppression. A low pre-transplant antibody titer along with maintenance anti-CD154 antibody therapy allowed normal kidney function greater than 125 days. Biopsies taken at day 100 showed no evidence of antibody- or cellular-mediated rejection. This study describes the longest survival of pig-toNHP kidney xenotransplantation to date. References 1. DENNER J, GARAHAM M. Xenotransplantation of islet cells: what can the non-human primate model bring for the evaluation of efficacy and safety? Xenotransplantation 2015; 22: 231–235.
2. DENVER J. Xenotransplantation and hepatitis E virus. Xenotransplantation 2015; 22: 167–173. 3. SAUTERMEISTER J. Xenotransplantation from the perspective of moral theology. Xenotransplantation 2015; 22: 183–191. 4. SAUTERMEISTER J, MATHIEU R, BOGNER V. Xenotransplantation-theological-ethical considerations in an interdisciplinary symposium. Xenotransplantation 2015; 22: 174– 182. 5. ESTRADA JL, MARTENS G, LI P et al. Evaluation of human and non-human primate antibody binding to pig cells lacking GGTA1/CMAH/Beta4GalNT2 genes. Xenotransplantation 2015; 22: 194–202. 6. PARIS LL, CHIHARA RK, REYES LM et al. ASGR1 expressed by porcine enriched liver sinusoidal endothelial cells mediates human platelet phagocytosis in vitro. Xenotransplantation 2011; 18: 245–251. 7. CHIHARA RK, PARIS LL, REYES LM et al. Primary porcine Kupffer cell phagocytosis of human platelets involves the CD18 receptor. Transplantation 2011; 92: 739–744. 8. PENG Q, YEH H, WEI L et al. Mechanisms of xenogeneic baboon platelet aggregation and phagocytosis by porcine liver sinusoidal endothelial cells. PLoS One 2012; 7: e47273. 9. PARIS LL, ESTRADA JL, PING L et al. Reduced human platelet uptake by pig livers deficient in the asialoglycoprotein receptor 1 protein. Xenotransplantation 2015; 22: 203– 210. 10. HIGGINBOTHAM L, MATHEWS D, BREEDEN CA et al. Pretransplant antibody screening and anti-CD154 costimulation blockade promote long-term xenograft survival in a pig to primate kidney transplant model. Xenotransplantation 2015; 22: 221–230.
327
Xenotransplantation 2015: 22: 325–327 doi: 10.1111/xen.12181
XENOTRANSPLANTATION
Literature Update
Xenotransplantation literature update, May–June 2015 Burlak C, Mueller KR, Beaton BP. Xenotransplantation literature update, May–June 2015. Xenotransplantation 2015: 22: 325–327. © 2015 John Wiley & Sons A–S. Published by John Wiley & Sons Ltd.
Christopher Burlak,1 Kate R. Mueller1 and Benjamin P. Beaton2 1
Department of Surgery, Schulze Diabetes Institute, University of Minnesota School of Medicine, Minneapolis, MN, , 2Division of Animal Sciences, University of Missouri-Columbia, Columbia, MO, USA Key words: genetic engineering – pig – xenotransplantation Abbreviations: NHP, non-human primate; aGal, galactose-a1,3-galactose; ASGR1, asialoglycoprotein receptor. Address reprint requests to Christopher Burlak, Department of Surgery, Schulze Diabetes Institute, University of Minnesota School of Medicine, 420 Delaware St. SE, Minneapolis 55455, MN, USA (E-mail: [email protected]) Received 23 June 2015; Accepted 26 June 2015
Commentary and review
The use of animal models in research is essential for the development and validation of methods for the diagnosis and treatment of diseases. Specific to diabetes research, several animal models are used for the development of treatments using islet cells. Denner and Graham [1] provided commentary on the efficacy and safety of nonhuman primates (NHPs) as a model for the evaluation of porcine-to-NHP islet cell transplantation. Their commentary gave a brief overview on the use of NHPs for the evaluation of islet cell transplantation, including limitations associated with using NHPs. New techniques (macro- and micro-encapsulated islet cell transplantation) and deficiencies (differences between human and NHP metabolic differences in glucose metabolism, zoonotic pathogens) associated with NHPs have recently called into question the efficacy of NHPs. The authors do grant that the pig-to-NHP xenotransplantation has provided advancements toward clinical translation but that they may no longer be required for all preclinical data. Joachim Denner has also
reviewed the impact and biology of hepatitis E virus on pigs and humans and the various genotypes of HEV that may pose a risk to specific pathogen-free or designated pathogen-free pigs [2]. Ethics of xenotransplanation
The ethics of xenotransplantation from a theological perspective take into account the status of man, the therapeutic intent, and the treatment of animals used for therapy [3]. Citing the Prospects for Xenotransplantation by the Pontifical Academy for Life and a collaborative statement of Catholic and Protestant point of view, Sautermeister considers the aspects of medicine, social ethics, and animal ethics in relation to xenotransplantation. Within the Catholic faith and theology, there is an intrinsic ethical imperative to heal; however, given the “relatively early stage of xenotransplantation,” all medical risks and expectations, both positive and negative, need to be considered. Theological ethics do not inherently prohibit xenotransplantation as long as certain conditions of personhood and autonomy are not violated and animals are treated responsibly and not merely as instruments. 325
Burlak et al. He acknowledges how the “pluralization of lifestyles” makes a unified societal agreement unlikely and that sociocultural conditions do not afford a general rejection of xenotransplantation. Theological ethics do not focus only on the religious aspect, but offer a perspective to the ethical and social discourse on xenotransplantation that takes into account the impact of biographical, psychosocial, culture-bound, and ideological preconditions and how they relate to a personal identity. He concludes that upon consideration, xenotransplantation is a permissible form of therapy, so long as biomedical ethics are followed, including a respectful treatment of animals. In a companion piece, Sautermeister et al. [4] address the theological ethical considerations of xenotransplantation from an interdisciplinary perspective. In a summary of lectures given at the symposium “Xenotransplantation – a challenge to theological ethics”, viewpoints on xenotransplantation from Christian, Jewish, and Muslim perspectives are given. Regarding Christian ethics, from an anthropocentric perspective, xenotransplantation cannot be definitively rejected as long as man’s identity, integrity, and dignity are not compromised. The use of animals is permissible, akin to use for agriculture or consumption, so long as the moral imperative that they are not treated arbitrarily is observed. Historically, there has been a progression of compassion for fellow mankind and animals such that in the context of xenotransplantation, there should be a transparency of motives other than a therapeutic intent. The core principle of Jewish ethics that human life should be preserved supersedes most other religious obligations, and with regard to xenotransplantation, using organs from animals that are considered impure is permissible because there is no “pleasure” (Hana’a) and they are being used for the benefit of man. Islam also ascribes to the notion that man has an “obligation to preserve one’s own health”. Animals are seen as a sign of God’s mercifulness and as moral objects that do not have the same dignity as man; however, exploitation or infliction of pain should be avoided. Hence, xenotransplantation is justifiable for therapeutic use in Islam. There is also a moral obligation to the human recipients of xenotransplantation. Building on the foundation of informed consent, there needs to be a transparent public discourse that addresses fair distribution, organ shortage, and animal welfare. While there are no fundamental religious prohibitions to xenotransplantation, the authors acknowledge that patient autonomy may lead to an individual’s refusal of treatment and should be respected. 326
Genetically engineered pigs
When discussing the possibility of generating a pig-to-human xenotransplantation model, the first barriers to overcome are antigens on the surface of pig cells/tissues. Two of the more characterized xenoantigens are the carbohydrate galactose-a1, 3-galactose (aGal, synthesized by GGTA1) and N-glycolylneuraminic acid (Neu5Gc, synthesized by CMAH), but additional xenoreactive antibodies are of concern such as b1,4 N-acetylgalactosaminyl transferase (b4GalNT2). Estrada et al. [5] examined human and NHP antibody binding to peripheral blood mononuclear cells (PBMCs) derived from genetically engineered pigs devoid of these carbohydrate-modulating genes (GGTA1, GGTA1/ CMAH, and GGTA1/CMAH/b4GalNT2). The cells from GGTA1/CMAH/b4GalNT2 null pigs exhibited a reduction in human IgM and IgG binding when compared to cells lacking both GGTA1 and CMAH. However in the NHP antibody reactivity experiments, cells lacking both GGTA1 and CMAH had an increase in NHP antibodies binding when compared to cells lacking either GGTA1 alone or to those null in GGTA1/CMAH/ b4GalNT2. The authors concluded that ablation of the b4GalNT2 gene reduces antibody binding in both humans and NHPs but that immunity in NHPs highlights the complexities of xenoantigens. These results overall were useful in human sera but demonstrated a potential limitation in the use of NHP as a model for examining genetic modifications in the pig for the use of human xenotransplantation. Using pig organs for liver xenotransplantation results in lethal thrombocytopenia due to the mechanism of human platelet removal in the donor pig organ. It has been suggested that the mechanism associated with platelet removal is partially dependent upon the asialoglycoprotein receptor (ASGR) [6–8]. Using the transcription activator-like effector nuclease (TALEN) platform, Paris et al. [9] have targeted the ASGR1 gene resulting in ASGR1 knockout pigs. The authors originally mutated the ASGR1 gene in adult porcine liver-derived cells and generated six cloned fetuses using somatic cell nuclear transfer (SCNT). Fibroblast cells were harvested from the cloned fetuses and SCNT was performed using these cells to generate 22 live piglets. The live piglets appeared normal and were evaluated for xenogeneic human platelet uptake. Compared to wild-type pigs (ASGR1 +/+), livers from ASGR1 null pigs (ASGR1 / ) exhibited a decrease in human platelet uptake. The authors concluded that the disruption of the ASGR1 gene does reduce platelet destruction, but additional
Xenotransplantation literature update modifications, such as the CD18 receptor, are likely required to completely reduce thrombocytopenia. Preclinical research
Higginbotham et al. [10] sought to enhance the survival of pig-to-primate kidney xenografts by screening potential NHP for low anti-pig antibody titers prior to xenotransplantation. The authors then transplanted NHP using GGTA1 KO / CD55 transgenic pig kidneys with T-cell depletion and immune suppression. As has been seen in allotransplantation, the NHP with a high pre-transplant antibody titer rejected the pig kidney in the first week despite the use of genetically modified pigs and immune suppression. A low pre-transplant antibody titer along with maintenance anti-CD154 antibody therapy allowed normal kidney function greater than 125 days. Biopsies taken at day 100 showed no evidence of antibody- or cellular-mediated rejection. This study describes the longest survival of pig-toNHP kidney xenotransplantation to date. References 1. DENNER J, GARAHAM M. Xenotransplantation of islet cells: what can the non-human primate model bring for the evaluation of efficacy and safety? Xenotransplantation 2015; 22: 231–235.
2. DENVER J. Xenotransplantation and hepatitis E virus. Xenotransplantation 2015; 22: 167–173. 3. SAUTERMEISTER J. Xenotransplantation from the perspective of moral theology. Xenotransplantation 2015; 22: 183–191. 4. SAUTERMEISTER J, MATHIEU R, BOGNER V. Xenotransplantation-theological-ethical considerations in an interdisciplinary symposium. Xenotransplantation 2015; 22: 174– 182. 5. ESTRADA JL, MARTENS G, LI P et al. Evaluation of human and non-human primate antibody binding to pig cells lacking GGTA1/CMAH/Beta4GalNT2 genes. Xenotransplantation 2015; 22: 194–202. 6. PARIS LL, CHIHARA RK, REYES LM et al. ASGR1 expressed by porcine enriched liver sinusoidal endothelial cells mediates human platelet phagocytosis in vitro. Xenotransplantation 2011; 18: 245–251. 7. CHIHARA RK, PARIS LL, REYES LM et al. Primary porcine Kupffer cell phagocytosis of human platelets involves the CD18 receptor. Transplantation 2011; 92: 739–744. 8. PENG Q, YEH H, WEI L et al. Mechanisms of xenogeneic baboon platelet aggregation and phagocytosis by porcine liver sinusoidal endothelial cells. PLoS One 2012; 7: e47273. 9. PARIS LL, ESTRADA JL, PING L et al. Reduced human platelet uptake by pig livers deficient in the asialoglycoprotein receptor 1 protein. Xenotransplantation 2015; 22: 203– 210. 10. HIGGINBOTHAM L, MATHEWS D, BREEDEN CA et al. Pretransplant antibody screening and anti-CD154 costimulation blockade promote long-term xenograft survival in a pig to primate kidney transplant model. Xenotransplantation 2015; 22: 221–230.
327
