Erythrocytes from GGTA1/CMAH knockout pigs

Implications for xenotransfusion and testing in non-human primates

Zheng Yu Wang, Christopher Burlak, Jose L. Estrada, Ping Li, Matthew F. Tector, A. Joseph Tector

Research output: Contribution to journalArticle

19 Citations (Scopus)

Abstract

Background Pig erythrocytes are potentially useful to solve the worldwide shortage of human blood for transfusion. Domestic pig erythrocytes, however, express antigens that are bound by human preformed antibodies. Advances in genetic engineering have made it possible to rapidly knock out the genes of multiple xenoantigens, namely galactose α1,3 galactose (aGal) and N-glycolylneuraminic acid (Neu5Gc). We have recently targeted the GGTA1 and CMAH genes with zinc finger endonucleases resulting in double knockout pigs that no longer express aGal or Neu5Gc and attract significantly fewer human antibodies. In this study, we characterized erythrocytes from domestic and genetically modified pigs, baboons, chimpanzees, and humans for binding of human and baboon natural antibody, and complement-mediated lysis. Methods Distribution of anti-Neu5Gc IgG and IgM in pooled human AB serum was analyzed by ELISA. Erythrocytes from domestic pigs (Dom), aGal knockout pigs (GGTA1 KO), aGal and Neu5Gc double knockout pigs (GGTA1/CMAH KO), baboons, chimpanzees, and humans were analyzed by flow cytometry for aGal and Neu5Gc expression. In vitro comparative analysis of erythrocytes was conducted with pooled human AB serum and baboon serum. Total antibody binding was accessed by hemagglutination; complement-dependent lysis was measured by hemolytic assay; IgG or IgM binding to erythrocytes was characterized by flow cytometry. Results The pooled human AB serum contained 0.38 μg/ml anti-Neu5Gc IgG and 0.085 μg/ml anti-Neu5Gc IgM. Both Gal and Neu5Gc were not detectable on GGTA1/CMAH KO erythrocytes. Hemagglutination of GGTA1/CMAH KO erythrocytes with human serum was 3.5-fold lower compared with GGTA1 KO erythrocytes, but 1.6-fold greater when agglutinated with baboon serum. Hemolysis of GGTA1/CMAH KO erythrocytes by human serum (25%) was reduced 9-fold compared with GGTA1 KO erythrocytes, but increased 1.64-fold by baboon serum. Human IgG binding was reduced 27-fold on GGTA1/CMAH KO erythrocytes compared with GGTA1 KO erythrocytes, but markedly increased 3-fold by baboon serum IgG. Human IgM binding was decreased 227-fold on GGTA1/CMAH KO erythrocytes compared with GGTA1 KO erythrocytes, but enhanced 5-fold by baboon serum IgM. Conclusions Removal of aGal and Neu5Gc antigens from pig erythrocytes significantly reduced human preformed antibody-mediated cytotoxicity but may have complicated future in vivo analysis by enhancing reactivity from baboons. The creation of the GGTA1/CMAH KO pig has provided the xenotransplantation researcher with organs and cells that attract fewer human antibodies than baboon and our closest primate relative, chimpanzee. These finding suggest that while GGTA1/CMAH KO erythrocytes may be useful for human transfusions, in vivo testing in the baboon may not provide a direct transplantation to the clinic.

Original languageEnglish
Pages (from-to)376-384
Number of pages9
JournalXenotransplantation
Volume21
Issue number4
DOIs
StatePublished - 2014

Fingerprint

Primates
Swine
Erythrocytes
Papio
Serum
Pan troglodytes
Antibodies
Immunoglobulin M
Sus scrofa
Immunoglobulin G
Hemagglutination
Galactose
Flow Cytometry
Heterophile Antigens
Antigens
Gene Knockout Techniques
Heterologous Transplantation
Genetic Engineering
Endonucleases
Zinc Fingers

Keywords

  • erythrocyte
  • Neu5Gc
  • xenoantigen
  • xenotransfusion

ASJC Scopus subject areas

  • Transplantation
  • Immunology

Cite this

Wang, Z. Y., Burlak, C., Estrada, J. L., Li, P., Tector, M. F., & Tector, A. J. (2014). Erythrocytes from GGTA1/CMAH knockout pigs: Implications for xenotransfusion and testing in non-human primates. Xenotransplantation, 21(4), 376-384. https://doi.org/10.1111/xen.12106

Erythrocytes from GGTA1/CMAH knockout pigs : Implications for xenotransfusion and testing in non-human primates. / Wang, Zheng Yu; Burlak, Christopher; Estrada, Jose L.; Li, Ping; Tector, Matthew F.; Tector, A. Joseph.

In: Xenotransplantation, Vol. 21, No. 4, 2014, p. 376-384.

Research output: Contribution to journalArticle

Wang, Zheng Yu ; Burlak, Christopher ; Estrada, Jose L. ; Li, Ping ; Tector, Matthew F. ; Tector, A. Joseph. / Erythrocytes from GGTA1/CMAH knockout pigs : Implications for xenotransfusion and testing in non-human primates. In: Xenotransplantation. 2014 ; Vol. 21, No. 4. pp. 376-384.
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abstract = "Background Pig erythrocytes are potentially useful to solve the worldwide shortage of human blood for transfusion. Domestic pig erythrocytes, however, express antigens that are bound by human preformed antibodies. Advances in genetic engineering have made it possible to rapidly knock out the genes of multiple xenoantigens, namely galactose α1,3 galactose (aGal) and N-glycolylneuraminic acid (Neu5Gc). We have recently targeted the GGTA1 and CMAH genes with zinc finger endonucleases resulting in double knockout pigs that no longer express aGal or Neu5Gc and attract significantly fewer human antibodies. In this study, we characterized erythrocytes from domestic and genetically modified pigs, baboons, chimpanzees, and humans for binding of human and baboon natural antibody, and complement-mediated lysis. Methods Distribution of anti-Neu5Gc IgG and IgM in pooled human AB serum was analyzed by ELISA. Erythrocytes from domestic pigs (Dom), aGal knockout pigs (GGTA1 KO), aGal and Neu5Gc double knockout pigs (GGTA1/CMAH KO), baboons, chimpanzees, and humans were analyzed by flow cytometry for aGal and Neu5Gc expression. In vitro comparative analysis of erythrocytes was conducted with pooled human AB serum and baboon serum. Total antibody binding was accessed by hemagglutination; complement-dependent lysis was measured by hemolytic assay; IgG or IgM binding to erythrocytes was characterized by flow cytometry. Results The pooled human AB serum contained 0.38 μg/ml anti-Neu5Gc IgG and 0.085 μg/ml anti-Neu5Gc IgM. Both Gal and Neu5Gc were not detectable on GGTA1/CMAH KO erythrocytes. Hemagglutination of GGTA1/CMAH KO erythrocytes with human serum was 3.5-fold lower compared with GGTA1 KO erythrocytes, but 1.6-fold greater when agglutinated with baboon serum. Hemolysis of GGTA1/CMAH KO erythrocytes by human serum (25{\%}) was reduced 9-fold compared with GGTA1 KO erythrocytes, but increased 1.64-fold by baboon serum. Human IgG binding was reduced 27-fold on GGTA1/CMAH KO erythrocytes compared with GGTA1 KO erythrocytes, but markedly increased 3-fold by baboon serum IgG. Human IgM binding was decreased 227-fold on GGTA1/CMAH KO erythrocytes compared with GGTA1 KO erythrocytes, but enhanced 5-fold by baboon serum IgM. Conclusions Removal of aGal and Neu5Gc antigens from pig erythrocytes significantly reduced human preformed antibody-mediated cytotoxicity but may have complicated future in vivo analysis by enhancing reactivity from baboons. The creation of the GGTA1/CMAH KO pig has provided the xenotransplantation researcher with organs and cells that attract fewer human antibodies than baboon and our closest primate relative, chimpanzee. These finding suggest that while GGTA1/CMAH KO erythrocytes may be useful for human transfusions, in vivo testing in the baboon may not provide a direct transplantation to the clinic.",
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T1 - Erythrocytes from GGTA1/CMAH knockout pigs

T2 - Implications for xenotransfusion and testing in non-human primates

AU - Wang, Zheng Yu

AU - Burlak, Christopher

AU - Estrada, Jose L.

AU - Li, Ping

AU - Tector, Matthew F.

AU - Tector, A. Joseph

PY - 2014

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N2 - Background Pig erythrocytes are potentially useful to solve the worldwide shortage of human blood for transfusion. Domestic pig erythrocytes, however, express antigens that are bound by human preformed antibodies. Advances in genetic engineering have made it possible to rapidly knock out the genes of multiple xenoantigens, namely galactose α1,3 galactose (aGal) and N-glycolylneuraminic acid (Neu5Gc). We have recently targeted the GGTA1 and CMAH genes with zinc finger endonucleases resulting in double knockout pigs that no longer express aGal or Neu5Gc and attract significantly fewer human antibodies. In this study, we characterized erythrocytes from domestic and genetically modified pigs, baboons, chimpanzees, and humans for binding of human and baboon natural antibody, and complement-mediated lysis. Methods Distribution of anti-Neu5Gc IgG and IgM in pooled human AB serum was analyzed by ELISA. Erythrocytes from domestic pigs (Dom), aGal knockout pigs (GGTA1 KO), aGal and Neu5Gc double knockout pigs (GGTA1/CMAH KO), baboons, chimpanzees, and humans were analyzed by flow cytometry for aGal and Neu5Gc expression. In vitro comparative analysis of erythrocytes was conducted with pooled human AB serum and baboon serum. Total antibody binding was accessed by hemagglutination; complement-dependent lysis was measured by hemolytic assay; IgG or IgM binding to erythrocytes was characterized by flow cytometry. Results The pooled human AB serum contained 0.38 μg/ml anti-Neu5Gc IgG and 0.085 μg/ml anti-Neu5Gc IgM. Both Gal and Neu5Gc were not detectable on GGTA1/CMAH KO erythrocytes. Hemagglutination of GGTA1/CMAH KO erythrocytes with human serum was 3.5-fold lower compared with GGTA1 KO erythrocytes, but 1.6-fold greater when agglutinated with baboon serum. Hemolysis of GGTA1/CMAH KO erythrocytes by human serum (25%) was reduced 9-fold compared with GGTA1 KO erythrocytes, but increased 1.64-fold by baboon serum. Human IgG binding was reduced 27-fold on GGTA1/CMAH KO erythrocytes compared with GGTA1 KO erythrocytes, but markedly increased 3-fold by baboon serum IgG. Human IgM binding was decreased 227-fold on GGTA1/CMAH KO erythrocytes compared with GGTA1 KO erythrocytes, but enhanced 5-fold by baboon serum IgM. Conclusions Removal of aGal and Neu5Gc antigens from pig erythrocytes significantly reduced human preformed antibody-mediated cytotoxicity but may have complicated future in vivo analysis by enhancing reactivity from baboons. The creation of the GGTA1/CMAH KO pig has provided the xenotransplantation researcher with organs and cells that attract fewer human antibodies than baboon and our closest primate relative, chimpanzee. These finding suggest that while GGTA1/CMAH KO erythrocytes may be useful for human transfusions, in vivo testing in the baboon may not provide a direct transplantation to the clinic.

AB - Background Pig erythrocytes are potentially useful to solve the worldwide shortage of human blood for transfusion. Domestic pig erythrocytes, however, express antigens that are bound by human preformed antibodies. Advances in genetic engineering have made it possible to rapidly knock out the genes of multiple xenoantigens, namely galactose α1,3 galactose (aGal) and N-glycolylneuraminic acid (Neu5Gc). We have recently targeted the GGTA1 and CMAH genes with zinc finger endonucleases resulting in double knockout pigs that no longer express aGal or Neu5Gc and attract significantly fewer human antibodies. In this study, we characterized erythrocytes from domestic and genetically modified pigs, baboons, chimpanzees, and humans for binding of human and baboon natural antibody, and complement-mediated lysis. Methods Distribution of anti-Neu5Gc IgG and IgM in pooled human AB serum was analyzed by ELISA. Erythrocytes from domestic pigs (Dom), aGal knockout pigs (GGTA1 KO), aGal and Neu5Gc double knockout pigs (GGTA1/CMAH KO), baboons, chimpanzees, and humans were analyzed by flow cytometry for aGal and Neu5Gc expression. In vitro comparative analysis of erythrocytes was conducted with pooled human AB serum and baboon serum. Total antibody binding was accessed by hemagglutination; complement-dependent lysis was measured by hemolytic assay; IgG or IgM binding to erythrocytes was characterized by flow cytometry. Results The pooled human AB serum contained 0.38 μg/ml anti-Neu5Gc IgG and 0.085 μg/ml anti-Neu5Gc IgM. Both Gal and Neu5Gc were not detectable on GGTA1/CMAH KO erythrocytes. Hemagglutination of GGTA1/CMAH KO erythrocytes with human serum was 3.5-fold lower compared with GGTA1 KO erythrocytes, but 1.6-fold greater when agglutinated with baboon serum. Hemolysis of GGTA1/CMAH KO erythrocytes by human serum (25%) was reduced 9-fold compared with GGTA1 KO erythrocytes, but increased 1.64-fold by baboon serum. Human IgG binding was reduced 27-fold on GGTA1/CMAH KO erythrocytes compared with GGTA1 KO erythrocytes, but markedly increased 3-fold by baboon serum IgG. Human IgM binding was decreased 227-fold on GGTA1/CMAH KO erythrocytes compared with GGTA1 KO erythrocytes, but enhanced 5-fold by baboon serum IgM. Conclusions Removal of aGal and Neu5Gc antigens from pig erythrocytes significantly reduced human preformed antibody-mediated cytotoxicity but may have complicated future in vivo analysis by enhancing reactivity from baboons. The creation of the GGTA1/CMAH KO pig has provided the xenotransplantation researcher with organs and cells that attract fewer human antibodies than baboon and our closest primate relative, chimpanzee. These finding suggest that while GGTA1/CMAH KO erythrocytes may be useful for human transfusions, in vivo testing in the baboon may not provide a direct transplantation to the clinic.

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