Double knockout pigs deficient in N-glycolylneuraminic acid and Galactose α-1,3-Galactose reduce the humoral barrier to xenotransplantation

Andrew J. Lutz; Ping Li; Jose L. Estrada; Richard A. Sidner; Ray K. Chihara; Susan M. Downey; Christopher Burlak; Zheng Yu Wang; Luz M. Reyes; Bess Ivary; Fuqin Yin; Ross L. Blankenship; Leela L. Paris; A. Joseph Tector

doi:10.1111/xen.12019

Double knockout pigs deficient in N-glycolylneuraminic acid and Galactose α-1,3-Galactose reduce the humoral barrier to xenotransplantation

Andrew J. Lutz, Ping Li, Jose L. Estrada, Richard A. Sidner, Ray K. Chihara, Susan M. Downey, Christopher Burlak, Zheng Yu Wang, Luz M. Reyes, Bess Ivary, Fuqin Yin, Ross L. Blankenship, Leela L. Paris, A. Joseph Tector

Surgery

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Abstract

Background: Clinical xenotransplantation is not possible because humans possess antibodies that recognize antigens on the surface of pig cells. Galα-1,3-Gal (Gal) and N-glycolylneuraminic acid (Neu5Gc) are two known xenoantigens. Methods: We report the homozygous disruption of the α1, 3-galactosyltransferase (GGTA1) and the cytidine monophosphate-N- acetylneuraminic acid hydroxylase (CMAH) genes in liver-derived female pig cells using zinc-finger nucleases (ZFNs). Somatic cell nuclear transfer (SCNT) was used to produce healthy cloned piglets from the genetically modified liver cells. Antibody-binding and antibody-mediated complement-dependent cytotoxicity assays were used to examine the immunoreactivity of pig cells deficient in Neu5Gc and Gal. Results This approach enabled rapid production of a pig strain deficient in multiple genes without extensive breeding protocols. Immune recognition studies showed that pigs lacking both CMAH and GGTA1 gene activities reduce the humoral barrier to xenotransplantation, further than pigs lacking only GGTA1. Conclusions This technology will accelerate the development of pigs for xenotransplantation research.

Original language	English (US)
Pages (from-to)	27-35
Number of pages	9
Journal	Xenotransplantation
Volume	20
Issue number	1
DOIs	https://doi.org/10.1111/xen.12019
State	Published - Jan 2013

Keywords

Neu5Gc
humoral
pig
xenotransplantation
zinc-finger nuclease

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Lutz, A. J., Li, P., Estrada, J. L., Sidner, R. A., Chihara, R. K., Downey, S. M., Burlak, C., Wang, Z. Y., Reyes, L. M., Ivary, B., Yin, F., Blankenship, R. L., Paris, L. L., & Tector, A. J. (2013). Double knockout pigs deficient in N-glycolylneuraminic acid and Galactose α-1,3-Galactose reduce the humoral barrier to xenotransplantation. Xenotransplantation, 20(1), 27-35. https://doi.org/10.1111/xen.12019

Lutz, AJ, Li, P, Estrada, JL, Sidner, RA, Chihara, RK, Downey, SM, Burlak, C, Wang, ZY, Reyes, LM, Ivary, B, Yin, F, Blankenship, RL, Paris, LL & Tector, AJ 2013, 'Double knockout pigs deficient in N-glycolylneuraminic acid and Galactose α-1,3-Galactose reduce the humoral barrier to xenotransplantation', Xenotransplantation, vol. 20, no. 1, pp. 27-35. https://doi.org/10.1111/xen.12019

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abstract = "Background: Clinical xenotransplantation is not possible because humans possess antibodies that recognize antigens on the surface of pig cells. Galα-1,3-Gal (Gal) and N-glycolylneuraminic acid (Neu5Gc) are two known xenoantigens. Methods: We report the homozygous disruption of the α1, 3-galactosyltransferase (GGTA1) and the cytidine monophosphate-N- acetylneuraminic acid hydroxylase (CMAH) genes in liver-derived female pig cells using zinc-finger nucleases (ZFNs). Somatic cell nuclear transfer (SCNT) was used to produce healthy cloned piglets from the genetically modified liver cells. Antibody-binding and antibody-mediated complement-dependent cytotoxicity assays were used to examine the immunoreactivity of pig cells deficient in Neu5Gc and Gal. Results This approach enabled rapid production of a pig strain deficient in multiple genes without extensive breeding protocols. Immune recognition studies showed that pigs lacking both CMAH and GGTA1 gene activities reduce the humoral barrier to xenotransplantation, further than pigs lacking only GGTA1. Conclusions This technology will accelerate the development of pigs for xenotransplantation research.",

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doi = "10.1111/xen.12019",

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AU - Sidner, Richard A.

AU - Chihara, Ray K.

AU - Downey, Susan M.

AU - Burlak, Christopher

AU - Wang, Zheng Yu

AU - Reyes, Luz M.

AU - Ivary, Bess

AU - Yin, Fuqin

AU - Blankenship, Ross L.

AU - Paris, Leela L.

AU - Tector, A. Joseph

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N2 - Background: Clinical xenotransplantation is not possible because humans possess antibodies that recognize antigens on the surface of pig cells. Galα-1,3-Gal (Gal) and N-glycolylneuraminic acid (Neu5Gc) are two known xenoantigens. Methods: We report the homozygous disruption of the α1, 3-galactosyltransferase (GGTA1) and the cytidine monophosphate-N- acetylneuraminic acid hydroxylase (CMAH) genes in liver-derived female pig cells using zinc-finger nucleases (ZFNs). Somatic cell nuclear transfer (SCNT) was used to produce healthy cloned piglets from the genetically modified liver cells. Antibody-binding and antibody-mediated complement-dependent cytotoxicity assays were used to examine the immunoreactivity of pig cells deficient in Neu5Gc and Gal. Results This approach enabled rapid production of a pig strain deficient in multiple genes without extensive breeding protocols. Immune recognition studies showed that pigs lacking both CMAH and GGTA1 gene activities reduce the humoral barrier to xenotransplantation, further than pigs lacking only GGTA1. Conclusions This technology will accelerate the development of pigs for xenotransplantation research.

AB - Background: Clinical xenotransplantation is not possible because humans possess antibodies that recognize antigens on the surface of pig cells. Galα-1,3-Gal (Gal) and N-glycolylneuraminic acid (Neu5Gc) are two known xenoantigens. Methods: We report the homozygous disruption of the α1, 3-galactosyltransferase (GGTA1) and the cytidine monophosphate-N- acetylneuraminic acid hydroxylase (CMAH) genes in liver-derived female pig cells using zinc-finger nucleases (ZFNs). Somatic cell nuclear transfer (SCNT) was used to produce healthy cloned piglets from the genetically modified liver cells. Antibody-binding and antibody-mediated complement-dependent cytotoxicity assays were used to examine the immunoreactivity of pig cells deficient in Neu5Gc and Gal. Results This approach enabled rapid production of a pig strain deficient in multiple genes without extensive breeding protocols. Immune recognition studies showed that pigs lacking both CMAH and GGTA1 gene activities reduce the humoral barrier to xenotransplantation, further than pigs lacking only GGTA1. Conclusions This technology will accelerate the development of pigs for xenotransplantation research.

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KW - xenotransplantation

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Double knockout pigs deficient in N-glycolylneuraminic acid and Galactose α-1,3-Galactose reduce the humoral barrier to xenotransplantation

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