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Xenotransplantation. 2015 May-Jun;22(3):194-202. doi: 10.1111/xen.12161. Epub 2015 Mar 1.

Evaluation of human and non-human primate antibody binding to pig cells lacking GGTA1/CMAH/β4GalNT2 genes.

Author information

1
Department of Surgery, Indiana University School of Medicine, Indianapolis, IN, USA.
2
Yerkes National Primate Research Center, Atlanta, GA, USA.
3
Emory Transplant Center and Department of Surgery, Emory University, Atlanta, GA, USA.
4
Department of Surgery, Indiana University Health, Indianapolis, IN, USA.
5
IU Health Transplant Institute, Indianapolis, IN, USA.

Abstract

BACKGROUND:

Simultaneous inactivation of pig GGTA1 and CMAH genes eliminates carbohydrate xenoantigens recognized by human antibodies. The β4GalNT2 glycosyltransferase may also synthesize xenoantigens. To further characterize glycan-based species incompatibilities, we examined human and non-human primate antibody binding to cells derived from genetically modified pigs lacking these carbohydrate-modifying genes.

METHODS:

The Cas9 endonuclease and gRNA were used to create pigs lacking GGTA1, GGTA1/CMAH, or GGTA1/CMAH/β4GalNT2 genes. Peripheral blood mononuclear cells were isolated from these animals and examined for binding to IgM and IgG from humans, rhesus macaques, and baboons.

RESULTS:

Cells from GGTA1/CMAH/β4GalNT2 deficient pigs exhibited reduced human IgM and IgG binding compared to cells lacking both GGTA1 and CMAH. Non-human primate antibody reactivity with cells from the various pigs exhibited a slightly different pattern of reactivity than that seen in humans. Simultaneous inactivation of the GGTA1 and CMAH genes increased non-human primate antibody binding compared to cells lacking either GGTA1 only or to those deficient in GGTA1/CMAH/β4GalNT2.

CONCLUSIONS:

Inactivation of the β4GalNT2 gene reduces human and non-human primate antibody binding resulting in diminished porcine xenoantigenicity. The increased humoral immunity of non-human primates toward GGTA1-/CMAH-deficient cells compared to pigs lacking either GGTA1 or GGTA1/CMAH/β4GalNT2 highlights the complexities of carbohydrate xenoantigens and suggests potential limitations of the non-human primate model for examining some genetic modifications. The progressive reduction of swine xenoantigens recognized by human immunoglobulin through inactivation of pig GGTA1/CMAH/β4GalNT2 genes demonstrates that the antibody barrier to xenotransplantation can be minimized by genetic engineering.

KEYWORDS:

CRISPR; Cas9; antibody; genetic engineering; primate; swine; xenoantigen; β4GalNT2

PMID:
25728481
PMCID:
PMC4464961
DOI:
10.1111/xen.12161
[Indexed for MEDLINE]
Free PMC Article

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