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

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

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Animals, Genetically Modified
  • Antigens, Heterophile / genetics
  • Antigens, Heterophile / immunology*
  • Galactosyltransferases / genetics*
  • Graft Rejection / genetics
  • Graft Rejection / immunology
  • Humans
  • Leukocytes, Mononuclear / immunology*
  • Mixed Function Oxygenases / genetics
  • Mixed Function Oxygenases / metabolism
  • Protein Binding
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Swine
  • Transplantation, Heterologous*

Substances

  • Antigens, Heterophile
  • Recombinant Proteins
  • Mixed Function Oxygenases
  • CMPacetylneuraminate monooxygenase
  • Galactosyltransferases
  • alpha-1,3-galactosyltransferase 1, porcine