Journal
XENOTRANSPLANTATION
Volume 22, Issue 3, Pages 194-202Publisher
WILEY
DOI: 10.1111/xen.12161
Keywords
antibody; Cas9; CRISPR; genetic engineering; primate; swine; xenoantigen; beta 4GalNT2
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Funding
- IU Health Transplant Institute
- Indiana University Health
- NIH [P51 RR00165]
- National Center for Research Resources via a Research Facilities Improvement Grant [C06RR10601-01]
- ORIP/OD [P51OD011132]
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BackgroundSimultaneous inactivation of pig GGTA1 and CMAH genes eliminates carbohydrate xenoantigens recognized by human antibodies. The 4GalNT2 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. MethodsThe Cas9 endonuclease and gRNA were used to create pigs lacking GGTA1, GGTA1/CMAH, or GGTA1/CMAH/4GalNT2 genes. Peripheral blood mononuclear cells were isolated from these animals and examined for binding to IgM and IgG from humans, rhesus macaques, and baboons. ResultsCells from GGTA1/CMAH/4GalNT2 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/4GalNT2. ConclusionsInactivation of the 4GalNT2 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/4GalNT2 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/4GalNT2 genes demonstrates that the antibody barrier to xenotransplantation can be minimized by genetic engineering.
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