Evaluation of human and non‐human primate antibody binding to pig cells lacking <scp>GGTA</scp>1/<scp>CMAH</scp>/β4Gal<scp>NT</scp>2 genes

<jats:title>Abstract</jats:title><jats:sec><jats:title>Background</jats:title><jats:p>Simultaneous inactivation of pig <jats:styled-content style="fixed-case">GGTA</jats:styled-content>1 and <jats:styled-content style="fixed-case">CMAH</jats:styled-content> genes eliminates carbohydrate xenoantigens recognized by human antibodies. The β4Gal<jats:styled-content style="fixed-case">NT</jats:styled-content>2 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.</jats:p></jats:sec><jats:sec><jats:title>Methods</jats:title><jats:p>The Cas9 endonuclease and <jats:styled-content style="fixed-case">gRNA</jats:styled-content> were used to create pigs lacking <jats:styled-content style="fixed-case">GGTA</jats:styled-content>1, <jats:styled-content style="fixed-case">GGTA</jats:styled-content>1/<jats:styled-content style="fixed-case">CMAH</jats:styled-content>, or <jats:styled-content style="fixed-case">GGTA</jats:styled-content>1/<jats:styled-content style="fixed-case">CMAH</jats:styled-content>/β4Gal<jats:styled-content style="fixed-case">NT</jats:styled-content>2 genes. Peripheral blood mononuclear cells were isolated from these animals and examined for binding to IgM and IgG from humans, rhesus macaques, and baboons.</jats:p></jats:sec><jats:sec><jats:title>Results</jats:title><jats:p>Cells from <jats:styled-content style="fixed-case">GGTA</jats:styled-content>1/<jats:styled-content style="fixed-case">CMAH</jats:styled-content>/β4Gal<jats:styled-content style="fixed-case">NT</jats:styled-content>2 deficient pigs exhibited reduced human IgM and IgG binding compared to cells lacking both <jats:styled-content style="fixed-case">GGTA</jats:styled-content>1 and <jats:styled-content style="fixed-case">CMAH</jats:styled-content>. 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 <jats:styled-content style="fixed-case">GGTA</jats:styled-content>1 and <jats:styled-content style="fixed-case">CMAH</jats:styled-content> genes increased non‐human primate antibody binding compared to cells lacking either <jats:styled-content style="fixed-case">GGTA</jats:styled-content>1 only or to those deficient in <jats:styled-content style="fixed-case">GGTA</jats:styled-content>1/<jats:styled-content style="fixed-case">CMAH</jats:styled-content>/β4Gal<jats:styled-content style="fixed-case">NT</jats:styled-content>2.</jats:p></jats:sec><jats:sec><jats:title>Conclusions</jats:title><jats:p>Inactivation of the β4Gal<jats:styled-content style="fixed-case">NT</jats:styled-content>2 gene reduces human and non‐human primate antibody binding resulting in diminished porcine xenoantigenicity. The increased humoral immunity of non‐human primates toward <jats:styled-content style="fixed-case">GGTA</jats:styled-content>1‐/<jats:styled-content style="fixed-case">CMAH</jats:styled-content>‐deficient cells compared to pigs lacking either <jats:styled-content style="fixed-case">GGTA</jats:styled-content>1 or <jats:styled-content style="fixed-case">GGTA</jats:styled-content>1/<jats:styled-content style="fixed-case">CMAH</jats:styled-content>/β4Gal<jats:styled-content style="fixed-case">NT</jats:styled-content>2 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 <jats:styled-content style="fixed-case">GGTA</jats:styled-content>1/<jats:styled-content style="fixed-case">CMAH</jats:styled-content>/β4Gal<jats:styled-content style="fixed-case">NT</jats:styled-content>2 genes demonstrates that the antibody barrier to xenotransplantation can be minimized by genetic engineering.</jats:p></jats:sec>