Human immune reactivity of GGTA1/CMAH/A3GALT2 triple knockout Yucatan miniature pigs

Abstract

In this study, we investigated the effect of a triple knockout of the genes alpha-1,3-galactosyltransferase (GGTA1), cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH), and alpha 1,3-galactosyltransferase 2 (A3GALT2) in Yucatan miniature pigs on human immune reactivity. We used the CRISPR/Cas9 system to create pigs lacking GGTA1 (GTKO) and GGTA1/CMAH/A3GALT2 triple gene knockout (TKO). The expression of all three xenoantigens was absent in TKO pigs, but there was no additional reduction in the level of Galα1,3Gal (αGal) epitopes expression in the A3GALT2 gene KO. Peripheral blood mononuclear cells (PBMCs), aorta endothelial cells (AECs), and cornea endothelial cells (CECs) were isolated from these pigs, and their ability to bind human IgM/IgG and their cytotoxicity in human sera were evaluated. Compared to wild type (WT) pigs, the level of human antibody binding of the PBMCs, AECs, and CECs of the transgenic pigs (GTKO and TKO) was significantly reduced. However, there were significant differences in human antibody binding between GTKO and TKO depending on the cell type. Human antibody binding of TKO pigs was less than that of GTKO on PBMCs but was similar between GTKO and TKO pigs for AECs and CECs. Cytotoxicity of transgenic pig (GTKO and TKO) PBMCs and AECs was significantly reduced compared to that of WT pigs. However, TKO pigs showed a reduction in cytotoxicity compared to GTKO pigs on PBMCs, whereas in AECs from both TKO and GTKO pigs, there was no difference. The cytotoxicity of transgenic pig CECs was significantly decreased from that of WT at 300 min, but there was no significant reduction in TKO pigs from GTKO. Our results indicate that genetic modification of donor pigs for xenotransplantation should be tailored to the target organ and silencing of additional genes such as CMAH or A3GALT2 based on GTKO might not be essential in Yucatan miniature pigs.

Keywords: A3GALT2; Antibody binding; CMAH; CRISPR/Cas9; Cytotoxicity; GGTA1.

Fig. 1

Workflow of the generation of GGTA1, CMAH, and A3GALT2 gene KO pigs using CRISPR/Cas9 technology. Ear fibroblast cells of Yucatan miniature pigs were transfected with a Cas9-GGTA1 vector. We produced cloned GGTA1 KO (GTKO) piglets using GS-IB4 lectin negative cells as nuclear donors for SCNT. We then simultaneously targeted the CMAH and A3GALT2 genes using CRISPR/Cas9 in ear fibroblast cells from a GTKO piglet. SCNT was performed with these transfected cells, and GGTA1/CMAH/A3GALT2 triple gene KO (TKO) piglets were produced.

Fig. 2

Generation of GGTA1, CMAH, and A3GALT2 gene KO transgenic cell lines. Schematic diagram of the CRISPR/Cas9 system targeting pigs. a GGTA1, b CMAH, and c A3GALT2 locus. The target site was in exon 4 of A3GALT2 and exon 9 of GGTA1 and CMAH genes. The red highlight indicates sgRNA targeting sites, and the blue underlined highlight indicates protospacer adjacent motif (PAM).

Fig. 3

Generation of CRISPR/Cas9-induced GTKO and TKO cloned piglets. DNA sequence analysis and photographs of a GTKO and b TKO piglets. The DNA sequences of the cloned piglets showed a mutation identical to the sequences of the nuclear donor cells. Deletion, insertion, and shift mutations of the base pairs are indicated by dots (∙), plus (+), and delta (Δ), respectively.

Fig. 4

Immunofluorescence analysis of GTKO, TKO, and WT pigs. Tissue sections of the heart, kidney, lung, and liver from GTKO, TKO, and WT pigs were stained with a GS-IB4 lectin b anti-Neu5Gc antibodies. Expression of αGal and Neu5Gc antigens was widespread in WT pig tissues. All tissues from GTKO pigs were negative for αGal, but TKO pigs were negative for both αGal and Neu5Gc antigens (nuclei, blue; Gal, red; Neu5Gc, green, magnification, × 400).

Fig. 5

Comparison of A3GALT2 expression in AECs from GTKO, TKO, and WT pigs. a Endothelial cell marker CD31 was expressed on AECs from all types of pigs. Gray histograms represent isotype control, and black histograms represent CD31. b The expression level of A3GALT2 on TKO pig AECs was significantly decreased from that of GTKO or WT pigs (MFI; mean fluorescence intensity).

Fig. 6

Comparison of αGal epitope expression on various pig cells. αGal epitope expression on a PBMCs, b AECs, and c CECs from all transgenic pigs was decreased from that of WT. However, there were no differences in the expression of αGal between GTKO and TKO pig cells (WT, red; GTKO, blue; TKO, black).

Fig. 7

Human IgM and IgG binding to GTKO, TKO, and WT pig PBMCs (a, b), AECs (c, d), and CECs (e, f). a, b Human IgM/IgG binding of PBMCs was significantly decreased in transgenic pigs when compared to WT (***P < 0.001 vs. WT). There were also significant differences in IgM/IgG binding between GTKO and TKO pigs (**P < 0.01; ***P < 0.001). c, d Human IgM/IgG binding of AECs was significantly reduced in all transgenic pigs compared to that in WT (***P < 0.001 vs. WT). However, there was no further reduction in IgM and IgG binding in TKO pigs when compared to that in GTKO pigs. e, f Human IgM/IgG binding of CECs in GTKO and TKO pigs was markedly reduced when compared to that in WT pigs (***P < 0.001 vs. WT), but there were no significant differences in IgM/IgG binding between GTKO and TKO pigs. Experiments were performed in quadruplicate.

Fig. 8

Cytotoxicity of PBMCs, AECs, and CECs from GTKO, TKO, and WT pigs. a The cytotoxicity of GTKO pigs on PBMCs was significantly lower than that on WT pigs at 60, 90, and 120 min (**P < 0.01; ***P < 0.001 vs. WT), and there was further significant reduction in the cytotoxicity of TKO pig compared to that of GTKO or WT pigs (**P < 0.01; ***P < 0.001) at all time points. b The cytotoxicity of transgenic pigs on AECs was significantly decreased compared to that of WT pigs at all time points (**P < 0.01; ***P < 0.001 vs. WT), but there was no difference in cytotoxicity between GTKO and TKO pig AECs. c The cytotoxicity of transgenic pigs on CECs was significantly reduced from WT at 300 min (*P < 0.05; ***P < 0.001 vs. WT), but there was similar cytotoxicity between GTKO and TKO pigs. Experiments were performed in quadruplicate.