An Efficacious Transgenic Strategy for Triple Knockout of Xeno-Reactive Antigen Genes GGTA1, CMAH, and B4GALNT2 from Jeju Native Pigs

Abstract

Pigs are promising donors of biological materials for xenotransplantation; however, cell surface carbohydrate antigens, including galactose-alpha-1,3-galactose (α-Gal), N-glycolylneuraminic acid (Neu5Gc), and Sd blood group antigens, play a significant role in porcine xenograft rejection. Inactivating swine endogenous genes, including GGTA1, CMAH, and B4GALNT2, decreases the binding ratio of human IgG/IgM in peripheral blood mononuclear cells and erythrocytes and impedes the effectiveness of α-Gal, Neu5Gc, and Sd, thereby successfully preventing hyperacute rejection. Therefore, in this study, an effective transgenic system was developed to target GGTA1, CMAH, and B4GALNT2 using CRISPR-CAS9 and develop triple-knockout pigs. The findings revealed that all three antigens (α-Gal, Neu5Gc, and Sd) were not expressed in the heart, lungs, or liver of the triple-knockout Jeju Native Pigs (JNPs), and poor expression of α-Gal and Neu5G was confirmed in the kidneys. Compared with the kidney, heart, and lung tissues from wild-type JNPs, those from GGTA1/CMAH/ B4GALNT2 knockout-recipient JNPs exhibited reduced human IgM and IgG binding and expression of each immunological rejection component. Hence, reducing the expression of swine xenogeneic antigens identifiable by human immunoglobulins can lessen the immunological rejection against xenotransplantation. The findings support the possibility of employing knockout JNP organs for xenogeneic transplantation to minimize or completely eradicate rejection using multiple gene-editing methods.

Keywords: CRISPR-CAS9 system; Jeju native pigs; immune rejection; xenotransplantation.

Figure 1

Schematic workflow of the generation of triple-knockout Jeju Native Pigs (JNPs), including gene editing strategies and steps required to obtain the modified JNPs. (A) Schematic diagram for triple-knockout development; (B) the sequences of GGTA1, CMAH, and B4GALNT2; (C) deep sequence analysis of the target region of genes in delivered piglets and details of the target sequences in exon 7 (GGTA1), exon 2 (CMAH), and exon 4 (B4GALNT2); (D) knockout piglets produced. SCNT, somatic cell nuclear transfer. (B) Red color nucleotide sequences: sgRNA and PAM sequences; (B) Red box indicate deleted nucleotide base sequence by RNP complex; (C) red highlighted sequences indicate mutated amino acid.

Figure 2

Procedure for triple-knockout development. (A) Schematic representation of designing gRNA (vector schematic diagram including Guide RNA); (B) preparation of vector expressing Cas9 and gRNA targeting triple gene; M: 1 kb DNA ladder, Lane 1: pSpCas9(BB)-2A-Puro vector treated with BpiI, Lane 2: GGTA1 target vector treated with BpiI, Lane 3: CMAH target vector treated with BpiI, Lane 4: B4GALNT2 target vector treated with BpiI; (C–E) RFLP analysis of GGTA1, CMAH, and B4GALNT2, where the PCR product from each single cell was treated with BsrI; (F) constructed TKO cell line; (G) preparing immature oocyte; (H) expanded cumulus cell–oocyte complex at day 2 after in vitro maturation; (I) blastocyst was developed at day 7 after SCNT; (J,K) embryo transfer and development of healthy offspring. (A) Red color nucleotide sequences: sgRNA and PAM sequence; (C–E) 023 indicate selected construct.

Figure 3

Histology of the primary organs of TKO JNPs. Paraffin-embedded kidney, heart, lung, and liver tissues from wild-type and TKO JNPs were sectioned and hematoxylin and eosin (H&E) staining was performed. Scale bar = 100 μm.

Figure 4

Expressions of α-Gal, Neu5Gc, and Sd(a) along with human IgM and IgG antibody binding assays in the kidney and heart of wild-type and TKO JNPs. Immunofluorescence analysis of α-Gal, Neu5Gc, and Sd(a) was performed using the kidney (A) and heart (B) of wild-type and TKO JNPs. In addition, human IgM and IgG binding assays were performed using the kidney and heart tissues. Represented images were overlaid with blue (DAPI) for nucleus and green (Alexa Fluor 488) for target proteins (α-Gal, Neu5Gc, and Sd(a)). Scale bar = 30 μm.

Figure 5

Expressions of α-Gal, Neu5Gc, and Sd(a) along with human IgM and IgG antibody binding assays in the lungs and liver of wild-type and TKO JNPs. Immunofluorescence analysis of α-Gal, Neu5Gc, and Sd(a) was performed using wild-type and TKO JNP lung (A) and liver (B) tissues. In addition, human IgM and IgG binding assay was performed in the lung and liver tissues. Represented images were overlaid with blue (DAPI) for nucleus and green (Alexa Fluor 488) for target proteins (α-Gal, Neu5Gc, and Sd(a)). Scale bar = 30 μm.