Animal Molecular Breeding 2024, Vol.14, No.1, 106-118 http://animalscipublisher.com/index.php/amb 109 Acute Rejection: This can occur days to weeks post-transplant and involves both T-cell-mediated rejection (TCMR) and antibody-mediated rejection (ABMR). TCMR is characterized by the direct attack of donor cells by recipient T cells, while ABMR involves the production of donor-specific antibodies that target the graft, leading to inflammation and tissue damage (Ronca et al., 2020; Teng et al., 2022). Genes such as SLAMF8 and TLR4 have been identified as playing roles in the inflammatory response during acute rejection. Chronic Rejection: This occurs over months to years and is a major cause of long-term graft failure. Chronic rejection involves a combination of immune and non-immune factors, including continuous low-level immune responses and fibrosis. M2 macrophages, which are involved in tissue repair and fibrosis, play a significant role in chronic rejection by contributing to graft vasculopathy and fibrosis (Zhang et al., 2021). Additionally, extracellular vesicles released from the graft can mediate immune responses and contribute to chronic rejection by presenting donor antigens to the recipient's immune system (Ravichandran et al., 2022). Understanding these mechanisms and the genetic factors involved is crucial for developing strategies to engineer immune-compatible organs and improve the outcomes of organ transplantation. 3 Genetic Modifications in Pigs to Reduce Rejection 3.1 Overview of genetic engineering techniques Genetic engineering techniques such as CRISPR/Cas9 and TALENs have revolutionized the field of xenotransplantation by enabling precise modifications in the pig genome to reduce immunogenicity and improve compatibility with human recipients. CRISPR/Cas9, in particular, has been widely used due to its high efficiency and specificity. This technique involves the use of guide RNAs (gRNAs) to direct the Cas9 nuclease to specific genomic loci, where it introduces double-strand breaks that are repaired by non-homologous end joining or homology-directed repair, leading to targeted gene modifications (Zhang et al., 2018; Fu et al., 2020; Tanihara et al., 2021; Yoon et al., 2022). TALENs, another genome editing tool, use engineered nucleases to create double-strand breaks at specific sites, although they are less commonly used compared to CRISPR/Cas9 due to their complexity and lower efficiency (Yoon et al., 2022). 3.2 Specific genes targeted for modification 3.2.1GGTA1 (alpha-gal knockout) to prevent hyperacute rejection The GGTA1 gene encodes the enzyme α1,3-galactosyltransferase, which is responsible for the synthesis of the α-Gal epitope, a major xenoantigen that triggers hyperacute rejection in human recipients. Knockout of the GGTA1 gene in pigs has been shown to significantly reduce the binding of human IgG and IgM antibodies, thereby preventing hyperacute rejection (Fu et al., 2020; Tanihara et al., 2021). Studies have demonstrated that GGTA1 knockout pigs exhibit reduced expression of α-Gal in various tissues, including the heart, lungs, liver, and kidneys, making them more suitable for xenotransplantation (Wang et al., 2018; Zhang et al., 2018; Yoon et al., 2022). 3.2.2CMAHandβ4GalNT2 to reduce xenoantigen expression In addition to GGTA1, the CMAHand β4GalNT2 genes are also targeted to reduce xenoantigen expression (Figure 2). The CMAH gene encodes CMP-Neu5Ac hydroxylase, which is involved in the synthesis of N-glycolylneuraminic acid (Neu5Gc), another xenoantigen that elicits immune responses in humans. Knockout of the CMAH gene in pigs has been shown to reduce Neu5Gc expression and decrease human antibody binding (Wang et al., 2018; Yoon et al., 2022). Similarly, the β4GalNT2 gene encodes β-1,4-N-acetyl-galactosaminyl transferase 2, which is responsible for the synthesis of the Sd(a) antigen. Knockout of β4GalNT2 in pigs further reduces xenoantigen expression and enhances immune compatibility (Zhang et al., 2018). Tanihara et al. (2021) demonstrates the successful generation of gene-edited piglets using CRISPR/Cas9 technology targeting GGTA1, CMAH, and β4GalNT2. Two piglets (#4 and #5) were born from zygotes electroporated with Cas9 and guide RNAs. Deep sequencing revealed piglet #4 had biallelic mutations in GGTA1 and β4GalNT2, while piglet #5 had mutations in all three target genes, including an inframe mutation in β4GalNT2. The high mutation frequencies and rates indicate effective gene editing. This study showcases the potential of CRISPR/Cas9 for creating genetically modified pigs for xenotransplantation research.
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