International Journal of Molecular Zoology 2024, Vol.14, No.2, 72-83 http://animalscipublisher.com/index.php/ijmz 75 generation of pigs with triple knockouts of GGTA1, β2M, and CIITA, which resulted in significantly prolonged survival of pig skin grafts in immunocompetent mice (Fu et al., 2020) (Figure 1). Additionally, genetically engineered pig hearts expressing human complement and coagulation regulatory genes have achieved long-term survival in baboon models, paving the way for potential clinical applications (Mohiuddin et al., 2014). These case studies underscore the potential of genetic modifications in overcoming immunological barriers and enhancing the success of xenotransplantation. 4 Key Genetic Targets for Immunotolerance 4.1 Knockout of xenoantigen genes (e.g., GGTA1, CMAH, β4GalNT2) The knockout of xenoantigen genes such as GGTA1, CMAH, and β4GalNT2 is a critical strategy to reduce immunogenicity in genetically modified pigs for xenotransplantation. These genes encode enzymes responsible for the synthesis of major carbohydrate antigens, including galactose-α1,3-galactose (αGal), N-glycolylneuraminic acid (Neu5Gc), and Sd(a) antigen, which are recognized by human antibodies and can trigger hyperacute rejection. Studies have demonstrated that the elimination of these antigens significantly reduces human IgG and IgM binding to porcine tissues, thereby minimizing immune responses and improving graft survival (Wang et al., 2018; Zhang et al., 2018; Yoon et al., 2022). For instance, triple knockout (TKO) pigs lacking GGTA1, CMAH, and β4GalNT2 showed minimal human antibody binding and maintained the physical properties of the tissues, making them promising candidates for bioprosthetic heart valves and other xenografts (Zhang et al., 2018; Yoon et al., 2022). 4.2 Expression of human regulatory proteins (e.g., CD46, CD55, CD59) The expression of human regulatory proteins such as CD46, CD55, and CD59 in genetically modified pigs is another essential approach to enhance immunotolerance. These proteins play a crucial role in regulating the complement system, which is part of the innate immune response that can lead to graft rejection. By incorporating these human proteins into pig tissues, researchers aim to inhibit complement activation and reduce immune-mediated damage. Studies have shown that genetically engineered pigs expressing these human complement regulatory proteins exhibit prolonged graft survival and reduced signs of hyperacute rejection in xenotransplantation models (Mohiuddin et al., 2014; Fischer et al., 2016; Burdorf et al., 2021). For example, pig hearts expressing human CD46 and other regulatory proteins have achieved long-term survival in baboon models, indicating the potential for clinical application (Mohiuddin et al., 2014). 4.3 Modification of MHC class I and II molecules to reduce immune recognition Modifying the major histocompatibility complex (MHC) class I and II molecules in pigs is a strategy to reduce immune recognition and subsequent rejection of xenografts. MHC molecules are critical for presenting antigens to T-cells, and their modification can help evade the human immune system. Genetic modifications such as the knockout of swine leukocyte antigen (SLA) class I and II genes, along with the introduction of human leukocyte antigen (HLA) molecules, have been explored to achieve this goal. Research has demonstrated that pigs with reduced or absent SLA expression show decreased T-cell activation and lower levels of human antibody binding, thereby enhancing immunotolerance (Martens et al., 2017; Xu et al., 2022). For instance, pigs with triple gene modifications (GGTA1, B2M, and CIITA) exhibited reduced expression of SLA-I and absence of SLA-II, leading to weaker human immune responses (Xu et al., 2022). 4.4 Combined genetic modifications Combining multiple genetic modifications, including the knockout of xenoantigen genes and the expression of human regulatory proteins, has shown promising results in enhancing immunotolerance. This approach aims to address various aspects of the immune response simultaneously, thereby providing a more comprehensive solution to xenograft rejection. Studies have reported that pigs with combined genetic modifications, such as the knockout of GGTA1 and the expression of human CD46, CD55, and CD59, exhibit significant protection against human complement-mediated lysis and reduced immune activation (Fischer et al., 2016; Yamamoto et al., 2019). These multi-modified pigs represent a significant advancement towards clinical xenotransplantation, demonstrating the potential to overcome the barriers of immune rejection and improve graft survival (Fischer et al., 2016).
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