International Journal of Molecular Zoology 2024, Vol.14, No.2, 72-83 http://animalscipublisher.com/index.php/ijmz 74 2.2 Importance of immunotolerance in the context of xenotransplantation Immunotolerance is vital for the success of xenotransplantation, as it helps prevent the rejection of transplanted organs. Genetically modified pigs have been engineered to express human complement regulatory proteins and to knock out pig-specific antigens, which helps in reducing the immune response from the human recipient (Fischer et al., 2016; Lei et al., 2022; Xi et al., 2023). Achieving immunotolerance can lead to longer graft survival and better overall outcomes, as demonstrated in studies where genetically modified pig organs showed reduced signs of acute and chronic rejection (Shimizu et al., 2012; Hawthorne et al., 2022). The ability to induce immunotolerance can also minimize the need for long-term immunosuppressive therapy, which has significant side effects (Shimizu et al., 2012; Hawthorne et al., 2022). 2.3 Comparison of immunotolerance in human-to-human versus pig-to-human transplants In human-to-human transplants, immunotolerance is primarily achieved through immunosuppressive drugs and, in some cases, through the induction of donor-specific tolerance via mixed chimerism or Treg therapy. The immune system's response to human antigens is generally less severe compared to xenogeneic antigens from pigs (Shimizu et al., 2012; Lei et al., 2022). In pig-to-human transplants, the immune response is more complex due to the presence of xenogeneic antigens, which can trigger hyperacute rejection, acute vascular rejection, and chronic rejection (Westall et al., 2013; Fischer et al., 2016; Lei et al., 2022). Genetically modified pigs help mitigate these responses by expressing human proteins and knocking out pig-specific antigens, but achieving complete immunotolerance remains a significant challenge (Westall et al., 2013; Fischer et al., 2016; Carvalho-Oliveira et al., 2021; Lei et al., 2022; Xi et al., 2023). Immunotolerance is a critical factor in the success of xenotransplantation. Advances in genetic engineering of pigs have shown promise in reducing immune responses and improving graft survival, but further research is needed to fully understand and achieve long-term immunotolerance in pig-to-human transplants. 3 Mechanisms for Enhancing Immunotolerance 3.1 Genetic modifications targeting specific immune pathways Genetic modifications in pigs have been pivotal in addressing the immunological barriers in xenotransplantation. One of the primary targets is the elimination of xenoantigens, such as α-Gal and Neu5Gc, which are responsible for hyperacute rejection. The knockout of genes like GGTA1 and CMAHhas been shown to significantly reduce the binding of human antibodies and subsequent immune responses (Fischer et al., 2016; Butler et al., 2016; Tanihara et al., 2021). Additionally, the insertion of human complement regulatory proteins, such as CD46, CD55, and CD59, has been effective in protecting pig tissues from human complement-mediated lysis (Fischer et al., 2016; Cooper et al., 2019; Lei et al., 2022). These genetic modifications collectively contribute to reducing both humoral and cellular immune responses, thereby enhancing immunotolerance. 3.2 Use of CRISPR/Cas9 and other gene editing technologies The advent of CRISPR/Cas9 technology has revolutionized the field of genetic engineering, allowing for precise and efficient modifications. This technology has been employed to create pigs with multiple gene knockouts, such as GGTA1, CMAH, and B4GALNT2, in a single step, thereby reducing the biosynthesis of xenoantigens (Mohiuddin et al., 2014; Tanihara et al., 2021; Eisenson et al., 2022). CRISPR/Cas9 has also been used to generate pigs with triple knockouts of GGTA1, β2M, and CIITA, which significantly alleviates xenogeneic immune responses by reducing the expression of major histocompatibility complex (MHC) antigens (Fu et al., 2020). The use of CRISPR/Cas9 not only accelerates the generation of genetically modified pigs but also enhances the precision and effectiveness of these modifications, making it a cornerstone technology in xenotransplantation research. 3.3 Case studies of successful genetic modifications for immunotolerance Several case studies have demonstrated the success of genetic modifications in enhancing immunotolerance in xenotransplantation. For instance, pigs with multiple genetic modifications, including the knockout of GGTA1 and the expression of human complement regulatory proteins, have shown complete protection against human complement-mediated lysis and reduced endothelial activation (Fischer et al., 2016). Another study reported the
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