International Journal of Molecular Medical Science, 2024, Vol.14, No.3, 177-192 http://medscipublisher.com/index.php/ijmms 182 suppress natural killer (NK) cell activity and promote immune tolerance (Obando et al., 2021; Lopez et al., 2022). Moreover, the introduction of porcine vascularized thymic grafts has shown promise in inducing tolerance by re-educating the recipient’s immune system to accept the xenograft (Yamada et al., 2020). One promising approach involves the use of immune-privileged cells, such as neonatal pig Sertoli cells (NPSC), which have been shown to create an immune modulatory environment. These cells can prolong the survival of co-transplanted cells by recruiting regulatory T cells (Tregs) and producing immunoregulatory factors like TGF-β and IL-10, which help in reducing inflammation and apoptosis (Kaur et al., 2020). Additionally, the deletion of genes such as β2-microglobulin (β2M) and CIITA in pigs has been shown to reduce the activation and proliferation of human T cells, thereby alleviating xenogeneic immune responses and prolonging graft survival (Fu et al., 2020). 4.2 Genetic modifications to reduce antigenicity One of the primary barriers to successful xenotransplantation is the presence of xenoantigens on pig tissues, which can trigger hyperacute rejection. The most significant xenoantigens include galactose-alpha-1,3-galactose (α-Gal), N-glycolylneuraminic acid (Neu5Gc), and the Sd(a) antigen. Genetic modifications to knock out these antigens have shown promising results. For instance, pigs with triple gene knockouts (GGTA1, CMAH, and β4GalNT2) exhibit significantly reduced antigenicity, as evidenced by decreased human IgG and IgM binding to their tissues (Wang et al., 2018; Yoon et al., 2022). This reduction in antigenicity is crucial for minimizing hyperacute rejection and improving graft survival. 4.3 Genes enhancing organ resistance to rejection and injury In addition to reducing antigenicity, enhancing the graft’s intrinsic resistance to immune-mediated damage is essential. Genes encoding human complement regulatory proteins, such as CD46, CD55, and CD59, have been introduced into pigs to protect against complement-mediated lysis. These modifications help in preventing acute vascular rejection and improving long-term graft survival (Singh et al., 2018; Lei et al., 2022). Moreover, genetic modifications to enhance the expression of anti-inflammatory and anti-apoptotic genes in pigs have shown to reduce tissue injury and improve graft durability. For example, pigs expressing human thrombomodulin have demonstrated significantly prolonged graft survival by reducing coagulation-related complications (Porrett et al., 2023). 5 CRISPR/Cas9 and Genetic Engineering in Xenotransplantation 5.1 Overview of CRISPR/Cas9 technology and its applications CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats associated with Cas9) is a revolutionary gene-editing technology that allows for precise modifications of DNA within organisms. The system consists of the Cas9 nuclease, which cuts DNA at specific locations guided by a synthetic RNA molecule that matches the target DNA sequence. This technology has transformed genetic research and biotechnology, enabling targeted gene knockouts, insertions, and modifications with high efficiency and accuracy. CRISPR/Cas9 has been widely adopted for various applications, including biomedical research, agricultural improvements, and the development of genetically modified organisms. In the context of xenotransplantation, CRISPR/Cas9 is employed to modify the genomes of donor pigs to make their organs more compatible with human recipients (Ryczek et al., 2021). In the context of xenotransplantation, CRISPR/Cas9 has been employed to modify the genomes of donor pigs to reduce the risk of immune rejection and improve graft survival. By knocking out specific genes that are responsible for hyperacute rejection, such as the alpha-1,3-galactosyltransferase (GGTA1) gene, researchers have been able to create genetically modified pigs that are more compatible with human immune systems (Firl and Markmann, 2022; Montgomery et al., 2022). 5.2 Case studies of CRISPR/Cas9-mediated genetic modifications in pigs Several case studies highlight the successful application of CRISPR/Cas9 in creating genetically modified pigs for xenotransplantation:
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