International Journal of Clinical Case Reports 2024, Vol.14, No.2, 94-106 http://medscipublisher.com/index.php/ijccr 97 2.2 Challenges in pig-to-human organ transplants While the use of pigs as organ donors offers significant potential, it also presents several challenges that must be addressed for successful xenotransplantation. The immune system's response to foreign tissues is a major barrier. Pigs express the alpha-1,3-galactosyltransferase (α-Gal) antigen, which triggers hyperacute rejection (HAR) in humans. Genetic modifications, such as the knockout of the α-Gal gene and the introduction of human complement regulatory proteins, have been developed to mitigate this issue (Klymiuk et al., 2010; Gock et al., 2011). Physiological differences between pigs and humans can lead to issues with organ functionality and longevity. For instance, differences in coagulation pathways can cause thrombotic microangiopathy in transplanted organs. Genetic engineering approaches aim to address these incompatibilities by incorporating human genes that regulate coagulation and immune responses (Lei et al., 2022; Wu et al., 2023). The potential transmission of porcine endogenous retroviruses (PERVs) to human recipients is a concern. Recent advances have made it possible to inactivate these viruses using CRISPR-Cas9 technology, reducing the risk of cross-species infections (Wolf et al., 2019). 2.3 Current status and advancements in pig organ transplantation The field of xenotransplantation has made significant strides in recent years, bringing the prospect of clinical application closer to reality. The creation of genetically modified pigs that lack α-Gal and express human complement regulatory proteins has greatly improved the survival rates of pig organs in non-human primate models. For instance, hearts and kidneys from genetically modified pigs have survived for several months in primates, demonstrating the potential for longer-term graft function (Ekser et al., 2009; Hryhorowicz et al., 2017; Längin et al., 2018) (Figure 2). Advances in immunosuppressive therapies have been crucial in extending the viability of xenografts. The development of novel immunosuppressive agents that target specific immune pathways has helped reduce the incidence of acute humoral xenograft rejection (AHXR) and other immune-mediated complications (Ekser et al., 2009; Zhang et al., 2020). Ongoing preclinical trials and regulatory discussions are paving the way for the first clinical trials of pig organ xenotransplantation. These trials will be critical in assessing the safety, efficacy, and long-term outcomes of xenotransplantation in human patients (Wolf et al., 2019; Ali et al., 2023). 3 CRISPR-Cas9 and Immunogenicity 3.1. Strategies for reducing immunogenicity through gene editing CRISPR-Cas9 has revolutionized the field of xenotransplantation by enabling precise genetic modifications to reduce the immunogenicity of pig organs. The primary strategy involves knocking out genes that encode for xenoantigens responsible for triggering immune responses in human recipients. The main antigens targeted include galactose-α1,3-galactose (α-Gal), N-glycolylneuraminic acid (Neu5Gc), and Sd(a) antigen. By using CRISPR-Cas9 to disrupt the genes responsible for these antigens (GGTA1, CMAH, and β4GalNT2), researchers have significantly reduced the immunogenicity of pig tissues (Wang et al., 2018; Yoon et al., 2022). Additionally, further strategies involve creating multi-gene knockouts to eliminate other immune targets such as the major histocompatibility complex (MHC) antigens SLA-I and SLA-II. This approach involves the simultaneous deletion of multiple genes, which can be efficiently achieved using CRISPR-Cas9 (Fu et al., 2020). 3.2 Specific genes targeted to minimize immune rejection This gene encodes α1,3-galactosyltransferase, which is responsible for the synthesis of the α-Gal antigen. Knockout of GGTA1 has been shown to significantly reduce hyperacute rejection in xenotransplantation (Tanihara et al., 2021). This gene encodes CMP-N-acetylneuraminic acid hydroxylase, which synthesizes Neu5Gc, another xenoantigen. Combined knockout of GGTA1 and CMAH has been shown to further decrease the immune response compared to GGTA1 knockout alone (Gao et al., 2016).
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