International Journal of Molecular Medical Science, 2024, Vol.14, No.3, 177-192 http://medscipublisher.com/index.php/ijmms 183 Tanihara et al. (2021) utilized CRISPR/Cas9 to simultaneously knock out three key genes involved in xenoantigen biosynthesis—GGTA1, CMAH, and B4GALNT2. This approach aimed to reduce the antigenicity of pig organs, thereby minimizing the risk of hyperacute rejection when transplanted into humans. The modified pigs exhibited a significant reduction in xenoantigens, demonstrating the potential of CRISPR/Cas9 for creating viable xenograft donors (Tanihara et al., 2021). Yue et al. (2020) conducted research involving the creation of pigs with multiple genetic modifications, including the inactivation of porcine endogenous retroviruses (PERVs) and the introduction of human transgenes to enhance immunological compatibility. These pigs showed normal physiology and improved resistance to human immune responses, highlighting the efficacy of CRISPR/Cas9 in generating complex, multi-gene modifications (Yue et al., 2020). Obando et al. (2021) focused their research on modifying genes involved in immune tolerance. For instance, pigs engineered to express human leukocyte antigen-G (HLA-G) and other immune regulatory proteins have shown promise in reducing natural killer (NK) cell activity and promoting tolerance of xenografts (Obando et al., 2021). 5.3 Potential and limitations of CRISPR/Cas9 for improving graft survival CRISPR/Cas9 technology allows for precise and efficient genetic modifications, enabling the elimination of immunogenic antigens and the introduction of human genes that can protect grafts from immune attack. This can significantly reduce the risk of hyperacute and acute rejection, potentially making xenotransplantation a viable solution to the organ shortage crisis (Cowan et al., 2019). Additionally, CRISPR/Cas9 can be used to deactivate PERVs, reducing the risk of zoonotic infections (Ross and Coates, 2018). Despite its advantages, CRISPR/Cas9 has limitations, including the risk of off-target effects, where unintended parts of the genome are edited, potentially leading to unforeseen consequences. Additionally, achieving complete and uniform gene edits across all cells in an organ remains challenging, which can result in mosaicism. Ethical concerns regarding genetic modifications also need to be addressed before widespread clinical application (Naeimi Kararoudi et al., 2018). While CRISPR/Cas9 technology holds great promise for improving the compatibility and survival of pig-to-human xenografts, further research and ethical considerations are essential to fully realize its potential in clinical settings. 6 Mechanisms of Genetic Modifications for Graft Survival 6.1 Mechanisms reducing hyperacute rejection Hyperacute rejection is a significant barrier in xenotransplantation, primarily driven by preformed antibodies against pig antigens in the human recipient. Genetic modifications in pigs have been pivotal in mitigating this response. One of the most critical modifications is the knockout of the alpha-1,3-galactosyltransferase (GTKO) gene, which eliminates the expression of the alpha-Gal epitope, a major target for human antibodies. Studies have shown that kidneys from GTKO pigs transplanted into non-human primates (NHPs) and brain-dead human recipients did not exhibit signs of hyperacute rejection, indicating the effectiveness of this genetic modification (Firl and Markmann, 2022; Montgomery et al., 2022; Lei et al., 2022). Additionally, the insertion of human complement regulatory proteins, such as CD46 and CD55, further protects the xenograft from complement-mediated damage, which is a crucial component of hyperacute rejection (Lei et al., 2022; Goerlich et al., 2020). 6.2 Mechanisms mitigating acute vascular and cellular rejection Acute vascular and cellular rejection occurs days to weeks post-transplantation and involves immune responses that target the endothelial cells of the graft. Genetic modifications to express human complement regulatory proteins (such as CD46, CD55, and CD59) and coagulation regulatory proteins (such as thrombomodulin) in pigs have shown to be effective in preventing complement activation and controlling coagulation, thereby protecting the graft from immune-mediated damage. The expression of these proteins helps maintain vascular integrity and reduces inflammatory responses within the graft (Burdorf et al., 2018; Singh et al., 2018).
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