International Journal of Molecular Medical Science, 2024, Vol.14, No.3, 155-166 http://medscipublisher.com/index.php/ijmms 156 xenotransplantation. Additionally, the study explores future development directions and proposes strategies to address the remaining scientific, ethical, and regulatory issues. Through this analysis, the study seeks to promote a broader understanding and acceptance of xenotransplantation as a life-saving medical intervention driven by innovations in CRISPR/Cas9 technology. 2 Current State of Xenotransplantation 2.1 Definition and historical background Xenotransplantation refers to the transplantation of living cells, tissues, or organs from one species to another. Historically, the concept of xenotransplantation dates back to the early 20th century, with initial attempts involving the transplantation of animal organs into humans. However, these early efforts were largely unsuccessful due to severe immunological rejection and other complications. It wasn't until the advent of immunosuppressive drugs and advances in genetic engineering that xenotransplantation began to show promise as a potential solution to the shortage of human donor organs (Ryczek et al., 2021). The advent of genetic engineering, particularly the development of CRISPR/Cas9 technology, has significantly advanced the field by enabling precise genetic modifications to donor animals, primarily pigs (Figure 1), to reduce immunogenicity and improve compatibility with human recipients (Ryczek et al., 2021; Kararoudi et al., 2018; Zhang et al., 2021). Figure 1 Two methods for creating genetically edited pigs for xenotransplantation using the CRISPR/Cas system (Adapted from Ryczek et al., 2021) Image caption: Panel A involves microinjecting CRISPR/Cas-modified constructs or RNP complexes into pig zygotes. Panel B uses somatic cell nuclear transfer (SCNT) technology. The diagram details the workflows of these two methods, including how to provide CRISPR/Cas9 components to fertilized eggs at the early pronuclear stage to minimize genetic mosaicism. Research indicates that the presence of genetic mosaics is one of the major limiting factors in the application of CRISPR/Cas9 in large animal models. To reduce mosaicism caused by the CRISPR/Cas9 system in large animals, it is recommended to inject CRISPR/Cas9 components during the early pronuclear stage and use the appropriate form of Cas9-sgRNA (ribonucleoprotein complex). These two methods demonstrate the potential and challenges of generating gene-edited pigs for xenotransplantation research (Adapted from Ryczek et al., 2021) 2.2 Current challenges in xenotransplantation Despite significant advancements, xenotransplantation faces several critical challenges: 1) Immunological Rejection: One of the primary obstacles is the acute and chronic rejection of xenografts by the human immune system. Hyperacute rejection occurs within minutes to hours and is triggered by pre-existing antibodies against xenoantigens, such as those produced by genes like GGTA1, CMAH, and B4GALNT2. Efforts to knock out these genes using CRISPR/Cas9 have shown promise in reducing immunogenicity, but issues like genetic mosaicism still need to be addressed (Tanihara et al., 2021; Cowan et al., 2019). Subsequent acute and chronic rejections are driven by cellular immune responses, which involve T-cells and other immune mechanisms (Kararoudi et al., 2018).
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