International Journal of Clinical Case Reports 2024, Vol.14, No.2, 94-106 http://medscipublisher.com/index.php/ijccr 98 Figure 2 Factors enabling consistent success in life-supporting pig-to-baboon cardiac xenotransplantation (Adopted from Längin et al., 2018) Image caption: In addition to genetically multimodified porcine donor hearts (lacking αGal epitopes and expressing human CD46 as well as human thrombomodulin) and appropriate immunosuppression, two steps were key to success: 1) nonischemic preservation of the donor hearts by perfusion with oxygenated hyperoncotic blood-based solution; and 2) prevention of detrimental xeno-heart overgrowth by early weaning of cortisone, lowering of blood pressure and treatment with the mTOR inhibiting prodrug temsirolimus (Adopted from Längin et al., 2018) This gene encodes β-1,4-N-acetyl-galactosaminyl transferase 2, responsible for the synthesis of Sd(a) antigen. Triple knockout of GGTA1, CMAH, and β4GalNT2 results in even lower human antibody binding and reduced immunogenicity (Zhang et al., 2018). These genes encode the swine leukocyte antigens, which are the porcine equivalents of human MHC molecules. Their knockout can reduce cellular immune responses against pig organs (Fu et al., 2020). 3.3 Case studies and experimental results on immunogenicity reduction Triple Gene Knockout Pigs Research has shown that pigs with knockouts in GGTA1, CMAH, and β4GalNT2 exhibit significantly reduced levels of human IgG and IgM binding. In a study by Wang et al. (2018), tissues from these triple knockout (TKO) pigs demonstrated minimal human antibody binding, indicating a substantial reduction in immunogenicity. These TKO pigs were produced using CRISPR-Cas9, which successfully eliminated the expression of α-Gal, Neu5Gc, and Sd(a) antigens. Another study by Yoon et al. (2022) developed triple knockout JNPs using CRISPR-Cas9, targeting GGTA1, CMAH, and B4GALNT2. These pigs showed no expression of the three antigens in major organs, and human IgM and IgG binding was significantly reduced compared to wild-type pigs, supporting the potential use of these organs in xenotransplantation (Figure 3). Zhang et al. (2018) investigated the use of TKO pigs for BHVs. Their findings demonstrated that valves from TKO pigs exhibited reduced human IgM/IgG binding and had similar collagen composition and physical properties compared to wild-type pigs, making them a promising alternative for reducing immunogenicity in heart valve replacements. Fu et al. (2020) generated pigs with triple knockout of GGTA1, β2M, and CIITA genes, reducing both humoral and cellular immune responses. These pigs showed prolonged survival of skin grafts in immunocompetent mice, demonstrating the effectiveness of multi-gene knockouts in reducing xenogeneic immune reactions.
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