In this study, we investigated the effect of a triple knockout of the genes alpha-1,3-galactosyltransferase (GGTA1), cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH), and alpha 1,3-galactosyltransferase 2 (A3GALT2) in Yucatan miniature pigs on human immune reactivity. We used the CRISPR/Cas9 system to create pigs lacking GGTA1 (GTKO) and GGTA1/CMAH/A3GALT2 triple gene knockout (TKO). The expression of all three xenoantigens was absent in TKO pigs, but there was no additional reduction in the level of Galα1,3Gal (αGal) epitopes expression in the A3GALT2 gene KO. Peripheral blood mononuclear cells (PBMCs), aorta endothelial cells (AECs), and cornea endothelial cells (CECs) were isolated from these pigs, and their ability to bind human IgM/IgG and their cytotoxicity in human sera were evaluated. Compared to wild type (WT) pigs, the level of human antibody binding of the PBMCs, AECs, and CECs of the transgenic pigs (GTKO and TKO) was significantly reduced. However, there were significant differences in human antibody binding between GTKO and TKO depending on the cell type. Human antibody binding of TKO pigs was less than that of GTKO on PBMCs but was similar between GTKO and TKO pigs for AECs and CECs. Cytotoxicity of transgenic pig (GTKO and TKO) PBMCs and AECs was significantly reduced compared to that of WT pigs. However, TKO pigs showed a reduction in cytotoxicity compared to GTKO pigs on PBMCs, whereas in AECs from both TKO and GTKO pigs, there was no difference. The cytotoxicity of transgenic pig CECs was significantly decreased from that of WT at 300 min, but there was no significant reduction in TKO pigs from GTKO. Our results indicate that genetic modification of donor pigs for xenotransplantation should be tailored to the target organ and silencing of additional genes such as CMAH or A3GALT2 based on GTKO might not be essential in Yucatan miniature pigs.

在这项研究中，我们研究了基因 alpha-1,3-galactosyltransferase ( GGTA1 )、胞苷单磷酸-N-乙酰神经氨酸羟化酶 ( CMAH ) 和 alpha 1,3-galactosyltransferase 2 ( A3GALT2 ) 基因三重敲除的影响。尤卡坦微型猪对人体免疫反应的影响。我们使用 CRISPR/Cas9 系统创建了缺乏GGTA1 (GTKO) 和GGTA1/CMAH/A3GALT2三重基因敲除 (TKO) 的猪。TKO 猪中不存在所有三种异种抗原的表达，但 A3GALT2 中的Galα1,3Gal (αGal) 表位表达水平没有额外降低基因KO。从这些猪中分离出外周血单个核细胞 (PBMC)、主动脉内皮细胞 (AEC) 和角膜内皮细胞 (CEC)，并评估了它们与人 IgM/IgG 结合的能力及其在人血清中的细胞毒性。与野生型 (WT) 猪相比，转基因猪 (GTKO 和 TKO) 的 PBMC、AEC 和 CEC 的人抗体结合水平显着降低。然而，根据细胞类型的不同，GTKO 和 TKO 之间的人抗体结合存在显着差异。TKO 猪对 PBMC 的人抗体结合低于 GTKO，但 GTKO 和 TKO 猪对 AEC 和 CEC 的结合率相似。与 WT 猪相比，转基因猪 (GTKO 和 TKO) PBMC 和 AEC 的细胞毒性显着降低。然而，与 PBMC 上的 GTKO 猪相比，TKO 猪的细胞毒性有所降低，而在 TKO 和 GTKO 猪的 AEC 中，没有区别。转基因猪 CECs 的细胞毒性在 300 分钟时较 WT 显着降低，但 GTKO 对 TKO 猪没有显着降低。我们的研究结果表明，供体猪的异种移植基因改造应针对目标器官和其他基因的沉默进行调整，例如基于 GTKO 的CMAH或A3GALT2在尤卡坦小型猪中可能不是必需的。