Wang Jiaoxiang, Xu Kaixiang, Liu Tao, Zhao Heng, Jamal Muhammad Ameen, Chen Gen, Huo Xiaoying, Yang Chang, Jiao Deling, Wei Taiyun, Huang Hanfei, Zhao Hongfang, Guo Jianxiong, Wang Fengchong, Zhang Xiong, Liu Kai, Qu Siming, Wang Gang, Guo Hui, Chen Gang, Zhao Hong-Ye, Zeng Zhong, Dou Kefeng, Wei Hong-Jiang
Yunnan Province Key Laboratory for Porcine Gene Editing and Xenotransplantation, Yunnan Agricultural University, Kunming, China.
Yunnan Province Xenotransplantation Research Engineering Center, Yunnan Agricultural University, Kunming, China.
Cell Prolif. 2025 Apr 6:e70028. doi: 10.1111/cpr.70028.
Gene-edited (GE) pig-to-human xenotransplantation continues to make breakthroughs, but which kind of gene combination is suitable for organ-specific transplantation remains unclear. In this study, we utilised CRISPR/Cas9 gene editing technology, PiggyBac transposon system, and serial somatic cell cloning technology to develop GTKO/CMAHKO/β4GalNT2KO/hCD46/hCD55/hCD59/hCD39/hTBM 8 gene-edited cloned (GEC) donor pigs and performed pig-to-non-human primate (NHP) transplantation to evaluate the effectiveness of these GEC pigs. The 8-GEC pigs were obtained by recloning with a 33-day-old 8-GEC fetus with O blood type, which was generated after cell transfection, screening of cell colonies, and somatic cell cloning. Molecular identification at DNA, mRNA, and protein levels confirmed successful 8-gene editing. Three copies of transgenes were identified by droplet digital polymerase chain reaction and whole genome sequencing, which were inserted into the introns of pig RFTN1 and MYO10 genes, as well as the intergenic region between PRLR and LOC110257300 genes of these 8-GEC pigs. The 8-GEC pigs also exhibited the ability of germline transmission when mated with our previously generated 4-GEC male pigs. Moreover, antigen-antibody binding assay and complement-dependent cytotoxicity assay demonstrated that 8-gene editing effectively reduced the immune incompatibility and kidney xenograft from 8-GEC pigs survived for 15 and 17 days in two NHPs, respectively. Postoperatively, the recipient serum antibodies IgA, IgG and IgM, complements C3 and C4, coagulation indicators PT, APTT, TT and FIB, as well as most electrolytes and liver function indicators remained relatively stable. Serum creatinine was normal within 10 days post operation. However, the kidney xenograft developed active antibody-mediated rejection at necropsy, characterised by the deposition of antibodies IgG and IgM, as well as complements C4d, C3c and C5b-C9, infiltration of CD68 macrophages, and micro-thrombotic embolism of glomerular capillaries, etc. In conclusion, we successfully developed fertile 8-GEC pigs, which effectively alleviated immune rejection and exerted life-supporting kidney function in the recipients.
基因编辑猪到人的异种移植不断取得突破，但哪种基因组合适合器官特异性移植仍不清楚。在本研究中，我们利用CRISPR/Cas9基因编辑技术、PiggyBac转座子系统和连续体细胞克隆技术，培育出GTKO/CMAHKO/β4GalNT2KO/hCD46/hCD55/hCD59/hCD39/hTBM 8基因编辑克隆（GEC）供体猪，并进行猪到非人灵长类动物（NHP）的移植，以评估这些GEC猪的有效性。这8头GEC猪是通过对一头33天大、血型为O型的8-GEC胎儿进行再克隆获得的，该胎儿是在细胞转染、细胞集落筛选和体细胞克隆后产生的。DNA、mRNA和蛋白质水平的分子鉴定证实了8基因编辑成功。通过液滴数字聚合酶链反应和全基因组测序鉴定出转基因的三个拷贝，它们插入到这8头GEC猪的猪RFTN1和MYO10基因的内含子以及PRLR和LOC110257300基因之间的基因间区域。当与我们之前培育的4-GEC公猪交配时，这8头GEC猪还表现出种系传递能力。此外，抗原-抗体结合试验和补体依赖性细胞毒性试验表明，8基因编辑有效地降低了免疫不相容性，来自GEC猪的肾脏异种移植在两只NHP中分别存活了15天和17天。术后，受体血清抗体IgA、IgG和IgM、补体C3和C4、凝血指标PT、APTT、TT和FIB，以及大多数电解质和肝功能指标保持相对稳定。术后10天内血清肌酐正常。然而，在尸检时，肾脏异种移植出现了活跃的抗体介导排斥反应，其特征是抗体IgG和IgM以及补体C4d、C3c和C5b-C9的沉积、CD68巨噬细胞浸润以及肾小球毛细血管微血栓栓塞等。总之，我们成功培育出了可育的8-GEC猪，其有效减轻了免疫排斥反应，并在受体中发挥了维持生命的肾脏功能。
