Research on xenotransplantation using genetic engineering

以猪为供体的临床异种移植的前景为解决人体器官短缺提供了一个潜在的解决方案。基因工程的技术进步促进了旨在预防异种移植排斥的各种策略的发展，包括（i）物种特异性人补体调节蛋白的表达，（ii）异种抗原表达的修饰和（iii）内皮活化的抑制。本研究利用竞争性抑制α 1，3半乳糖基转移酶（α 1，3 GT）的α 1，2岩藻糖基转移酶（α 1，2 FT）基因转染猪培养细胞，导致α Gal抗原表达降低，从而获得α 1，2 FT转基因猪。然而，由于这些猪死于恶性肿瘤或发育不完全，因此无法建立该菌株。我们还发现，这种基因转移 关于我们 nsfer不仅降低了alphaGal表达，而且降低了培养的内皮细胞中的唾液酸化，并抑制了管形成的能力。我们还证明了使用腺病毒载体转移α 1，2 FT基因或α 1，3 GT核酶在体外降低α Gal表达。本研究通过对猪α 1，3 GT基因的结构分析，构建了α 1，3 GT基因的靶向表达载体，旨在利用猪胚胎干细胞或核移植技术，尽快诱导α 1，3 GT基因敲除猪。一些不含外显子8或突变cDNA（催化区中的两个突变;外显子9）的选择性剪接变体也缺乏酶活性。这些观察结果提供了一种以显性负效应降低α Gal抗原表达的新策略。关于非α Gal抗原，发现Hanganutziu-Deicher（HD）抗原在猪和狒狒肾的血管内皮细胞上表达，但在人肾中不表达，表明HD抗原可以在猪-人移植中引发强烈的体液应答，即使避免了由于α Gal表位引起的HAR。猪-狒狒移植模型不能证明这种可能性，因为狒狒表达HD抗原，并且似乎不产生抗HD抗体。一些狒狒血清显示阳性疱疹B病毒使用ELISA，然而，这是由于与人类单纯疱疹病毒的交叉反应。血液检查显示，所有人都在正常范围内。所有狒狒血清中含有与人血清一样多的抗猪（抗α Gal）IgM抗体，尽管狒狒血清中的抗猪IgG和伊加低于人血清中的抗猪IgG和IgA。证明了猪-狒狒移植模型的可行性和安全性。少

The prospect of clinical xenotransplantation using pigs as donors holds out a potential solution to the shortage of human organs. The technical advance of genetic engineering has prompted the development of various strategies aimed at preventing xenograft rejection, including (i) expression of species-specific human complement regulatory proteins, (ii) modification of xenogeneic antigen expression and (iii) suppression of endothelial activation. The purpose of this study is to provide the useful information for clinical application of xenotransplantation.As gene transfection of alpha1,2 fucosyl transferase (alpha1,2 FT), which was capable of competitive inhibition of alpha1,3 galactosyltransferase (alpha1,3 GT), caused the decreased expression of alphaGal antigens in pig cultured cells, we produced alpha1,2 FT transgenic pigs. However, this strain could not be established, since these pigs died of malignant tumor or incomplete development. We also showed the findings that such gene tra … More nsfer decreased not only alphaGal expression but also sialylation in cultured endothelial cells and inhibited the capacity for tube formation. We also demonstrated that alpha1,2 FT gene or alpha1,3 GT ribozyme transfer using adenoviral vectors decreased alphaGal expression in vitro. We are now exploring direct gene replacement of alpha1,3 GT gene with human alpha1,2 FTgene, namely knock-in method.We analyzed the genomic structure of alpha1,3 GT in pigs and constructed a targeting vector with the aim of inducing alphaGal knockout pigs as soon as porcine embryonic stem cells or nuclear transplantation are available. Some alternative splicing variants without exon 8 or mutant cDNA (two mutations in the catalytic region ; exon 9) were also devoid of enzymatic activity. These observations provides a new strategy for reducing alphaGal antigen expression in a dominant-negative effect. With regard to non-alphaGal antigens, Hanganutziu-Deicher (HD) antigens were found to be expressed on the vascular endothelial cells of pig and baboon kidneys, but not in the human kidney, suggesting that HD antigens could elicit a strong humoral response in pig-to-human transplantation, even if HAR due to alphaGal epitopes is avoided. The pig-to-baboon transplantation model cannot demonstrate this possibility, because the baboon expresses HD antigens and does not appear to produce anti-HD antibodies.We examined baboon sera to establish pig-to-baboon transplantation model. Some baboon sera showed the positivity for herpes B-virus using ELISA,however, this was ascribed to crossreactivity with human herpes simplex virus. Blood test revealed that all were within normal limits. All baboon sera contained anti-pig (anti-alphaGal) IgM antibodies as much as human sera, although anti-pig IgG and IgA in baboon sera were less than those in human sera. Usefulness and safety of pig-to-baboon transplantation model was demonstrated for clinical application. Less