Transgenic pigs as organ donors in xenotransplantation

转基因猪作为异种移植的器官捐献者

• 批准号: 08557069
• 负责人: TAKAGI Hiroshi
• 金额: $ 12.8万
• 依托单位: Nagoya University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (A)
• 财政年份: 1996
• 资助国家: 日本
• 起止时间: 1996 至 1997
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08557069/
• 关键词: Transplantation, Heterologous; Animals, Transgenic; Complement Inactivators; Antigens, Heterophile; Galactosylransferases; Fucosyltransferases; 遺伝子制御; トランスジェニックブタ; ノックアウト動物; α1.3GT; α1.2FT

Xenotransplantation is considered to be promising as an alternative method to solve the shortage of donor organs. As molecular biological technique has recently advanced, attempts to overcome the problem of antigen-antibody interaction has been directed towards the modification of the donors. Attention has been directed toward removal or replacement of alphaGal epitopes, since transgenic pigs expressing DAF (decay accelerating factor) could not suppress hyperacte rejection completely. alphaGal epitopes are synthesized by alphagalactosyltransferase (GT). Unfortunately, gene knockout is at present not achievable in the pig due to the anavailability of porcine embryonic stem cells. An alternative approach, which is called "enzymatic remodeling of the carbohydrate surface" has been attempted. That is to say, this concept is that the alphaGal molecules would be replaced with another oligosaccharides such as fucose by indtoruction of alpha1,2 fucosyltransferase (FT). We have produced alpha1, … More 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 observation that such gene transfer decreased not only alphaGal expression but also sialylation in cultured endothelial cells and inhibited the capacity for tube formation. Our results suggests that extreme modification of carbohydrate antigens on cell surfaces may have a detrimental effect on developement or differentiation. We also demonstrated that alpha1,2 FT gene or 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 FT gene, namely knock-in method. However, further experiment will be necessary to examine if modification of carbohydrate antigens in cell surface is valid for xenotransplantation.We analyzed the genomic structure of alpha1,3 GT in pigs and constructing a targeting vector with the aim of inducing alphaGal knockout pigs as soon as porcine embryonic stem cells or nuclear transplantation are available. We have shown several alternative splicing variants in pig alpha1,3 GT,which are related to functional differences in the enzyme activity. Some splicing variants, which are defective in exon 5,6 and 7, have been found to have less enzymatic activity, indicating the importance of the stem region in the enxyme activity. Furthermore, splicing varlants without exon 8 or mutant cDNA (two mutations in the catalytic region ; exon 9) were also devoid of enzymatic activity. These observations suggest new strategles for reducing alphaGal antigen expression in pigs, that is to say, the introduction of cDNA without exon 7,8 or of mutant cDNA that can inhibit alpha1,3 GT enzymatic activity in a dominant negative effect. Less

异种移植被认为是解决供体器官短缺的一种有前途的替代方法。随着分子生物学技术最近的发展，克服抗原-抗体相互作用问题的尝试已经指向了对供体的修饰。由于表达衰变加速因子(DAF)的转基因猪不能完全抑制超活性排斥反应，人们的注意力集中在移除或替换AlphaGal表位上。α-半乳糖基转移酶(GT)合成α-Gal表位。不幸的是，由于猪胚胎干细胞的缺乏，目前还不能在猪身上实现基因敲除。另一种被称为“碳水化合物表面的酶重塑”的方法也被尝试过。也就是说，这个概念是通过α1，2岩藻糖基转移酶(FT)的作用，将α-半乳糖分子替换为另一种低聚糖，如岩藻糖。我们已经生产了Alpha1，…又有2头FT转基因猪。然而，由于这些猪死于恶性肿瘤或发育不全，该菌株无法建立。我们还观察到，这样的基因转移不仅减少了培养的内皮细胞中α-Gal的表达，还减少了唾液酸化，并抑制了血管形成的能力。我们的结果表明，细胞表面糖类抗原的过度修饰可能会对发育或分化产生不利影响。我们还证实了用腺病毒载体转导α-1，2-FT基因或GT核酶在体外降低了α-Gal的表达。我们正在探索用人的α1，2 FT基因直接替代α1，3 GT基因的方法，即敲入法。然而，细胞表面碳水化合物抗原的修饰是否适用于异种移植还需要进一步的实验。我们分析了猪α1，3 GT的基因组结构，并构建了靶向载体，目的是在猪胚胎干细胞或核移植出现后，尽快诱导αGal基因敲除猪。我们已经在猪α1，3 GT中发现了几种选择性剪接变异体，它们与酶活性的功能差异有关。一些外显子5、6和7有缺陷的剪接变异体的酶活性较低，这表明茎区域在酶活性中的重要性。此外，没有外显子8或突变基因的剪接变异体(催化区的两个突变；外显子9)也没有酶活性。这些观察结果提示了降低猪α-Gal抗原表达的新策略，即引入不含外显子7、8或突变的、能以显性负效应抑制α-1，3-GT酶活性的基因。较少

项目成果

高木弘: "異種移植臨床への研究" BIO Clinica. 10(14). 1064-1066 (1995)

Hiroshi Takagi：“异种移植的临床研究”BIO Clinica 10(14)。

高木弘: "異種臓器移植におけるトランスジェニックブタ作製の重要性" 細胞工学. 15(1). 81-89 (1996)

Hiroshi Takagi：“异种器官移植中转基因猪的重要性”，Cell Engineering 15(1)。

S Hayashi, H Takagi: "Protection of guinea pig cell from rat complement-mediated lysis by the transfection of rat complement regulatory factor512 geneXenotransplantation" Xenotransplantation. 3. 217-221 (1996)

S Hayashi、H Takagi：“通过转染大鼠补体调节因子 512 基因来保护豚鼠细胞免受大鼠补体介导的裂解”异种移植。

T Kobayashi, H Takagi, D. K. C Cooner: "In vitro and in vivo investigation of anticomplement agents FUT-175 and K76COOH,in the prevention of hyperacute rejection following discordant xenotransplantation in a nonhuman primate model" Transplantation Proceed

T Kobayashi、H Takagi、D. K. C Cooner：“抗补体剂 FUT-175 和 K76COOH 的体外和体内研究，在非人灵长类动物模型中预防不一致异种移植后的超急性排斥反应”

Y Tominaga, Takagi H: "Clonal Analysis of Nodular Parathyroid Hyperplasia in Ranal Hyperparathroidism" World journal of Surgery. 20. 744-752 (1996)

Y Tominaga、Takagi H：“肛甲状旁腺功能亢进症中结节性甲状旁腺增生的克隆分析”世界外科杂志。