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希望的替代方法，近年来，随着分子生物学技术的发展，克服抗原抗体相互作用问题的尝试已转向对供体的修饰。去除或替换αGal表位，因为表达DAF（腐烂加速因子）的转基因猪不能完全抑制αGal表位是由α半乳糖基转移酶（GT）合成的。由于猪胚胎干细胞的可用性，目前在猪中无法实现基因敲除，已经尝试了一种替代方法，即“碳水化合物表面的酶促重塑”，也就是说，这个概念是αGal。通过α1,2岩藻糖基转移酶（FT）的诱导，分子将被另一种寡糖（例如岩藻糖）取代。我们已经生产了α1,…更多2 FT转基因。然而，由于这些猪死于恶性肿瘤或发育不全，我们还观察到这种基因转移不仅降低了培养的内皮细胞中的αGal表达，还降低了唾液酸化，并抑制了管形成的能力。我们的结果表明，细胞表面碳水化合物抗原的极端修饰可能对发育或分化产生不利影响。我们还证明，使用腺病毒载体进行 α1,2 FT 基因或 GT 核酶转移会减少 αGal。我们现在正在探索用人类α1,2 FT基因直接替换α1,3 GT基因，即敲入方法，但还需要进一步的实验来检验细胞表面糖类抗原的修饰是否有效。我们分析了猪体内 alpha1,3 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可用时诱导 alphaGal 敲除猪。我们已经展示了几种替代方案。猪α1,3 GT中的剪接变异体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被发现具有较低的酶活性，表明茎的重要性。此外，没有外显子 8 或突变 cDNA（催化区域中的两个突变；外显子 9）的剪接变体也缺乏酶活性。降低猪体内αGal抗原表达的新策略，即引入无外显子7,8的cDNA或可以以显性负效应抑制α1,3 GT酶活性的突变体cDNA。

项目成果

Koike C,Takagi H: "Reduction of alpha-gal epitopes in transgenic pig by introduction of human alpha1-2 fucosyltransferase." Transplantation Proceedings. 29-1/2. 894 (1997)

Koike C、Takagi H：“通过引入人 α1-2 岩藻糖基转移酶减少转基因猪中的 α-gal 表位。”

Takagi H: "Organ Transplants Still Too in Japan and Asian Countries" Transplantation Proceedings. 29. 1580-1583 (1997)

高木 H：“日本和亚洲国家的器官移植仍然如此”移植论文集。

Negita M, Takagi H: "Protective effect of human suoeroxide dismutase cDNA transfection in the prevention of cold preservation injury" Transplantation Proceedings. 29. 1363- (1997)

Negita M、Takagi H：“人硫氧化物歧化酶 cDNA 转染对预防冷保存损伤的保护作用”移植论文集。

S,Hayashi, H Takagi: "Protection of guinea pig cell from rat complement-mediated lysis by the transfection of rat complement regulatory factor512 gene" 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。

Hayashi S,Takagi H: "Protection of guinea pig cells transfected with the 512 gene for complement dependent cytolysis in rats." Xenotransplantation. 3. 237-245 (1996)

Hayashi S、Takagi H：“用 512 基因转染的豚鼠细胞对大鼠补体依赖性细胞溶解的保护。”