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捐助组织短缺的替代方法。随着分子生物学技术最近进步，克服抗原抗体相互作用问题的尝试已针对供体的修饰。由于表达DAF的转基因猪（衰减加速度因子）无法完全抑制超级分离的排斥反应，因此注意力集中在去除或替换字母表位。字母表位由字母乳糖基转移酶（GT）合成。不幸的是，由于猪胚胎干细胞的吻合性，基因敲除目前无法在猪中实现。已经尝试了一种称为“碳水化合物表面的酶促重塑”的替代方法。也就是说，这个概念是，字母分子将被另一种寡糖（例如fucose）代替，例如fucose fucose（ft）。我们已经产生了α1，…更多2英尺的转基因猪。但是，由于这些猪死于恶性肿瘤或不完整的发育，因此无法确定这种菌株。我们还展示了这样的观察结果，即这种基因不仅降低了字母表达，而且在培养的内皮细胞中也降低了溶解作用，并抑制了管形成的能力。我们的结果表明，细胞表面上碳水化合物抗原的极端修饰可能对发育或分化有害。我们还证明了使用腺病毒载体转移α1,2英尺基因或GT核酶在体外降低字母表达。我们现在正在用人α1,2ft基因（即敲入方法）探索α1,3GT基因的直接基因替代。但是，需要进一步的实验来检查细胞表面中的碳含抗原抗原是否有效。我们分析了猪中α1,3GT的基因组结构，并构建靶向载体，目的是诱导的α型敲除猪的目的是在猪敲除猪的目的中，因为猪的胆量型干细胞或核转置均可使用。我们已经显示了猪α1,3gt中的几种替代剪接变体，这与酶活性的功能差异有关。在外显子5,6和7中有缺陷的某些剪接变体的酶促活性较少，表明茎区域在Enxyme活性中的重要性。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）的剪接清晰度也没有酶活性。这些观察结果表明，降低猪字母抗原表达的新策略，也就是说，引入没有外显子7,8的cDNA或突变cDNA或突变cDNA可以抑制α1,3GT酶促活性在显性负面影响中。较少的

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。”