GalT-KO Pigs Expressing Primate CD47 to Facilitate Organ Xenografts

描述(申请人提供)：猪器官异种移植为满足人体器官短缺带来的限制提供了最好的近期希望。这项研究项目旨在将转基因猪作为一种产品来生产，它将是异种移植到人类身上的最好的潜在捐赠者。 尽管灵长类受体猪器官的超急性排斥反应已经通过培育半乳糖基转移酶基因敲除(GALT-KO)猪来克服，但迄今为止关于GALT-KO器官的实验强烈表明，临床上与受体相关的慢性免疫抑制不足以克服异种移植的排斥反应。通过混合造血细胞嵌合体诱导耐受是目前诱导这种耐受的最好方法，最近的研究表明，这种方法可以在没有慢性免疫抑制的情况下接受人类同种异体移植。跨越猪到灵长类的障碍实现混合嵌合体的主要问题是难以实现持久的嵌合体。 我们建议的产品将包括具有增强的异种造血嵌合体潜力的转基因猪，我们将向研究界提供商业应用，从而为推动固体器官异种移植走向临床提供一种策略。我们将通过克服CD47(IAP，整合素相关蛋白)细胞表面分子的物种不相容来实现这一目标，CD47似乎是猪到灵长类骨髓移植后建立嵌合体的关键障碍。CD47广泛表达，与巨噬细胞上的SIRP(受体)结合，从而抑制吞噬作用。灵长类CD47在猪细胞上的表达大大降低了猪细胞对人巨噬细胞吞噬的敏感性。 在这个STTR提案的第一阶段，我们开发并验证了通过核移植生产在Galt-KO背景下表达灵长类CD47的猪所需的工具。我们成功地开发了CD47的靶向/表达载体和鉴定靶向细胞克隆的分子检测方法。我们还验证了原代细胞克隆的分离，并分离出了用于生产CD47转基因猪的原代胎儿成纤维细胞系。在第二阶段，我们打算使用这些工具通过核移植培育表达灵长类CD47的Galt-KO猪，分析这些猪的CD47表达，使用体外系统评估CD47表达的有效性，并进行概念验证移植实验，以验证该产品在实现混合异种嵌合体和耐受性方面的有效性。

DESCRIPTION (provided by applicant): Xenotransplantation with pig organs offers the best near term hope for satisfying the limitation imposed by shortage of human organs. This research project is directed toward producing as a product a genetically modified pig that will be the best potential donor for xenotransplants to humans. Although hyperacute rejection of pig organs in primate recipients has been overcome by the production of (-1,3-galactosyltransferase knockout (GalT-KO) pigs, experiments to date with GalT-KO organs strongly suggest that clinically relevant chronic immunosuppression of recipients will be insufficient to overcome rejection of xenotransplants. Tolerance induction through mixed hematopoietic chimerism is currently the best means for inducing such tolerance, and this methodology has recently been shown to allow human allograft acceptance without chronic immunosuppression. The major problem in achieving mixed chimerism across the pig-to- primate barrier has the difficulty in achieving durable chimerism. Our proposed product will consist of genetically modified pigs with enhanced xenogeneic hematopoietic chimerism potential that we will make available commercially to the research community, thereby providing a strategy for advancing solid organ xenotransplantation toward the clinic. We will achieve this goal by overcoming the species incompatibility of the CD47 (IAP, integrin associated protein) cell surface molecule, which appears to be the key barrier to the establishment of chimerism following pig-to-primate bone marrow transplantation. Ubiquitously expressed, CD47 binds SIRP( receptors on macrophages and thereby inhibits phagocytosis. Expression of primate CD47 on pig cells greatly reduces their susceptibility to phagocytosis by human macrophages. In Phase I of this STTR proposal we developed and verified the tools necessary for production, via nuclear transfer, of pigs expressing primate CD47 on a GalT-KO background. We successfully developed targeting/expression vectors for CD47 and molecular assays for identifying targeted cell clones. We have also verified a primary cell clone isolation and have isolated primary fetal fibroblast lines for use in the production of CD47 transgenic pigs. In Phase II, we now intend to use these tools to produce GalT-KO pigs expressing primate CD47 via nuclear transfer, analyze CD47 expression in these pigs, evaluate the efficacy of CD47 expression using in vitro systems, and perform proof-of-concept transplant experiments to validate the usefulness of this product in achieving mixed xenogeneic chimerism and tolerance.