GalT-KO Pigs Expressing Primate CD47 to Facilitate Organ Xenografts

描述（由申请人提供）：猪器官异种移植为满足人体器官短缺所带来的限制提供了最好的近期希望。尽管通过生产β-1，3-半乳糖基转移酶敲除（GalT-KO）猪已经克服了灵长类受体中猪器官的超急性排斥，但是迄今为止用GalT-KO器官进行的实验强烈表明，受体的临床相关慢性免疫抑制将不足以克服异种移植物的排斥。通过造血嵌合体诱导耐受，最近在临床上显示允许人类同种异体移植物接受而没有慢性免疫抑制，可能在允许异种器官接受方面也发挥关键作用。 我们提出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化的产品，从而为临床推进实体器官异种移植提供策略。 最近的证据表明，猪至灵长类动物骨髓移植后嵌合体建立的关键障碍是CD47（IAP，整合素相关蛋白）细胞表面分子的种属不相容性。普遍表达的CD47结合巨噬细胞上的SIRP受体，从而抑制吞噬作用。灵长类动物CD47在猪细胞上的表达大大降低了它们对人巨噬细胞吞噬作用的敏感性。 本STTR提案的I期目标是开发和验证通过核转移生产在GalT-KO背景下表达灵长类CD47的猪所需的工具。这包括开发CD47的靶向/表达载体，开发用于鉴定靶向细胞克隆的分子测定，验证原代细胞克隆分离工艺和分离用于生产CD47转基因猪的原代胎儿成纤维细胞系。在第二阶段，我们将使用这些工具通过核转移生产表达灵长类动物CD47的GalT-KO猪，分析这些猪中的CD47表达，使用体外系统评估CD47表达的有效性，并进行有限数量的概念验证移植实验。 公共卫生相关性：对可移植人体器官的需求远远超过目前的供应，供需之间的差距继续扩大。猪的心脏、肾脏和其他器官的移植提供了极大地减少或消除这种短缺的机会。该项目的总体目标是生产转基因猪作为商业产品，这将进一步发展猪到人的器官移植。

DESCRIPTION (provided by applicant): Xenotransplantation with pig organs offers the best near term hope for satisfying the limitation imposed by shortage of human organs. 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 hematopoietic chimerism, which has recently been shown clinically to allow human allograft acceptance without chronic immunosuppression could potentially play a key role in allowing acceptance of xenogeneic organs as well. 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. Recent evidence suggests that a key barrier to the establishment of chimerism following pig-to-primate bone marrow transplantation is species incompatibility of the CD47 (IAP, integrin associated protein) cell surface molecule. 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. The goal of Phase I of this STTR proposal is to develop and verify the tools necessary for production, via nuclear transfer, of pigs expressing primate CD47 on a GalT-KO background. This includes development of targeting/expression vectors for CD47, development of molecular assays for identifying targeted cell clones, verification of a primary cell clone isolation process and isolation of primary fetal fibroblast lines for use in the production of CD47 transgenic pigs. In Phase II, we will 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 a limited number of proof-of-concept transplant experiments. PUBLIC HEALTH RELEVANCE: The demand for transplantable human organs far exceeds the current supply and the gap between demand and supply continues to grow. Transplantation of pig hearts, kidneys and other organs offers the opportunity to greatly reduce or eliminate this shortage. The overall goal of this project is to produce genetically modified pigs as a commercial product that will further the development of pig-to-human organ transplantation.