Cloned pigs for Xenotransplantation

描述(申请人提供)：建议研究的目的是开发用于异种移植的免疫保护通用供体克隆转基因猪的方法。根据器官共享联合网络(UNOS)的数据，仅在美国就进行了超过3万例器官移植，但目前有超过9.5万人在等待接受捐赠者的器官，以治疗终末期器官衰竭。目前增加可供移植的捐献器官池的战略只导致微不足道的增加，不足以满足对捐献器官和组织日益增长的需求。许多临床研究人员、大型国际制药公司以及几家生物技术公司都在寻求开发转基因猪，作为通用捐赠者组织的来源。因此，这项研究计划的第一阶段的目标是将来自先前克隆的含有人H-转移酶基因的原代细胞系和猪半乳糖转移酶基因的纯合子靶向突变(GT-/-HT)核移植到体外培养的猪卵母细胞中。将测试原代胚胎成纤维细胞和胚胎神经干细胞，通过评估囊胚发育来确定哪种细胞类型是体外核移植实验中最好的核供体(实验1)。随后，实验1中最有效地产生胚泡的细胞类型将被基因改造为双功能嵌合分子(CD152-CD59)，该分子既能抑制对移植组织的体液免疫反应，也能抑制细胞免疫反应。我们将选择转基因细胞克隆，并在体外对细胞进行分析，以评估转染基因产物的表达水平和功能。然后，将选择不同的转基因细胞克隆进行后续的核移植实验，以证明转基因细胞在体外形成囊胚。证明来自先前克隆的转基因猪的细胞的体外囊胚发育，然后进一步用设计来抵抗免疫破坏的分子进行转基因，将提供必要的数据来支持第二阶段STTR研究方案，该方案旨在开发克隆的活动物，然后用于评估体内移植模型的存活率，这最终可能导致临床移植应用。

DESCRIPTION (provided by applicant): The purpose of the proposed research is to develop methods to create immune protected universal donor cloned transgenic pigs for xenotransplantation. According to the United Network of Organ Sharing (UNOS) more than 30,000 organ transplants are performed in the US alone, yet greater than 95,000 people are currently on the waiting list to receive donors organs for the treatment of end-stage organ failure. Current strategies to increase the pool of donor organs available for transplantation has only led to marginal increases that are insufficient to satisfy the growing need for donor organs and tissues. Many clinical investigators, large international pharmaceutical companies as well as several biotechnology companies have looked to develop transgenic pigs as a source of universal donor tissue. Therefore, the goal of this research plan for the Phase I STTR, is to perfect nuclear transfer with primary cell lines derived from previously cloned animals containing the human H-transferase gene and a homozygous targeted mutation of the porcine Gal-alpha1,3Gal transferase gene (GT-/-HT) into in vitro cultured pig oocytes. Primary embryonic fibroblasts and embryonic neural stem cells will be tested to determine which cell type is the best nuclear donor in in vitro nuclear transfer experiments by assessing blastocyst development (Experiment 1). Subsequently, the cell type from Experiment 1 that is most efficacious in producing blastocysts will be genetically modified with a bi-functional chimeric molecule (CD152-CD59) that inhibits both humoral and cellular immune responses to transplanted tissues. We will select transfected cell clones and analyze the cells in vitro to asses expression levels and the function of the transfected gene product. Different transfected cell clones will then be selected for follow-on nuclear transfer experiments to demonstrate blastocyst formation in vitro from the transfected cells. Demonstrating blastocyst development in vitro from cells derived from a previously cloned transgenic pig that are then genetically modified further with a molecule designed to resist immune destruction will provide the necessary data to support a Phase II STTR research proposal for developing cloned living animals that could then be used to assess survival in in vivo transplant models, which could eventually lead to clinical transplantation applications.