Developing second generation SCID pig models: filling the gaps to improve translation of therapeutics in regenerative medicine

项目摘要/摘要：干细胞疗法的前景尚未完全实现，部分原因是 缺乏合适的临床前模型。由于相似之处，猪是人类生物学的极好模型。 在大小、生理和遗传学方面，在某些情况下可能优于啮齿动物模型，后者往往无法提供 有效转化为人体临床试验的数据。因此，更准确地模拟人类的猪模型 是改善再生医学疗法的研究成果和最大限度地提高安全性的关键 翻译到诊所。随着SCID猪模型在异种移植研究中显示出希望，这建立了进一步的需求 将人源化猪作为第二代模型和改进的方法来常规饲养免疫缺陷猪 用于测量植入的治疗细胞的存活率、安全性和潜在疗效。我们专注于 产生这样的新知识，并展示SCID猪在临床前再生医学研究中的应用。 我们已经开发了生物遏制设施和方案来饲养Artemis(ART)突变的SCID猪。我们 通过成功的人诱导异位移植，验证了该模型的意义 多能干细胞(IPSC)和人类癌细胞系以及原位移植的存活 人的皮肤。为了改进模型，我们在一名男性ART患者中突变了白细胞介素2受体伽马基因(IL2RG) 背景为空。ART零IL2RG零基因猪具有预期的T-B-NK表型，我们已经成功地 在这些ART-IL2RG猪身上展示了部分人类免疫系统的发育。因此，我们采取了 建立猪SCID模型的开创性步骤，但异种移植安全性/有效性测试和 人性化仍然是利用这些转化医学研究模式的先决条件。 本应用程序的具体目标是a)验证用于临床前细胞测试的SCID猪模型以及 组织异种移植，b)使用多种新的人性化方法来确定人类 SCID猪的造血，以及c)建立第二代SCID猪模型管理方案 多个生物控制设施。这项拟议研究的基本原理是，我们远缘繁殖的SCID猪可能会比 准确地反映出与小鼠相比，拟议的干细胞衍生疗法在人类中将如何发挥作用。这 该项目是创新的，因为我们将使用一种综合的方法来结合对模型的异种移植的研究 潜力，研究重点是改善免疫缺陷猪的使用方案，包括 人性化的方法。我们期待这个项目的成功完成将创造出遗传资源， 关于异种移植和人性化比率的数据，以及相关的动物程序，将是非常理想的 基于SCID的干细胞疗法安全性和有效性研究的建模。这些独特的资源 预计将对加速将再生医学研究转化为 诊所。

Project Summary/Abstract: The promise of stem cell therapies has not been fully realized, due in part to a paucity of appropriate pre-clinical models. The pig is an excellent model of human biology, due to similarities of size, physiology, and genetics, and in some cases may be superior to rodent models, which often fail to provide data which effectively translates to human clinical trials. Thus, pig models that more accurately model humans are critically needed to improve research outcomes of regenerative medicine therapeutics and maximize safe translation to the clinic. As SCID pig models show promise in xenograft studies, this establishes further needs for humanized pigs as second-generation models and improved methods to routinely rear immunodeficient pigs for measurement of survival, safety, and potential efficacy of implanted therapeutic cells. We are focused on generating such new knowledge and demonstrating SCID pig use in preclinical regenerative medicine research. We have developed biocontainment facilities and protocols to raise Artemis (ART)-mutant SCID pigs. We have demonstrated this model's significance through successful ectopic engraftment with human induced pluripotent stem cells (iPSC) and human cancer cell lines, as well as survival of orthotopically transplanted human skin. To improve the model, we mutated the Interleukin 2 Receptor gamma gene (IL2RG) in a male ART null background. The ART null IL2RG null pig has the expected T-B-NK- phenotype, and we have successfully demonstrated partial human immune system development in these ART-IL2RG pigs. Thus we have taken pioneering steps to establish a pig SCID model, but improvements in xenograft safety/efficacy testing and humanization remain prerequisites to harness these research models for translational medicine. The specific objectives of this application are to a) validate SCID pig models for preclinical testing of cell and tissue xenografts, b) use multiple novel humanization approaches to determine the extent of human hematopoiesis in SCID pigs, and c) establish second-generation SCID pig model management protocols at multiple biocontainment facilities. The rationale for the proposed research is that our outbred SCID pig may more accurately reflect how proposed stem cell derived therapies will function in humans compared to mice. This project is innovative because we will use an integrated approach to combine research on the model's xenograft potential with research focused on protocols for improving the use of immunodeficient pigs, including humanization methods. We expect that the successful completion of this project will create genetic resources, data on xenograft and humanization rates, and associated animal procedures that will be highly desirable for SCID based modeling for research on the safety and efficacy of stem cell therapeutics. These unique resources are expected to have a significant impact in accelerating the translation of regenerative medicine research into the clinic.