Modeling and correcting human SCID using patient-derived iPS cells

描述（由申请方提供）：严重联合免疫缺陷（SCID）包括多种遗传缺陷，其特征为T淋巴细胞的严重数量和功能缺陷，与B和NK淋巴细胞缺陷相关。小鼠和人类之间存在差异，在直系同源物SCID相关基因中存在突变。此外，对于某些形式的SCID，目前还没有可用的动物模型。最后，SCID提供了基因治疗可以治愈人类疾病的原则证据;然而，在5例接受基因治疗的SCID患者中报告了由于插入突变导致的白血病增殖。总之，这些概念强调了研究人类模型的重要性，以深入了解病理性人类T细胞的发育，并开发新的和更安全的基因治疗方法。人造血干细胞（HSC）与经工程化以表达Notch 1配体δ样1（Dll 1）或δ样4（Dll 4）的小鼠0 P9基质细胞的共培养已用于研究T细胞分化。此外，严重免疫缺陷的小鼠可以作为一个体内系统来研究T细胞从人类HSC的发展。然而，这些方法很难应用于人类SCID的研究，因为这种疾病的罕见性限制了对SCID的研究。 从患者新鲜分离的HSC。诱导多能干细胞（iPSC）代表了一种用于疾病建模和校正的新工具，可用于克服迄今为止阻碍人类SCID中T细胞发育研究的技术和可行性障碍。 我们已经组建了一个研究团队，他们在免疫缺陷，iPSC生物学，T细胞发育和基因转移后的整合位点分析方面具有特定和互补的专业知识。我们已经建立了一个来自各种形式的SCID患者的成纤维细胞系的大型储存库。我们现在打算从这些患者中产生iPSC。我们将表征干细胞和多能性特征，并验证患者来源的iPSC的遗传完整性。为了模拟SCID，我们将在体外和体内将iPSC分化为T淋巴细胞。我们还打算使用患者来源的iPSC作为平台，以探索自失活慢病毒载体在免疫缺陷小鼠中在体外和体内纠正各种形式的SCID的有效性和安全性。我们预计，SCID患者来源的iPSC将代表一种新的和强大的平台，用于研究人类T细胞发育和临床前评估这些疾病的基因治疗的有效性和安全性。

DESCRIPTION (provided by applicant): Severe combined immune deficiency (SCID) includes a variety of genetic defects characterized by severe numerical and functional defects of T lymphocytes, variably associated with defects of B and NK lymphocytes. Differences exist between mice and humans with mutations in orthologue SCID- associated genes. Moreover, for some forms of SCID no animal models are currently available. Finally, SCID have offered proof of principle that gene therapy can cure human diseases; however, leukemic proliferation due to insertional mutagenesis has been reported in 5 SCID patients treated by gene therapy. Altogether, these notions emphasize the importance of studying human models to gain insights into pathological human T cell development and to develop novel and safer approaches to gene therapy. Co-culture of human hematopoietic stem cells (HSCs) with mouse OP9 stromal cells engineered to express the Notch1 ligands Delta-like 1 (Dll1) or Delta-like 4 (Dll4) has been used to study T cell differentiation. Moreover, severely immunodeficient mice can serve as an in vivo system to study T cell development from human HSCs. However, these approaches can be hardly applied to the study of human SCID, given the rarity of this condition that limits access to freshly isolated HSCs from the patients. Induced pluripotent stem cells (iPSCs) represent a novel tool for disease modeling and correction and may be used to overcome technical and feasibility barriers that have so far hampered the study of T cell development in human SCID. We have assembled a team of investigators with specific and complementary expertise in immunodeficiency, iPSC biology, T cell development and integration site analysis upon gene transfer. We have created a large repository of fibroblast cell lines from patients with various forms of SCID. We now intend to generate iPSCs from these patients. We will characterize the stemness and pluripotency profile and verify genetic integrity of patient-derived iPSCs. To model SCID, we will differentiate iPSCs into T lymphocytes both in vitro and in vivo. We also intend to use patient-derived iPSCs as a platform to explore the efficacy and safety of self-inactivating lentiviral vectors in the correction of various forms of SCID both in vitro and in viv in immunodeficient mice. We anticipate that SCID patients-derived iPSCs will represent a novel and robust platform to study human T cell development and for preclinical assessment of efficacy and safety of gene therapy for these diseases.