Modeling and correcting human SCID using patient-derived iPS cells

描述(申请人提供)：严重联合免疫缺陷(SCID)包括多种遗传缺陷，其特征是T淋巴细胞严重的数量和功能缺陷，不同程度地与B和NK淋巴细胞缺陷有关。同源SCID相关基因突变的小鼠和人类之间存在差异。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理证据；然而，有报道称，在5名接受基因治疗的SCID患者中，由于插入突变而导致的白血病增殖。总之，这些概念强调了研究人类模型的重要性，以获得对人类T细胞病理性发育的洞察，并开发出新的、更安全的基因治疗方法。人造血干细胞(HSCs)与表达Notch1配体Delta-like 1(DLL1)或Delta-like 4(DLL4)的小鼠OP9基质细胞共培养用于T细胞分化的研究。此外，严重免疫缺陷的小鼠可以作为体内系统来研究人类HSCs的T细胞发育。然而，这些方法几乎不能应用于人类SCID的研究，因为这种情况很罕见，限制了获得 从患者身上新分离的HSCs。诱导多能干细胞(IPSCs)代表了一种新的疾病建模和纠正工具，可用于克服迄今阻碍人类SCID中T细胞发育研究的技术和可行性障碍。我们组建了一支在免疫缺陷、IPSC生物学、T细胞发育和基因转移整合部位分析方面具有特定和互补专业知识的研究团队。我们已经建立了来自不同形式SCID患者的成纤维细胞系的大型资料库。我们现在打算从这些患者身上培养出IPSCs。我们将鉴定干细胞的干性和多能性，并验证患者来源的IPSCs的遗传完整性。为了建立SCID模型，我们将在体外和体内将IPSCs分化为T淋巴细胞。我们还打算使用患者来源的IPSCs作为平台，探索自失活慢病毒载体在体外和免疫缺陷小鼠体内纠正各种形式的SCID方面的有效性和安全性。我们预计，SCID患者来源的IPSCs将代表一个新的和强大的平台，用于研究人类T细胞发育和对这些疾病的基因治疗的有效性和安全性的临床前评估。

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.