Modeling and correcting human SCID using patient-derived iPS cells

描述（由申请人提供）：严重联合免疫缺陷（SCID）包括以T淋巴细胞严重的数量和功能缺陷为特征的各种遗传缺陷，与B淋巴细胞和NK淋巴细胞的缺陷有不同的相关性。在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明；然而，在5例接受基因治疗的SCID患者中报道了由于插入突变引起的白血病增殖。总之，这些概念强调了研究人类模型以深入了解病理人类T细胞发育和开发新的更安全的基因治疗方法的重要性。人造血干细胞（hsc）与表达Notch1配体Delta-like 1 （Dll1）或Delta-like 4 （Dll4）的小鼠OP9基质细胞共培养已被用于研究T细胞分化。此外，严重免疫缺陷小鼠可以作为体内系统来研究人类造血干细胞的T细胞发育。然而，这些方法很难应用于人类SCID的研究，因为这种情况的罕见性限制了获取

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.