Reconstitution of T-lineage differentiation potential of induced pluripotent stem cells derived from severe combined immunodeficiency patients by retroviral gene transfer.

严重联合免疫缺陷（SCID）是一种异质性疾病，由不同的单基因突变引起，导致T细胞分化的完全阻断。免疫重建可以通过造血干细胞移植来实现，受供体可用性和潜在严重副作用的限制，或者基因转移到患者的造血细胞中，目前仅可用于ADA和XSCID。由于发生了几例插入突变，因此创建了缺乏病毒增强子序列的自失活载体，以改善正在进行的试验的安全性问题。该项目的目的是测试，如果T细胞重建也可以通过基因转移在其他形式的SCID中实现。因此，通过替换适当的转基因修饰的相同载体骨架将用于转导携带ADA、RAG 2、ILR 2G、JAK 3、LIG 4、DCLRE 1C和AK 2突变的患者来源的诱导多能干细胞（iPSC）。读数将是使用0 P9-DL 4共培养系统的体外T细胞分化的评估。作为疾病模型的IPSC将从患者的成纤维细胞系通过逆转录病毒转移四种重编程因子OCT 4、KLF 4、SOX 2和c-MYC与酶可切除的病毒产生。了解使用安全载体系统的逆转录病毒基因转移是否能够恢复这些其他SCID疾病的T细胞分化潜力具有重要意义，因为这将是未来基因治疗试验的基础。

Severe combined immunodeficiency (SCID) is a heterogenous disease caused by different monogenic mutations that result in a complete block of T-cell differentiation. Immune reconstitution can be achieved either by hematopoietic stem cell transplantation, limited by donor availability and potential severe side-effects, or gene transfer into the patients¿ hematopoietic cells, which is currently only available for ADA- and XSCID. As several cases of insertional mutagenesis occured, a self-inactivating vector lacking viral enhancer sequences was created to improve safety issues for ongoing trials. The aim of this project is to test, if T-cell reconstitution can be achieved by gene transfer also in other forms of SCID. Therefore, the same vector backbone modified by replacement of the appropriate transgene will be used for transduction of patient-derived induced pluripotent stem cells (iPSC) carrying mutations in ADA, RAG2, ILR2G, JAK3, LIG4, DCLRE1C, and AK2. The read-out will be the assessment of T-cell differentiation in vitro using the OP9-DL4 co-culture system. IPSCs, functioning as a disease model, will be generated from patient’s fibroblast lines by retroviral transfer of the four reprogramming factors OCT4, KLF4, SOX2 and c-MYC with enzymatically excisable viruses. It is of great significance to know, wether the retroviral gene transfer using a safe vector system is able to restore T-cell differentiation potential in these other SCID diseases, as this will be the basis for future gene therapy trials.