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）是一种由不同的单基因突变引起的异质性疾病，导致t细胞分化完全受阻。免疫重建既可以通过造血干细胞移植来实现，但受供体可用性和潜在严重副作用的限制，也可以通过将基因转移到患者的造血细胞中来实现，这目前仅适用于ADA和XSCID。由于发生了几例插入突变，因此创建了一种缺乏病毒增强子序列的自灭活载体，以改善正在进行的试验的安全性问题。这个项目的目的是测试，如果t细胞重组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主干将用于转导携带ADA、RAG2、ILR2G、JAK3、LIG4、DCLRE1C和AK2突变的患者源性诱导多能干细胞（iPSC）。读数将是使用OP9-DL4共培养系统体外t细胞分化的评估。IPSCs作为一种疾病模型，将由患者成纤维细胞系通过酶切可切除的病毒将四种重编程因子OCT4、KLF4、SOX2和c-MYC逆转录病毒转移产生。使用安全的载体系统进行逆转录病毒基因转移是否能够恢复其他SCID疾病的t细胞分化潜力，这将是未来基因治疗试验的基础，具有重要意义。

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.