Functional genetic correction of the X-linked Severe Combined Immunodeficiency using engineered nucleases (TALENs or CRISPR/Cas)

严重联合免疫缺陷（SCID）是免疫系统的破坏性疾病，大多数疾病的致病基因已经被确定。最常见的形式是SCID-X1，由IL2RG基因突变引起。同种异体造血干细胞移植可以治愈这种疾病，但当没有匹配的供体可用或儿童获得性感染时，结果会受到损害。一种理想的治疗遗传性疾病的方法是在自体造血细胞中纠正遗传缺陷。先前的基因治疗试验显示了总体的可行性和有效性，但也揭示了与所使用的导致插入突变的病毒载体的半随机整合相关的风险。最近，已经开发了新的方法，可以在其原始位置对突变基因进行靶向校正。它们依赖于创造一个特定位点的DNA断裂，并引入非突变的基因。细胞可以通过同源重组将该模板引入断裂位点。这用正确的版本取代了基因的突变版本，同时保留了基因调节的自然机制。这种基因编辑的工具是锌指核酸酶、TAL效应核酸酶（TALENs）和CRISPR/Cas系统。在初步工作中，基于TALEN和基于CRISPR/Cas的靶向外显子1下游基因的功能基因编辑方法已被确定为最有希望的IL2RG基因校正平台。已经使用了一系列的试验来比较这种方法与最可行的基因编辑竞争对手（基于外显子5的方法，锌指核酸酶），并确定外显子1 TALENs和CRISPR/Cas核酸酶将是更大比例的SCID-X1患者的治疗选择，并且这些试剂比基于zfn的方法具有更好的活性和毒性谱。本项目将通过TALENs或CRISPR/Cas系统在人造血干细胞和祖细胞中纠正IL2RG基因位点的缺陷，建立具有功能信号转导的多系造血和淋巴系统，并改进方法使翻译进入临床试验成为可能。其目的是通过优化功能基因校正方法来完善这一策略，以增加靶上活性，同时使用先进的技术进行广泛的安全性测试。其应用将增加到大规模GMP条件，以便准备首次人体试验。SCID-X1是应用新基因编辑方法的理想候选疾病，因为相对较少的校正细胞就足够了，但是，如果成功，这个项目的影响将是广泛的，因为它将确定组织特异性干细胞的基因编辑可以治愈破坏性疾病，从而将促进其他造血遗传疾病的类似项目的发展。

Severe combined immunodeficiencies (SCID) are devastating diseases of the immune system and for most of them disease causing genes have been identified. The most common form is SCID-X1 that is caused by mutations of the IL2RG gene. Allogeneic hematopoietic stem cell transplantation can cure the disease but the outcomes are impaired when no matched donor is available or the child has acquired infections. An ideal therapy for a genetic disease would be the correction of the genetic defect in autologous hematopoietic cells. Previous trials of gene therapy have shown general feasibility and efficacy but also uncovered the risks associated with the semi-random integration of the viral vectors that were used which lead to insertional mutagenesis. Recently, new methods have been developed that enable targeted correction of the mutated gene at its original site. They rely on the creation of a site-specific DNA break and introduction of a non-mutated version of the gene. The cell can introduce this template into the site of the break by homologous recombination. This replaces the mutated version of the gene by its correct version while the natural mechanisms of gene regulation are preserved. Tools for this gene editing are zinc finger nucleases, TAL effector nucleases (TALENs) and the CRISPR/Cas system. In preliminary work both TALEN based and CRISPR/Cas based functional gene editing approaches targeting the gene from exon 1 downstream have been identified as the most promising platforms for correction of the IL2RG gene. A series of assays has been used to compare this approach against the most viable gene editing competitors (exon 5 based approach, zinc finger nucleases) and determined that the exon 1 TALENs and CRISPR/Cas nucleases would be a treatment option for a much larger proportion of patients with SCID-X1 and that the reagents have better activity and toxicity profiles than ZFN-based approaches. In this project the defects of the IL2RG gene locus will be corrected by TALENs or the CRISPR/Cas system in human hematopoietic stem and progenitor cells to establish a multilineage hemato- and lymphopoiesis with functional signal transduction and the methods will be advanced to make translation into a clinical trial possible. It aims to refine this strategy by optimizing the functional gene correction methods in order to increase on-target activity while using advanced techniques for extensive safety testing. Its application will be increased to large-scale GMP conditions in order to prepare a first trial in humans. SCID-X1 is an ideal candidate disease for the application of the new gene editing methods because relatively small number of corrected cells are sufficient, but, if successful, the impact of this project will be broad because it will establish that gene editing of tissue specific stem cells can cure devastating diseases and thus will prompt the development of similar programs for other genetic diseases of hematopoiesis.