In vitro differentiation of RAG1-mutated iPS cells and correction by meganuclease

描述(申请人提供)：严重联合免疫缺陷(SCID)包括一组异质性遗传疾病，除非通过造血细胞移植(HCT)治疗，否则是致命的。RAG1和RAG2基因突变是人类T-B-NK+SCID最常见的原因。同一基因的亚型缺陷可能导致渗漏SCID或Omenn综合征(OS)，这是一种严重的免疫缺陷，与T细胞浸润性和寡克隆性T细胞的多器官损害有关。RAG缺乏症患者经HCT治疗后的长期存活率仅为50%，渗漏的SCID和OS患者的远期存活率更低。最近，病毒介导的CD34+造血祖细胞基因转移已被用于治疗某些形式的SCID，当缺乏匹配的供者时。然而，这种方法与插入突变有关。因此，寻求新的、更安全的基因校正技术是最重要的。归巢核酸内切酶(HE)以一种特定的方式识别大的(&gt；12个碱基)DNA目标序列，并可用于尝试基因校正。我们的合作者弗雷德里克·帕克斯设计了一种RAG1特有的HE。旨在探索免疫缺陷患者基因转移有效性的临床前研究的主要局限性之一是患者来源的靶细胞有限。然而，通过病毒介导的转录因子组合的转导，成纤维细胞在体外可以被重新编程为诱导多能干细胞(IPSCs)。这些IPSCs可以在体外定向分化为多系细胞(包括T淋巴细胞)。我们已经从SCID或OS患者那里建立了一个成纤维细胞系的储存库，携带不同的RAG1突变。我们现在打算从RAG1突变的患者中产生IPSCs，并表征它们的干性和多能性特征、染色体完整性和患者特有的派生。在Zuniga-Pflucker博士的合作下，我们将研究这些患者来源的IPSCs在体外沿着T细胞分化的能力。我们还将探索RAG1特异性HE纠正患者来源的IPSCs中突变的RAG1位点的能力，以及在体外支持V(D)J重组和T细胞分化的能力。我们将比较经过基因矫正和未经矫正的患者来源的IPSCs在体外支持T细胞发育的能力。这项研究将允许确定各种RAG1突变支持T淋巴细胞分化的特异性能力，从而可能有助于解释人类RAG缺陷表型多样性的基础。此外，本项目还将探索基于基因特异性内切酶介导的同源重组的一种新的SCID基因治疗方法的有效性和安全性。 公共卫生相关性：人类RAG1和RAG2基因缺陷导致一系列严重的免疫缺陷，从淋巴细胞完全缺失(SCID)到以免疫缺陷和自身免疫为特征的疾病(Omenn综合征)。这种多样性的基础仍然界定不清。为了解决这个问题，我们将RAG1基因缺陷患者的皮肤细胞(成纤维细胞)诱导为多能干细胞(IPSCs)，然后在试管中研究它们分化为淋巴细胞的能力。此外，我们将使用一种新的蛋白质来修复患者来源干细胞中的RAG1基因缺陷。这可能为通过基因修复来纠正遗传病的新的、更安全的方法铺平道路。

DESCRIPTION (provided by applicant): Severe combined immunodeficiency (SCID) comprises a group of heterogeneous genetic disorders that are fatal, unless treated by hematopoietic cell transplantation (HCT). Mutations of RAG1 and RAG2 genes are the most common cause of T-B-NK+ SCID in humans. Hypomorphic defects in the same gene may cause leaky SCID or Omenn syndrome (OS), a severe immunodeficiency associated with multiorgan damage due to infiltrating and oligoclonal T cells. Long-term survival rate after HCT for RAG deficiency is only 50%, and is even worse in leaky SCID and OS. Recently, virus-mediated gene transfer into hematopoietic CD34+ progenitor cells has been used to treat some forms of SCID, when HLA-matched donors are lacking. However, this approach has been associated with insertional mutagenesis. Therefore, the pursue of novel and safer technologies for gene correction is of outmost importance. Homing endonucleases (HE) recognize large (>12 bp) DNA target sequences in a specific manner, and could be used to attempt gene correction. Our collaborator Frederic Paques has engineered a RAG1-specific HE. One of the major limitations of the preclinical studies that aim at exploring the efficacy of gene transfer in humans with immunodeficiency is the limited availability of patient-derived target cells. However, fibroblasts can be reprogrammed in vitro into induced pluripotent stem cells (iPSCs) through virus-mediated transduction of a combination of transcription factors. These iPSCs can be targeted multilineage differentiation (including T lymphocytes) in vitro. We have generated a repository of fibroblast cell lines from patients with SCID or OS, carrying different RAG1 mutations. We now intend to generate iPSCs from RAG1-mutated patients, and to characterize their stemness and pluripotency profile, chromosomal integrity and patient-specific derivation. In collaboration with Dr. Zuniga-Pflucker, we will investigate the ability of these patient-derived iPSCs to proceed along T-cell differentiation in vitro. We will also explore the ability of the RAG1-specific HE to correct the mutant RAG1 locus in patient-derived iPSCs, and to support V(D)J recombination and T cell differentiation in vitro. We will compare the ability of gene-corrected corrected and uncorrected patient-derived iPSCs to support T-cell development in vitro. This study will permit to define the specific ability of various RAG1 mutations to support T-lymphocyte differentiation, and may thus help explain the basis of the phenotypic diversity of RAG defects in humans. Furthermore, this project will also explore the efficacy and safety of a novel approach to gene therapy of SCID, based on gene-specific endonuclease-mediated homologous recombination. PUBLIC HEALTH RELEVANCE: Defects of the RAG1 and RAG2 genes in humans cause a spectrum of severe immunodeficiencies that range from complete absence of lymphocytes (SCID) to a condition characterized by immunodeficiency and autoimmunity (Omenn syndrome). The basis for this diversity remains poorly defined. To address this issue, we will induce skin cells (fibroblasts) from patients with RAG1 gene defects, to become pluripotent stem cells (iPSCs), and we will then study their ability to differentiate into lymphocytes in a test tube. Moreover, we will use a novel protein to repair the RAG1 gene defect in patient-derived stem cells. This may pave the way to novel and safer approaches to correct genetic diseases by gene repair.