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

描述（由申请人提供）：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，否则这些疾病是致命的。RAG 1和RAG 2基因突变是人类T-B-NK+ SCID的最常见原因。同一基因的亚型缺陷可能导致渗漏性SCID或Omenn综合征（OS），这是一种与浸润性和寡克隆T细胞引起的多器官损伤相关的严重免疫缺陷。RAG缺陷HCT后的长期存活率仅为50%，在渗漏性SCID和OS中甚至更差。最近，当缺乏HLA匹配的供体时，病毒介导的基因转移到造血CD 34+祖细胞中已被用于治疗某些形式的SCID。然而，这种方法与插入诱变有关。因此，寻求新的和更安全的基因校正技术是至关重要的。归巢核酸内切酶（HE）以特异性方式识别大的（>12 bp）DNA靶序列，并且可以用于尝试基因校正。我们的合作者Frederic Paques设计了一种RAG 1特异性HE。旨在探索基因转移在免疫缺陷患者中的功效的临床前研究的主要限制之一是患者来源的靶细胞的有限可用性。然而，成纤维细胞可以通过病毒介导的转录因子组合的转导在体外重编程为诱导多能干细胞（iPSC）。这些iPSC可以在体外靶向多谱系分化（包括T淋巴细胞）。 我们已经从携带不同RAG 1突变的SCID或OS患者中产生了成纤维细胞系的储存库。我们现在打算从RAG 1突变的患者中产生iPSC，并表征其干细胞和多能性特征，染色体完整性和患者特异性衍生。 与Zuniga-Pflucker博士合作，我们将研究这些患者来源的iPSC在体外进行沿着T细胞分化的能力。我们还将探索RAG 1特异性HE纠正患者来源的iPSC中突变RAG 1基因座的能力，以及支持V（D）J重组和体外T细胞分化的能力。我们将比较基因校正的校正和未校正的患者来源的iPSC支持体外T细胞发育的能力。 这项研究将允许定义各种RAG 1突变的特定能力，以支持T淋巴细胞分化，从而有助于解释人类RAG缺陷的表型多样性的基础。此外，本项目还将探索一种基于基因特异性核酸内切酶介导的同源重组的基因治疗SCID的新方法的有效性和安全性。 公共卫生相关性：人类RAG 1和RAG 2基因的缺陷会导致一系列严重的免疫缺陷，从完全缺乏淋巴细胞（SCID）到以免疫缺陷和自身免疫为特征的病症（Omenn综合征）。这种多样性的基础仍然没有明确界定。为了解决这个问题，我们将诱导RAG 1基因缺陷患者的皮肤细胞（成纤维细胞）成为多能干细胞（iPSC），然后我们将研究它们在试管中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源性干细胞中的RAG 1基因缺陷。这可能为通过基因修复来纠正遗传疾病的新的和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.