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，否则是致命的。RAG1和RAG2基因突变是人类T-B-NK+ SCID的最常见原因。同一基因的半形缺陷可能导致漏性SCID或Omenn综合征（OS），这是一种严重的免疫缺陷，与浸润性和寡克隆T细胞引起的多器官损伤有关。RAG缺乏的HCT术后长期生存率仅为50%，漏SCID和OS的长期生存率更低。最近，病毒介导的基因转移到造血CD34+祖细胞已被用于治疗某些类型的SCID，当hla匹配供体缺乏。然而，这种方法与插入突变有关。因此，追求新颖和更安全的基因校正技术是至关重要的。归巢内切酶（HE）以特定的方式识别大的（bb0 - 12bp） DNA靶序列，并可用于尝试基因校正。我们的合作者Frederic Paques设计了一个rag1特异性HE。旨在探索人类免疫缺陷基因转移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转导在体外重编程为诱导多能干细胞（iPSCs）。这些iPSCs可以在体外靶向多系分化（包括T淋巴细胞）。我们从SCID或OS患者身上获得了携带不同RAG1突变的成纤维细胞系库。我们现在打算从rag1突变的患者中生成iPSCs，并表征它们的干性和多能性、染色体完整性和患者特异性来源。在与Zuniga-Pflucker博士的合作中，我们将研究这些患者来源的iPSCs在体外进行t细胞分化的能力。我们还将探索RAG1特异性HE在患者来源的iPSCs中纠正RAG1突变位点的能力，以及在体外支持V(D)J重组和T细胞分化的能力。我们将比较基因校正和未校正的患者来源的iPSCs在体外支持t细胞发育的能力。这项研究将允许定义各种RAG1突变支持t淋巴细胞分化的特定能力，并可能因此有助于解释人类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SCID基因治疗新方法的有效性和安全性。

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.