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

RAG1 突变 iPS 细胞的体外分化和大范围核酸酶校正

• 批准号: 7947212
• 负责人: Luigi Daniele Notarangelo
• 金额: $ 21.02万
• 依托单位: BOSTON CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-06-04 至 2012-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7947212
• 关键词: Address; Autoimmunity; B-Lymphocytes; Boston; CD34 gene; Canada; Cell Line; Cell Transplantation; Cells; Cleaved cell; Collaborations; Complex; DNA; DNA Repair; DNA Sequence; Data; Defect; Derivation procedure; Development; Disease; Engineering; Family; Fibroblasts; France; Gene Transfer; Generations; Genes; Hematopoietic; Hematopoietic stem cells; Hereditary Disease; Homing; Human; IL2RG gene; Immune; Immunologic Deficiency Syndromes; In Vitro; Insertional Mutagenesis; Location; Lymphocyte; Malignant - descriptor; Mediating; Mus; Mutate; Mutation; Names; Patients; Phenotype; Pluripotent Stem Cells; Proteins; RAG1 gene; Rag1 Mouse; SCID Mice; Safety; Severe Combined Immunodeficiency; Skin; Stem cells; Stromal Cells; Survival Rate; Syndrome; System; T cell differentiation; T-Cell Development; T-Lymphocyte; Technology; Testing; Therapeutic; Therapeutic Intervention; Tissues; Tube; V(D)J Recombination; Virus; X-Linked Severe Combined Immunodeficiency; adenosine deaminase deficiency; base; embryonic stem cell; endonuclease; gene correction; gene repair; gene therapy; homologous recombination; in vitro Model; in vivo; induced pluripotent stem cell; lentiviral-mediated; mouse model; multiorgan damage; mutant; novel; novel strategies; novel therapeutics; null mutation; pluripotency; pre-clinical; preclinical study; protein expression; public health relevance; reconstitution; repaired; repository; stemness; therapeutic gene; transcription factor

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.

描述（由申请人提供）：严重的合并免疫缺陷（SCID）包括一组致命的异质遗传疾病，除非受造血细胞移植（HCT）治疗。 RAG1和RAG2基因的突变是人类T-B-NK+ SCID的最常见原因。同一基因中的型缺陷可能导致SCID或OMENN综合征（OS），这是由于浸润和寡克隆T细胞引起的多项式损伤的严重免疫缺陷。 HCT后的RAG缺乏症后的长期存活率仅为50％，在SCID和OS中甚至更糟。最近，当缺乏HLA匹配的供体时，病毒介导的基因转移到造血CD34+祖细胞中已被用来治疗某些形式的SCID。但是，这种方法与插入诱变有关。因此，追求新颖和更安全的基因校正技术至关重要。归巢核酸内切酶（HE）以特定方式识别大型（> 12 bp）的DNA靶序列，可用于尝试基因校正。我们的合作者弗雷德里克·帕克斯（Frederic Paques）设计了一个特定于RAG1的HE。临床前研究的主要局限性之一是探索具有免疫缺陷的人类基因转移疗效的是患者衍生的靶细胞的有限可用性。然而，通过病毒介导的转录因子组合的转导，可以在体外重编程成纤维细胞在诱导的多能干细胞（IPSC）中重编程。这些IPSC可以在体外被靶向多琳分化（包括T淋巴细胞）。 我们已经产生了来自SCID或OS患者的成纤维细胞系的存储库，并带有不同的RAG1突变。现在，我们打算从RAG1突变患者中产生IPSC，并表征其干性和多能性特征，染色体完整性和患者特异性推导。 与Zuniga-Pflucker博士合作，我们将研究这些患者衍生的IPSC在体外沿T细胞分化的能力。我们还将探讨他在患者衍生的IPSC中校正突变体RAG1基因座的能力，并在体外支持V（d）J重组和T细胞分化的能力。我们将比较经过基因校正的校正和未校正的患者衍生的IPSC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，因此可以帮助解释人类抹布缺陷的表型多样性的基础。此外，基于基因特异性核酸内切酶介导的同源重组，该项目还将探索一种新型SCID基因治疗方法的功效和安全性。 公共卫生相关性：人类RAG1和RAG2基因的缺陷会导致一系列严重的免疫缺陷，范围从完全不存在淋巴细胞（SCID）到以免疫缺陷和自身免疫性（OMENN综合征）为特征的疾病。这种多样性的基础仍然很差。为了解决这个问题，我们将诱导患有RAG1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，我们将使用一种新型蛋白质来修复患者衍生的干细胞中的RAG1基因缺陷。这可能为通过基因修复纠正遗传疾病的新颖和更安全的方法铺平了道路。