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

基本信息

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
项目类别：
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

项目摘要

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）：严重联合免疫缺陷（SCID）包括一组异质遗传性疾病，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。同一基因的亚形缺陷可能会导致渗漏 SCID 或 Omenn 综合征 (OS)，这是一种与浸润性寡克隆 T 细胞导致的多器官损伤相关的严重免疫缺陷。 RAG 缺陷的 HCT 后长期生存率仅为 50%，漏漏 SCID 和 OS 则更差。最近，当缺乏 HLA 匹配供体时，病毒介导的基因转移至造血 CD34+ 祖细胞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，寻求新颖且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 (>12 bp) DNA 靶序列，可用于尝试基因校正。我们的合作者 Frederic Paques 设计了 RAG1 专用的 HE。旨在探索基因转移在免疫缺陷人类中的功效的临床前研究的主要局限性之一是来自患者的靶细胞的可用性有限。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（iPSC）。这些 iPSC 可在体外进行靶向多谱系分化（包括 T 淋巴细胞）。我们已经建立了来自 SCID 或 OS 患者的成纤维细胞系库，这些细胞系携带不同的 RAG1 突变。我们现在打算从 RAG1 突变患者身上产生 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衍生。我们将与 Zuniga-Pflucker 博士合作，研究这些源自患者的 iPSC 在体外进行 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 iPSC 中突变 RAG1 位点的能力，并支持体外 V(D)J 重组和 T 细胞分化。我们将比较基因校正的校正和未校正的患者来源 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，因此可能有助于解释人类 RAG 缺陷表型多样性的基础。此外，该项目还将探索基于基因特异性核酸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。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，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不明确。为了解决这个问题，我们将诱导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且更安全的方法铺平道路。