Immune repertoire and function in typical and atypical SCID

典型和非典型 SCID 的免疫组库和功能

基本信息

批准号：
9027475
负责人：
JOHN P MANIS
金额：
$ 46.08万
依托单位：
BOSTON CHILDREN'S HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-07-01 至 2021-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9027475
关键词：
Accounting Affect Apoptosis Autoantibodies Autoimmunity Avidity B cell repertoire B-Cell Development B-Lymphocytes CRISPR/Cas technology Cell Differentiation process Cell Line Cell physiology Clinical Clone Cells Code Data Development Disease Disease model Enzyme-Linked Immunosorbent Assay Frequencies Functional disorder Funding Future Gene Rearrangement Generations Genes Graft Rejection Granuloma Hematopoietic Stem Cell Transplantation Hematopoietic stem cells High-Throughput Nucleotide Sequencing Human Human Engineering IGH@ gene cluster Immune Immunologic Deficiency Syndromes In Vitro Individual Lymphocyte Mediating Modeling Molecular Monoclonal Antibodies Mus Mutate Mutation Natural Killer Cells Outcome Patients Phenotype Pre-Clinical Model Production Rag1 Mouse Receptors, Antigen, B-Cell Recombinants Role SCID Mice Sampling Severities Shapes Specificity Syndrome T cell differentiation T-Cell Development T-Cell Receptor T-Lymphocyte Techniques Testing Work autoreactive B cell base clinical phenotype expression cloning fitness gene correction gene therapy human stem cells induced pluripotent stem cell innovation insight mouse model next generation sequencing novel novel strategies pre-clinical progenitor public health relevance recombinase repaired repository tool

项目摘要

DESCRIPTION (provided by applicant): This competing renewal application seeks funding for studies aimed at elucidating how and why mutations in the recombinase activating gene 1 (RAG1) or RAG2 cause a spectrum of immune phenotypes, including severe combined immune deficiency (SCID), Omenn syndrome (OS), "leaky" SCID (LS), and delayed onset combined immunodeficiency with granuloma and/or autoimmunity (CID-G/A). We propose to test the overall hypothesis that hypomorphic RAG mutations associated with distinct clinical phenotypes differentially shape composition of the T and B cell antigen receptor repertoire, and perturb NK cell phenotype and function. We will continue studies of the currently funded project and use gene editing to model faulty T cell differentiation and to develop an in vitro pre-clinical model o correction of human RAG1 deficiency using human induced pluripotent stem cells (iPSCs). In Aim 1, we will test the disease-causing role of naturally occurring human RAG1 and RAG2 mutations, and analyze mechanisms of immune dysregulation in this disease. We will use next generation sequencing (NGS) to study the diversity and composition of the TCR and BCR repertoire in patients with diverse clinical phenotypes. We will characterize the spectrum and avidity of the autoantibodies produced, and we will use single cell cloning to study the frequency and specificity of circulating autoreactive B cells. To test the hypothesis that RAG mutations affect cellular fitness of NK cell progenitors, we will perform an extensive phenotypic and functional characterization of NK lymphocytes. In Aim 2, in order to gain mechanistic insights into the immune dysregulation of RAG deficiency, we propose to characterize a new mouse model that we have generated, and that is homozygous for the Rag1 F971L mutation. The equivalent mutation in humans is associated with CID-G/A. We will analyze T, B and NK cell development and function, and mechanisms of immune dysregulation in this model, and results will be compared to those observed in Rag1S723C/S723C and Rag2R229Q/R229Q mice, which are models of LS and OS, respectively. In Aim 3, we propose to model and correct RAG1 deficiency with iPSCs. We will use CRISPR/Cas9 to generate isogenic iPSCs that harbor different RAG1 mutations, and we will investigate the ability of these mutations to support in vitro T cell differentiation and generation of a diversified T cell receptor repertoire. We will alo investigate the ability of CRISPR/Cas9 gene editing approach to correct RAG1 mutations in patient-derived iPSCs and restore T cell differentiation in vitro. Overall, these studies will provide novel insights into the pathophysiology of human RAG deficiency and may provide the basis for future development of innovative forms of treatment based on gene editing.

描述（由申请人提供）：此竞争性续展申请寻求资助研究，旨在阐明重组酶激活基因 1 (RAG1) 或 RAG2 的突变如何以及为何导致一系列免疫表型，包括严重联合免疫缺陷 (SCID)、Omenn综合征 (OS)、“渗漏”SCID (LS) 和迟发性联合免疫缺陷伴肉芽肿和/或自身免疫 (CID-G/A)。测试总体假设，即与不同临床表型相关的低等位 RAG 突变会影响 T 细胞和 B 细胞抗原受体库的组成，并扰乱 NK 细胞表型和功能。我们将继续研究当前资助的项目，并使用基因编辑来模拟错误。 T 细胞分化并开发体外临床前模型，使用人类诱导多能干细胞 (iPSC) 纠正人类 RAG1 缺陷。在目标 1 中，我们将测试天然存在的致病作用。我们将使用下一代测序 (NGS) 来研究具有不同临床表型的患者的 TCR 和 BCR 库的多样性和组成。产生的自身抗体的亲和力，我们将使用单细胞克隆来研究循环自身反应性 B 细胞的频率和特异性。为了检验 RAG 突变影响 NK 细胞祖细胞的细胞适应性的假设。对 NK 淋巴细胞进行广泛的表型和功能表征在目标 2 中，为了深入了解 RAG 缺陷的免疫失调，我们建议表征我们生成的新小鼠模型，该模型与 Rag1 F971L 纯合。人类中的等效突变与 CID-G/A 相关。我们将分析该模型中的 T、B 和 NK 细胞的发育和功能以及免疫失调的机制，并将结果与这些结果进行比较。在 Rag1S723C/S723C 和 Rag2R229Q/R229Q 小鼠（分别是 LS 和 OS 模型）中观察到。在目标 3 中，我们建议使用 iPSC 来建模和纠正 RAG1 缺陷，我们将使用 CRISPR/Cas9 生成含有不同 RAG1 的同基因 iPSC。突变，我们将研究这些突变支持体外 T 细胞分化和生成多样化 T 细胞受体库的能力。我们将利用 CRISPR/Cas9 基因编辑方法来纠正患者来源的 iPSC 中的 RAG1 突变并恢复体外 T 细胞分化。总体而言，这些研究将为人类 RAG 缺陷的病理生理学提供新的见解，并可能提供基础。基于基因编辑的创新治疗形式的未来发展。