Immune repertoire and function in typical and atypical SCID

典型和非典型 SCID 的免疫组库和功能

• 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
• 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

 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）：该竞争性更新申请旨在为旨在阐明重组酶激活基因1（RAG 1）或RAG 2突变如何以及为何导致一系列免疫表型的研究寻求资金，包括严重联合免疫缺陷（SCID），Omenn综合征（OS），“渗漏”SCID（LS）和迟发型联合免疫缺陷伴肉芽肿和/或自身免疫（CID-G/A）。我们建议测试的总体假设，即与不同的临床表型相关联的亚型RAG突变的差异形状组成的T和B细胞抗原受体库，并扰乱B NK细胞的表型和功能。我们将继续研究目前资助的项目，并使用基因编辑来模拟错误的T细胞分化，并开发一种使用人类诱导多能干细胞（iPSC）纠正人类RAG 1缺陷的体外临床前模型。在目标1中，我们将测试自然发生的人类RAG 1和RAG 2突变的致病作用，并分析这种疾病中免疫失调的机制。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中TCR和BCR库的多样性和组成。我们将描述产生的自身抗体的谱和亲合力，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，我们将对NK淋巴细胞进行广泛的表型和功能表征。在目标2中，为了获得对RAG缺陷的免疫失调的机制性见解，我们提出表征我们已经产生的新小鼠模型，并且该模型对于Rag 1 F971 L突变是纯合的。人类中的等效突变与CID-G/A相关。我们将分析该模型中T、B和NK细胞的发育和功能以及免疫失调的机制，并将结果与分别作为LS和OS模型的Rag 1 S723 C/S723 C和Rag 2 R229 Q/R229 Q小鼠中观察到的结果进行比较。在目标3中，我们建议用iPSC建模和纠正RAG 1缺陷。我们将使用CRISPR/Cas9来产生具有不同RAG 1突变的同基因iPSC，我们将研究这些突变支持体外T细胞分化和产生多样化T细胞受体库的能力。我们还将研究CRISPR/Cas9基因编辑方法在体外纠正患者来源的iPSC中的RAG 1突变并恢复T细胞分化的能力。总的来说，这些研究将为人类RAG缺乏症的病理生理学提供新的见解，并可能为未来开发基于基因编辑的创新治疗形式提供基础。