GENETIC, IMMUNOLOGIC AND MECHANISTIC BASIS OF HUMAN NK CELL DEFICIENCY

人类 NK 细胞缺陷的遗传、免疫学和机制基础

• 批准号: 9003675
• 负责人: Jordan Scott Orange
• 金额: $ 72.63万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-01-19 至 2020-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9003675
• 关键词: Affect; Basic Science; Biological; Biological Assay; Biological Models; Biology; Bypass; CD34 gene; CRISPR/Cas technology; Candidate Disease Gene; Cell Count; Cell Differentiation process; Cell Line; Cell physiology; Cells; Cellular biology; Child; Clinical; Collaborations; Data; Defect; Development; Diagnostic; Disease; Etiology; Evaluation; Flow Cytometry; Genes; Genetic; Genetic study; Gold; Guidelines; Health; Human; Image; Immune; Immune system; Immunologics; Immunologist; In Vitro; Individual; Inflammatory; Journals; Laboratories; Lytic; MCM10 gene; Malignant Neoplasms; Medicine; Mutation; Natural Killer Cells; Patients; Phenotype; Predisposition; Process; Publishing; Recruitment Activity; Resolution; Role; Sampling; Stem cells; Synapses; Techniques; Therapeutic; Time; Validation; Viral Cancer; Virus Diseases; Western Blotting; Work; base; cohort; college; congenital immunodeficiency; cytokine; design; exome sequencing; field study; humanized mouse; immunological synapse; in vivo Model; induced pluripotent stem cell; innovation; insight; killings; knock-down; knockout gene; meetings; novel; programs; public health relevance; receptor; reconstitution; research study; segregation; small hairpin RNA; tool; transcription factor; whole genome

 DESCRIPTION (provided by applicant): Natural Killer (NK) cell deficiency (NKD) is a recently appreciated subset of primary immunodeficiency (PID) in which NK cell deficits represent the main immunological defect. These diseases are typically severe; patients suffer from profound and often lethal susceptibility to viral infection and cancer. To date, only three NKD genes have been identified and little is known of the biological mechanisms by which they interfere with NK cell numbers and/or function. At the same time, no good therapeutic options are available for those affected. For over a decade, our laboratory has sought genetic, immunologic, and therapeutic solutions to these rare, but deadly diseases. In the process, we have established a robust worldwide referral base, assembled an expert team of geneticists, immunologists and clinicians, and are now poised to dramatically advance our understanding of NKD and the role of NK cells in health and disease. Through two Aims, we will define genes responsible for human NKD (Aim 1), and determine their relevance through intensive biological characterization (Aim 2). In Aim 1, we use whole exome sequencing and tandem chromosomal microarray to identify NKD mutations in the world's largest cohort of NKD patients. We have already identified 7 novel gene candidates using this approach. In Aim 2, we will rigorously evaluate our candidate NKD genes, defining their roles in NK cell cytolytic functions, developmental maturation, receptor repertoire, and immunoregulatory and inflammatory function using a carefully thought out progression of cutting-edge immunologic experiments. Tools to be used include "gold-standard" 51Cr-release killing assays, Western blots, shRNA knockdown, gene knockout using CRISPR/Cas technology, extended multiparameter flow cytometry-based phenotypic and functional analysis, high- and super-resolution quantification of the lytic immunological synapse, imaging flow cytometry quantification of transcription factor activation and lytic synapse dynamics, and in vitro NK cell differentiation from CD34+ hematopoetic stem cells. Specifically, we will prove that these genes cause NKD in ex vivo NKD patient cells, ex vivo healthy donor NK cells and NK cell lines ("knock-outs" and "knock-ins"), and patient-derived induced pluripotent stem cells (iPSC) and reconstituted humanized mice. Additionally, the potential therapeutic benefit of clinically available cytokines will be evaluated for each candidate. Overall our approach represents iterative cycles of genetic discovery, confirmation, biological validation, and immunologic insight. We are confident that these innovative approaches, combined with our established NK cell and genetic expertise, and privileged NKD patient pipeline, will drive meaningful diagnostic, therapeutic, and basic science progress in this burgeoning field. We provide preliminary data from two examples of our discoveries, ATP6V0A2 and MCM10, as concrete examples of how the proposed work will substantively advance the field.

 描述（由申请人提供）：自然杀伤（NK）细胞缺陷（NKD）是最近认识到的原发性免疫缺陷（PID）的子集，其中NK细胞缺陷代表主要的免疫缺陷。这些疾病通常很严重；患者对病毒感染和癌症具有严重且往往致命的易感性。迄今为止，仅鉴定了三个 NKD 基因，并且对它们干扰 NK 细胞数量和/或功能的生物学机制知之甚少。与此同时，对于受影响的人来说，没有好的治疗选择。十多年来，我们的实验室一直在寻找针对这些罕见但致命疾病的遗传、免疫和治疗解决方案。在此过程中，我们建立了强大的全球转诊基础，组建了由遗传学家、免疫学家和临床医生组成的专家团队，现在准备大幅推进我们对 NKD 以及 NK 细胞在健康和疾病中的作用的理解。通过两个目标，我们将定义与人类 NKD 相关的基因（目标 1），并通过深入的生物学表征确定它们的相关性（目标 2）。在目标 1 中，我们使用全外显子组测序和串联染色体微阵列来识别世界上最大的 NKD 患者群体中的 NKD 突变。我们已经使用这种方法鉴定了 7 个新的候选基因。在目标 2 中，我们将严格评估候选 NKD 基因，使用经过深思熟虑的尖端免疫学实验进展，定义它们在 NK 细胞溶细胞功能、发育成熟、受体库以及免疫调节和炎症功能中的作用。使用的工具包括“金标准”51Cr释放杀伤测定、Western印迹、shRNA敲低、使用CRISPR/Cas技术的基因敲除、基于扩展多参数流式细胞术的表型和功能分析、裂解性免疫突触的高分辨率和超分辨率定量、转录因子激活和裂解性突触动力学的成像流式细胞术定量以及体外NK细胞分化 CD34+造血干细胞。具体来说，我们将证明这些基因在离体 NKD 患者细胞、离体健康供体 NK 细胞和 NK 细胞系（“敲除”和“敲入”）、患者来源的诱导多能干细胞 (iPSC) 和重组人源化小鼠中引起 NKD。此外，还将针对每个候选者评估临床上可用的细胞因子的潜在治疗益处。总的来说，我们的方法代表了遗传发现、确认、生物验证的迭代循环， 和免疫学见解。我们相信，这些创新方法，结合我们成熟的 NK 细胞和遗传专业知识，以及独特的 NKD 患者管道，将推动这一新兴领域有意义的诊断、治疗和基础科学进展。我们提供了我们发现的两个例子（ATP6V0A2 和 MCM10）的初步数据，作为拟议工作将如何实质性推进该领域的具体例子。