Molecular Analysis Of Leukocyte Activation By Chemoattractants

这个项目的目的是确定血液白细胞迁移到炎症或感染的特定组织部位的分子机制和生物学背景。我们一直专注于介导这一过程的趋化蛋白，并鉴定了部署在白细胞表面的趋化受体大家族的成员。我们还鉴定了一组不同的趋化物质和趋化物质受体模拟物，包括疱疹病毒、痘病毒和艾滋病毒。我们使用基因组学、分子生物学、细胞生物学和流行病学作为分析这些分子的主要方法。一个主要的目标是确定单个趋化物质和趋化物质受体的特定疾病关联，以确定潜在的新的治疗靶点。一个关键的策略是分析疾病模型中基因敲除小鼠的表型，以及人类化学吸引系统基因突变患者的表型。 1)在23财年，我们报告了一例6岁男童严重的中性粒细胞减少症。患者有典型的CXCR4突变，与突发事件综合征一致。这个病例说明了这种疾病的表现形式的异质性。 2.)在2013财年，我们报告了在Whim患者和Whim模型小鼠中，Whim突变导致的CD8比CD4淋巴细胞减少更严重。小鼠的机制研究显示，由于胸腺内滞留时间的延长，成熟的CD8单阳性细胞在胸腺内以细胞固有的方式选择性地和突发的等位基因依赖的积聚，与CD8单阳性胸腺细胞对CXCR4配体CXCL12的体外趋化反应增加有关。此外，成熟的Whim CD8+T细胞优先归宿于小鼠的骨髓，并以细胞固有的方式保留在骨髓中。在小鼠体内注射特异性CXCR4拮抗剂AMD3100(Plerixafor)，可迅速和瞬时纠正T细胞淋巴细胞减少症和CD4/CD8比率。在淋巴细胞脉络膜脑膜炎病毒感染后，我们发现野生型和突发模型小鼠在记忆、CD8+T细胞分化和病毒载量方面没有差异。因此，突发综合征的淋巴细胞减少可能涉及严重的依赖CXCR4的CD8+T细胞缺乏，部分原因是初级淋巴器官、胸腺和骨髓中的隔离。 3.)在2013财年，我们在《血液》中报道了一种涉及自体HSPC移植的两步临床前基因治疗方案。首先，用不区分突发和野生型CXCR4等位基因的单一引导RNA(SgRNA)在体外通过CRISPR/Cas9编辑使HSCs中CXCR4的一个拷贝失活。然后，通过体内自然选择，奇想等位基因失活的细胞比野生型等位基因失活的细胞更丰富。突变等位基因失活的造血干细胞保留了长期多能性和选择性造血重建的优势。这篇论文还附带了一份《血色透视》。据我们所知，这是使用疾病等位基因失活策略取代效率较低的疾病等位基因修复方法对常染色体显性显性功能获得疾病进行基因治疗的第一个例子。本文将我们先前发表的CXCR4+/o造血干细胞在同基因小鼠移植中比野生型造血干细胞具有更好的植入潜力的发现扩展到一个基因治疗平台，以及我们通过在单个造血干细胞中缺失疾病等位基因来表征对突发综合征的发色疗法的特征。这些结果提供了原理证据，即抑制患者HSCs的突发等位基因沉默是一种可行且比纠正突发等位基因更可行的基因治疗策略。

The aim of this project is to define the molecular mechanisms and biological contexts for blood leukocyte migration to specific tissue sites that are inflamed or infected. We have focused on chemoattractant proteins that mediate this process and have identified members of a large family of chemoattractant receptors that are deployed on the leukocyte cell surface. We have also identified members of a diverse group of chemoattractant and chemoattractant receptor mimics made by viruses, including herpesviruses, poxviruses and HIV. We use genomics, molecular biology, cell biology and epidemiology as the principle methods for analyzing these molecules. A major goal is to identify specific disease associations of individual chemoattractants and chemoattractant receptors, in order to identify potential new therapeutic targets. A key strategy is to analyze phenotypes of gene knockout mice in disease models as well as phenotypes in patients with mutations in human chemoattractant system genes. 1.) In FY23, we reported a case of severe neutropenia in a 6-year-old boy. The patient has a classic CXCR4 mutation consistent with WHIM syndrome. The case illustrates the heterogeneity of presentation in this disorder. 2.) In FY23, we reported that WHIM mutations cause more severe CD8 than CD4 lymphopenia in WHIM patients and WHIM model mice. Mechanistic studies in mice revealed selective and WHIM allele dose-dependent accumulation of mature CD8 single-positive cells in thymus in a cell-intrinsic manner due to prolonged intrathymic residence, associated with increased CD8 single-positive thymocyte chemotactic responses in vitro toward the CXCR4 ligand CXCL12. In addition, mature WHIM CD8+ T cells preferentially home to and are retained in the bone marrow in mice in a cell-intrinsic manner. Administration of the specific CXCR4 antagonist AMD3100 (plerixafor) in mice rapidly and transiently corrected T cell lymphopenia and the CD4/CD8 ratio. After lymphocytic choriomeningitis virus infection, we found no difference in memory CD8+ T cell differentiation or viral load between wild-type and WHIM model mice. Thus, lymphopenia in WHIM syndrome may involve severe CXCR4-dependent CD8+ T cell deficiency resulting in part from sequestration in the primary lymphoid organs, thymus, and bone marrow. 3.) In FY23, we reported in Blood a two-step preclinical gene therapy protocol involving autologous HSPC transplantation. First, one copy of Cxcr4 in HSCs was inactivated ex vivo by CRISPR/Cas9 editing with a single guide RNA (sgRNA) that does not discriminate between WHIM and wildtype Cxcr4 alleles. Then, through in vivo natural selection, WHIM allele-inactivated cells were enriched over wildtype allele-inactivated cells. The WHIM allele-inactivated HSCs retained long-term pluripotency and selective hematopoietic reconstitution advantages. The paper was accompanied by a Perspective in Blood. To our knowledge this is the first example of gene therapy for an autosomal dominant gain-of-function disease using a disease allele inactivation strategy in place of the less efficient disease allele repair approach. This paper extends to a gene therapy platform our previously published discovery that Cxcr4+/o HSCs have superior engraftment potential in congenic mouse transplantation over wild type HSCs as well as our characterization of chromothriptic cure of WHIM syndrome by deletion of the disease allele in a single HSC. The results provide proof of principle that WHIM allele silencing of patient HSCs is a viable and more feasible gene therapy strategy than WHIM allele correction.