Role of Lyn in Systemic lupus erythematosus-like autoimmune disease in mice

描述(由申请人提供)： Lyn是B细胞中主要的Src家族激酶，在BCR信号的反馈调节中具有独特的作用。LYN基因敲除小鼠具有高度活跃的B细胞，其表型与人类相似，即系统性红斑狼疮(SLE)并伴有严重的肾脏疾病。本研究的目的是确定Lyn介导的抑制信号在B细胞中的物理位置，并利用基因疗法改善Lyn缺陷小鼠的自身免疫性肾脏疾病。通过设计适当的嵌合结构，LYN将被靶向于质膜的脂筏或非脂筏间隔。这些嵌合蛋白将在体外通过在Lyn缺乏的B细胞中异位表达来测试它们重建信号的能力。通过建立表达RAFT限制性或RAFT排除的LYN而不是野生型LYN的敲入小鼠，将在体内测试LYN的脂筏定位对自身免疫性疾病发展的贡献。最后，试图通过用野生型Lyn重组的自体造血祖细胞重新填充LYN缺陷小鼠来改善狼疮性肾炎的表型。 我在研究免疫细胞信号方面有丰富的经验，重点是B细胞激活过程中信号中间产物的亚细胞定位。这项拟议的研究将把我的体外信号和细胞生物学专业知识扩展到人类自身免疫性疾病的活体小鼠模型和逆转录病毒介导的干细胞移植作为基因治疗手段的研究。我将与加州大学旧金山分校内外的调查人员合作，执行研究计划。拟议的研究将产生关键的分子和细胞工具，以建立一个强大的研究计划，旨在研究SLE样肾脏疾病的免疫细胞缺陷，并使用基因治疗作为干预手段。最终，这个项目将使我能够发展成为一名独立的调查员，并追求终身教职。

DESCRIPTION (provided by applicant): Lyn is the predominant Src family kinase in B cells and has a unique role in the feedback regulation of BCR signaling. Lyn knockout mice have hyperactive B cells and a phenotype resembling the human condition, systemic lupus erythematosus (SLE) with severe kidney disease. The goals of the proposed research are to identify the physical site of inhibitory signaling mediated by Lyn in B cells, and to use gene therapy to ameliorate the autoimmune kidney disease in Lyn-deficient mice. Lyn will be targeted to the lipid raft or non-raft compartment of the plasma membrane by engineering appropriate chimeric constructs. The chimeric proteins will be tested for their ability to reconstitute signaling by ectopic expression in Lyn-deficient B cells in vitro. The contribution of lipid raft localization of Lyn towards development of autoimmune disease will be tested in vivo by creating knock-in mice expressing raft-restricted or raft-excluded Lyn instead of wild type Lyn. Finally, an attempt will be made to ameliorate the lupus-nephritis phenotype of Lyn-deficient mice by repopulating them with autologous hematopoeitic progenitors reconstituted with wild type Lyn. I have extensive experience in studying immune cell signaling with emphasis on subcellular localization of signaling intermediates during B cell activation. The proposed research will expand my in vitro signaling and cell biology expertise to the study of in vivo mouse models of human autoimmune disease and retrovirus-mediated stem cell transplantation as a means of gene therapy. I will collaborate with investigators within and outside UCSF to execute the research plan. The proposed studies will generate critical molecular and cellular tools to establish a strong research program that aims to study the immune cell defect in SLE-like kidney disease and use gene therapy as a means of intervention. Ultimately, this project will enable me to develop into an independent investigator and pursue a tenure-track faculty position