Role of Lyn in SLE-like autoimmune disease in mice

描述（由申请人提供）： 林恩是B细胞中主要的Src家族激酶，在BCR信号传导的反馈调节中具有独特的作用。林恩基因敲除小鼠具有过度活跃的B细胞和类似于人类状况的表型，即伴有严重肾脏疾病的系统性红斑狼疮（SLE）。该研究的目的是确定林恩在B细胞中介导的抑制性信号传导的物理位点，并使用基因治疗来改善Lyn缺陷小鼠的自身免疫性肾病。通过工程化适当的嵌合构建体，林恩将靶向细胞膜的脂筏或非筏区室。将测试嵌合蛋白在体外通过在Lyn-deficient B细胞中异位表达重建信号传导的能力。通过产生表达筏限制性或筏排除性林恩而不是野生型林恩的敲入小鼠，在体内测试林恩的脂筏定位对自身免疫性疾病发展的贡献。最后，将尝试通过用野生型林恩重建的自体造血祖细胞对其进行再增殖来改善Lyn缺陷小鼠的狼疮肾炎表型。 我在研究免疫细胞信号方面有丰富的经验，重点是B细胞活化过程中信号中间体的亚细胞定位。拟议的研究将扩大我在体外信号和细胞生物学专业知识的研究，在体内小鼠模型的人类自身免疫性疾病和逆转录病毒介导的干细胞移植作为基因治疗的一种手段。我将与UCSF内外的研究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样肾病中的免疫细胞缺陷，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个独立的调查员，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