Cellular and Genetic Basis of Systemic Lupus

 描述(申请人提供)：系统性红斑狼疮(SLE)，一种全身性自身免疫性疾病，影响多个器官系统，导致显著的发病率和死亡率。狼疮性肾炎是系统性红斑狼疮的一种破坏性并发症，死亡率增加，肾脏损害进行性增加，导致终末期肾病(ESRD)。到目前为止，仍然没有明确的途径来确定狼疮性肾炎患者的群体，这些患者最终将死于终末期肾病。识别预测系统性红斑狼疮ESRD风险的基因将允许在这一亚组中进行更有力的治疗，尽管存在潜在的显著副作用。在我们对狼疮易感小鼠模型NZM2328的遗传学研究中，我们发现了控制ANA和抗dsDNA抗体产生的单独基因(第4染色体上的Adaz1)，以及控制急性和慢性肾小球肾炎(GN)的基因(第1染色体上的Cgnz1控制慢性GN，第1染色体上的Agnz1控制慢性GN，染色体上的H-2复合体和第17染色体上的Agnz2控制急性GN)。NZM2328.Lc1(Lc1)和Lc1染色体内重组同源系以及NZM2328.Lc4的表型证实了我们的遗传分析。提出了一种新的SLE发病机制模型，将狼疮的致病基因分为影响自身免疫反应的基因和影响终末器官损害的基因两类。这两类基因相互作用，导致临床疾病。控制系统性红斑狼疮终末器官抵抗的基因是一个新的概念。其他数据显示，急性和慢性肾炎受到不同的基因控制。小鼠1号染色体上的1.34MB区域(Cgnz1位点)已被证明含有抵抗急性肾炎(AgN)向慢性肾炎(CGN)进展的基因(S)。在这次竞争性更新申请中，我们希望继续我们的研究计划，以确定Cgnz1，该基因(S)位于1.34Mb区域，使末端器官(肾脏)对损伤易感。具体目标1：以NZM2328和NZM2328.Lc1R27(R27)为材料，建立足细胞和管状细胞系，利用CRISPR/Cas方法进行体外基因缺失，筛选出S抵抗细胞凋亡或在自噬中起重要作用的候选基因(S)。足细胞和管状细胞都将被使用。这些候选基因将针对特定目标2；特定目标2：通过CRISPR/Cas方法在(NZM2328XR27)F1胚胎中建立靶基因缺失的突变系，并产生纯合突变以验证对终末期肾损伤的易感性，并产生足细胞特异性缺失突变以证明对足细胞起作用的基因(S)；以及特定目标3：鉴定决定狼疮向终末期肾损害进行性GN进展的候选基因(S)。拟议研究的结果将在未来涉及更大样本的研究中得到验证和改进。我们期望它们能为终末期肾病风险管理提供有预后价值和实用价值的生物标志物。因此，该应用程序将产生关于狼疮性肾炎发病机制的重要信息，并将具有重大的翻译潜力。

 DESCRIPTION (provided by applicant): Systemic Lupus Erythematosus (SLE), a systemic autoimmune disorder, affects multiple organ systems resulting in significant morbidity and mortality. Lupus nephritis is a devastating complication of SLE, with an increased mortality and risk of progressive renal damage leading to end-stage renal disease (ESRD). To date, there remains no definitive pathway to identify the population of patients with lupus nephritis who are ultimately destined to ESRD. Identification of genes predicting ESRD risk in SLE would permit more vigorous treatment in this subgroup despite the potentially significant side effects. In our study of the genetics of lupus- prone mouse model NZM2328, we have identified separate genes controlling ANA and anti-dsDNA Ab production (Adaz1 on chromosome 4), and controlling both acute and chronic glomerulonephritis (GN) (Cgnz1 on chromosome 1 for controlling chronic GN and Agnz1 on chromosome 1, H-2 complex and Agnz2 on chromosome 17 for controlling acute GN). The phenotypes of NZM2328.Lc1 (Lc1) and Lc1 intrachromosomal recombinant congenic lines and NZM2328.Lc4 confirm our genetic analysis. A novel model for pathogenesis of SLE was proposed in which the genes contributing to lupus are classified into two classes - those affecting autoimmune responsiveness and those affecting end organ damage. These two classes of genes interact, resulting in clinical disease. The concept of genes controlling end organ resistance in SLE is novel. Additional data show that acute and chronic GN are under separate genetic control. A 1.34MB region on mouse chromosome 1 (the Cgnz1 locus) has been shown to contain gene(s) that confer resistance to the progression of acute GN (aGN) to chronic GN (cGN). In this competitive renewal application we wish to continue our research program to identify Cgnz1, the gene(s) within the 1.34Mb regions that confers end organ (kidney) susceptibility to damage. Three specific aims are proposed: Specific Aim 1: To generate podocytes and tubular cell lines from NZM2328 and NZM2328.Lc1R27 (R27) to carryout in vitro gene deletion by the CRISPR/Cas method to identify candidate gene(s) that confer(s) resistance to apoptosis or play an important role in autophagy. Both podocytes and tubular cells will be used. These candidate genes will be targeted in Specific Aim 2; Specific Aim 2: To generate mutant lines with deficiency in targeted genes in (NZM2328XR27)F1 embryos by CRISPR/Cas and to generate homozygous mutants to validate susceptibility to end stage renal damage and to generate podocyte specific deletion mutants to demonstrate the gene(s) that exert(s) effects on podocytes; and Specific Aim 3: To identify candidate gene(s) on the human Cgnz1 1.6Mb locus (Hu Cgnz1) that determine the progression of progressive GN in lupus to ESRD. The results of the proposed studies will be validated and refined in future studies involving larger samples. We anticipate that they may provide biomarkers that are of prognostic value and utility for ESRD risk management. Thus the application will generate significant information regarding the pathogenesis of lupus GN and will have significant translational potentials.