Systems-level transcriptomic analyses to Identify mouse models of asthma

 描述(由申请人提供)：过敏性呼吸道疾病(AADS)包括哮喘和过敏性鼻炎的高流行率使确定新的疾病预防和治疗策略成为优先事项。最近的研究表明，气道上皮细胞中的基因表达与AAD有关，并可能是不同AAD亚型的基础。除了少数例外，AAD中上皮基因表达的关键转录调节因子尚不清楚。它们的识别代表着开发新的治疗策略的重要一步。AAD(尤其是哮喘的亚型)小鼠模型的缺乏也阻碍了新的治疗靶点的确定。我们假设，使用保守的基因表达模式作为决策标准，我们可以识别出比目前存在的更接近人类AAD亚型的AAD小鼠模型。此外，我们假设这些新模型可以用于识别和测试新的疾病靶点。在目标1中，我们将建立AAD转录调控的预测模型，在该模型中，基因表达的主要调控因子，即转录因子(TF)和microRNAs(MiRNAs)与其靶基因偶联。我们将首先对人类AAD的6项转录研究(N=218名受试者：118例哮喘、26例过敏性鼻炎和74名对照)进行荟萃分析，以确定AAD中持续变化的基因，以及哪些基因区分AAD亚型。然后，我们将使用调控基序(转录因子结合位点和miRNA靶点)的基因集浓缩分析来确定差异表达基因(包括共表达的亚集)的推测的主要调控因子。在目标2中，我们将鉴定新的菌株 与现有的小鼠遗传资源相比，合作杂交(CC)种群的小鼠具有更强的遗传多样性，更好地模仿人类AAD亚型。我们将对40株CC菌株进行表型，以标记AAD表型对急性和慢性过敏原暴露的反应，并通过RNA测序来测量肺基因的表达。使用RNA-SEQ数据的创新统计方法，我们将识别哪些CC菌株表现出与人类AAD亚型最相似的基因表达模式。同时，我们将对差异表达(和共表达)的基因进行基因集浓缩分析，以确定人和小鼠共享的关键转录因子和miRNAs。在目标3中，我们进行赠款的验证部分。我们将测试在小鼠和人之间保守的新的候选主控调节因子(在目标1和2中确定)是否代表新的AAD治疗靶点。我们将在小鼠体内使用基因敲除和基因敲除方法扰乱两个候选的主调控因子。这些扰动的影响将在急性和慢性过敏原模型中进行评估，以测试改变主调节器是否可以影响AAD的开始和/或改变已建立的AAD。因此，总的来说，我们将(1)确定AAD表型的保守主调控子，(2)建立更好地模拟人类疾病的新的AAD小鼠模型，以及(3)评估基因表达的主调控子是否代表新的药物靶点。

 DESCRIPTION (provided by applicant): The high prevalence of allergic airway diseases (AADs) including asthma and allergic rhinitis make identifying new disease prevention and treatment strategies a priority. Recent studies have shown that gene expression in airway epithelia is associated with AAD and may underlie different AAD sub-types. With few exceptions, the key transcriptional regulators of epithelial gene expression in AAD are unknown. Their identification represents an important step in the development of novel therapeutic strategies. The identification of novel therapeutic targets has also been hindered by the lack of validated mouse models for AAD (in particular, sub-types of asthma). We hypothesize that we can identify mouse models of AAD that more closely mimic human AAD sub- types than currently exists using conserved patterns of gene expression as decision criteria. Further, we hypothesize that these new models can be used to identify and test novel disease targets. In Aim 1, we will build predictive models of transcriptional regulation in AAD in which master regulators of gene expression, namely transcription factors (TFs) and microRNAs (miRNAs), are coupled to their target genes. We will first perform a meta-analysis of six transcriptomic studies of human AAD (N = 218 subjects: 118 asthma cases, 26 allergic rhinitis cases, and 74 controls) to identify genes consistently altered in AAD and which genes distinguish AAD sub-types. We will then identify putative master regulators of the differentially expressed genes (including subsets that are co-expressed) using gene set enrichment analysis for regulatory motifs (transcription factor binding sites and miRNA target sites). In Aim 2, we will identify new strains of mice from the Collaborative Cross (CC) population, which features enhanced genetic diversity compared to existing mouse genetics resources, that better mimic human AAD sub-types. We will phenotype 40 CC strains for hallmark AAD phenotypes in response to acute and chronic allergen exposure, and measure lung gene expression by RNA-sequencing. Using an innovative statistical approach of the RNA-seq data, we will identify which CC strains exhibit patterns of gene expression most akin to human AAD subtypes. At the same time, we will conduct gene set enrichment analysis of differentially expressed (and co-expressed) genes to identify key TFs and miRNAs that are shared between human and mouse. In Aim 3, we conduct the validation component of the grant. We will test whether novel candidate master regulators that are conserved between mouse and man (identified in Aims 1 and 2) represent new AAD therapeutic targets. We will perturb two candidate master regulators in vivo using gene knockdown and gene knockout approaches in mice. The effect of these perturbations will be evaluated in acute and chronic allergen models to test whether altering the master regulator can affect AAD onset and/or modify established AAD. Thus in total we will (1) identify conserved master regulators of AAD phenotypes, (2) establish new mouse models of AAD that better mimic human disease, and (3) evaluate whether master regulators of gene expression represent novel drug targets.