A novel cell-based platform to study human circadian disorders

项目总结/摘要 遗传干扰，如生物钟基因中的致病性单核苷酸多态性（SNP），可以干扰 昼夜节律并导致睡眠障碍。例如，CK 1 ε基因中的tau突变导致了显著的 缩短动物的觉醒-睡眠周期，约20小时而不是正常的24小时。同样的突变 报告为人类的SNP。由于生物钟机制在哺乳动物中是保守的， 物种，受影响的人类将被预测具有相同的睡眠周期改变。结构和生化 分析预测时钟基因存在许多潜在的致病突变。然而，我们还没有 了解这些潜在致病突变的体内意义。学习中的一个关键瓶颈 遗传破坏的发病机理是缺乏有效的体内系统， 资源密集型活体动物模型 目标1。开发一个有效的平台来研究哺乳动物的生物钟机制， 突变。为了验证可以在人类细胞中研究时钟基因中SNP功能的假设， 基于平台，我们在U2 OS细胞中产生了内源性Per 1-luc和Per 2-luc报告基因，其中不同的SNP 将被准确地生成和评估。我们通过确认一致性敲除验证了该系统 我们的报告细胞和小鼠之间的表型，并复制具有已知突变的相似表型。 我们的系统将通过比较两种细胞模型U2 OS之间的SNP表型来进一步验证 和mPer 2Luc MEF。 目标2.阐明CK 1 δ、CK 1 ε、Clock和Bmal 1关键突变的潜在病理生理学 基因.我们将使用我们的平台来测试关于先前提出的具体可量化变化的假设。 编码CK 1 δ/ε、CLOCK和BMAL 1的时钟基因突变。这些突变中的大多数还没有 在体内模型中得到验证和量化。我们将重点关注这些突变和几十个SNP， 在这些位点附近，可能会像以前的研究所确定的突变一样具有破坏性。 目标3。开发一种新的，特异性的治疗方法，用于与以下疾病相关的昼夜睡眠障碍： 病理性SNP。目前治疗时差或重置睡眠周期的方法包括光疗，褪黑激素， 还有一些实验性药物，没有一种是专门针对生物钟的，也没有一种被证明对昼夜节律有效。 紊乱我们假设使用PROTAC快速降解限制性时钟蛋白可以抵消 SNP突变的致病性，使得时钟被调节以补偿致病性。 总之，正如我们现在所知道的，人类生理学受到有缺陷的时钟机制的极大影响， 与时钟基因中的病理性SNP相关，CRISPR允许有效的基因组编辑， 及时开发一个有效的基于细胞的平台来研究这些临床SNP的致病性。

Project Summary/Abstract Genetic disruptions such as pathogenic single-nucleotide polymorphisms (SNPs) in clock genes can perturb circadian rhythms and cause sleep disorders. For example, the tau mutation in the CK1ε gene causes dramatic shortening of the wake-sleep cycle in animals, ~20 hrs instead of normal 24 hrs. The same mutation has been reported as a SNP in humans. Because the circadian clock mechanism is conserved across mammalian species, affected humans would be predicted to have the same altered sleep cycle. Structural and biochemical assays have predicted many potentially pathogenic mutations exist for clock genes. However, we do not yet understand the in vivo significance of these potentially pathogenic mutations. A critical bottleneck in studying the pathogenesis of genetic disruptions is lack of an efficient in vivo system that uses cell models instead of resource-intensive, live animal models. Aim 1. Develop an efficient platform to study mammalian clock mechanisms and identify pathological mutations. To test the hypothesis that functionality of SNPs in clock genes can be studied in a human cell- based platform, we generated endogenous Per1-luc and Per2-luc reporters in U2OS cells where diverse SNPs will be generated and assessed accurately. We have validated the system by confirming consistent knockout phenotypes between our reporter cells and mice, and reproducing similar phenotypes with known mutations. Our system will be further validated by comparing phenotypes of the SNPs between two cell models, U2OS and mPer2Luc MEFs. Aim 2. Elucidate the underlying pathophysiology of critical mutations in CK1δ, CK1ε, Clock and Bmal1 genes. We will use our platform to test hypotheses on quantifiable changes previously proposed for specific mutations in clock genes encoding CK1δ/ε, CLOCK and BMAL1. The majority of these mutations have not yet been validated and quantified in in vivo models. We will focus on these mutations and dozens of SNPs at or near these sites that could be as disruptive as the mutations identified by previous studies. Aim 3. Develop a novel, specific treatment approach for circadian sleep disorders associated with pathological SNPs . Current approaches to treat jet-lag or reset sleep cycles include light therapy, melatonin and a few experimental drugs, none of which are specific to the clock and proven to be effective for circadian disorders. We hypothesize that rapid degradation of limiting clock proteins using PROTACs can counteract the pathogenicity of SNP mutations such that the clock is modulated to compensate the pathogenicity. In summary, as we are now aware that human physiology is greatly impacted by defective clock mechanisms associated with pathological SNPs in clock genes and CRISPR allows efficient genome editing, it is imperative and timely to develop an efficient cell-based platform to study pathogenicity of these clinical SNPs.