A novel cell-based platform to study human circadian disorders

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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.