The circadian rhythm as a lentiviral vector restriction factor

摘要： 昼夜节律（CR）定义了控制主要生理途径的基因表达的每日振荡 在真核细胞体内和体外。这些生物过程包括DNA修复活动，先天和适应性 免疫功能、炎症和代谢途径。CR在自主外围设备上安装 振荡核心，存在于大多数器官以及培养细胞中。在体内，中央主时钟位于 在视交叉上核（SCN），通过交感神经支配周围振荡核团 系统和神经内分泌剂，如褪黑激素（Mel）。外周器官也被各种 代谢/激素和物理输入（例如地塞米松、食物摄入、温度）。但只有 主SCN核心通过视网膜下丘脑束接收光输入。所有振荡核心都基于类似的 正/负转录/翻译反馈环。最近的研究表明，CR对 病毒感染在小鼠模型中的过程。改变病毒应用的时间并敲除基因 编码振荡核心蛋白增强病毒感染高达10倍。迄今为止，CR对疗效的影响 慢病毒载体（LVV）转导的研究。我们假设CR是高水平的 限制因子对病毒载体转导的影响。我们提出了三个具体目标来检验这一假设。在目标1中，我们 将表征正常和破坏的CR行为对LVV肝转导效率的影响。 我们将采用三种小鼠品系，显示出不同的昼夜节律行为和褪黑激素（Mel）的生产，包括 C3 H（Mel+）、C57 B6（Mel-）和BMAL 1（振荡核心蛋白）缺陷型小鼠。矢量的时间效应 施用，以及暴露于Mel或Mel受体拮抗剂对肝转导的影响将是有利的。 测定改变的CR行为模式对载体转导的影响将被确定a）以下 在C57 B6和C3 H小鼠中通过急性阿片样物质戒断方案的CR破坏，和B）通过基因递送至未处理的小鼠， C57 B6和C3 H小鼠在正常白天睡眠时间段被破坏后。在目标2中，我们将测试 假设正常和破坏的CR影响体外人和小鼠细胞中LVV转导效率。 具体地，我们将量化地塞米松夹带对人和人的LVV转导的影响。 包含正常或突变BMAL 1基因的小鼠细胞。在目标3中，我们将使用F2杂交， 确定CC 036和CC 057小鼠品系之间存在遗传关联的效率 LVV介导的肝脏基因传递和CR模式，并将确定有助于上述的遗传位点 主机和LVV特性。

Abstract: Circadian rhythm (CR) defines the daily oscillation in gene expression that controls major physiologic pathways in eukaryotic cells in vivo and in vitro. These biological processes include DNA repair activity, innate and adaptive immune functions, inflammation, and metabolic pathways. CRs are premised on autonomous peripheral oscillatory cores, which exist in most organs as well as in cultured cells. In vivo, a central master clock located in the suprachiasmatic nucleus (SCN) synchronizes the peripheral oscillatory cores via the sympathetic nervous system and neuroendocrine agents such as melatonin (Mel). Peripheral organs are also entrained by various metabolic/hormonal and physical inputs (e.g. Dexamethasone, food intake, temperature). However, only the master SCN core receives light inputs via the retinal hypothalamic tract. All oscillatory cores are based on similar positive/negative transcriptional/translational feedback loops. Recent studies demonstrated major CR effects on the course of viral infections in murine models. Altering the time of viral application and knocking out genes encoding oscillatory core proteins enhanced viral infection by up to 10-fold. To date, CR effects on the efficacy of Lentiviral vector (LVV) transduction have not been studied. We hypothesize that the CR is a high-level restriction factor to viral-vector transduction. We propose three specific aims to test this hypothesis. In aim 1, we will characterize the effects of normal and disrupted CR behavior on hepatic transduction efficiency by LVVs. We will employ three mouse strains showing distinct circadian behavior and melatonin (Mel) production, including C3H(Mel+), C57B6 (Mel-), and BMAL1 (oscillatory core protein)-deficient mice. The effects of time of vector administration, and exposure to either Mel or the Mel- receptor antagonist on hepatic transduction will be determined. The effects of altered CR behavior patterns on vector transduction will be determined a) following CR disruption by an acute opioid withdrawal protocol in C57B6 and C3H mice, and b) by gene delivery to naïve C57B6 and C3H mice following disruption of the normal day-sleep time period. In aim 2, We will test the hypothesis that normal and disrupted CRs affect LVV transduction efficiency in human and mouse cells in vitro. Specifically, we will quantify the effects of dexamethasone entrainment on LVV transduction of human and mouse cells comprising either normal or mutant BMAL1 gene. In aim 3, We will employ an F2 cross between the CC036 and CC057 mouse strains to determine the existence of a genetic association between the efficiency of LVV mediated hepatic gene delivery and CR patterns and will identify genetic loci contributing to the above host and LVV characteristics.