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 在体内和体外的真核细胞中。这些生物过程包括先天的和适应性的DNA修复活动 免疫功能、炎症和代谢途径。CRS以自主外围设备为前提 振荡核，存在于大多数器官和培养细胞中。在活体中，一个中央主时钟位于 在视交叉上核(SCN)通过交感神经同步化外周振荡核 系统和神经内分泌剂，如褪黑素(MEL)。外周器官也被各种不同的 代谢/荷尔蒙和身体输入(如地塞米松、食物摄入量、体温)。然而，只有 主控SCN核心通过视网膜下丘脑束接收光输入。所有的振荡核心都基于类似的 正/负转录/翻译反馈循环。最近的研究表明，CR对 病毒在小鼠模型中的感染过程。改变病毒应用的时间和敲除基因 编码振荡核心蛋白使病毒感染增加了10倍。到目前为止，CR对疗效的影响 关于慢病毒载体(LVV)转导的研究尚未见报道。我们假设CR是一个高级别的 病毒载体转导的制约因素。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 将表征正常和紊乱的CR行为对LVV肝脏转导效率的影响。 我们将使用三个显示出不同的昼夜行为和褪黑素(MEL)产生的小鼠品系，包括 C3H(Mel+)、C57B6(Mel-)和BMAL1(振荡核心蛋白)缺陷小鼠。矢量的时间效应 给药，在肝脏转导中暴露于MEL或MEL受体拮抗剂将是 下定决心。CR行为模式的改变对载体转导的影响将在以下方面确定 通过急性阿片类药物戒断方案对C57B6和C3H小鼠的CR破坏，以及b)通过将基因传递给幼稚 C57B6和C3H小鼠的正常白天睡眠时间被打乱。在目标2中，我们将测试 假设CRS正常和受损在体外会影响人和小鼠细胞的LVV转导效率。 具体地说，我们将量化地塞米松对人和 含有正常或突变BMAL1基因的小鼠细胞。在目标3中，我们将使用F2杂交 确定CC036和CC057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模式的研究，并将确定与上述相关的遗传位点 主机和LVV特征。

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.