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 nave 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.
The overall goal of the proposed studies is to characterize the effects of the circadian rhythm on lentiviral vector transduction with the aim to improve lentiviral vector-based gene therapy protocols.
