Antagonizing miRNAs in a strategy to cure HSV latency

描述（由申请人提供）：本项目的长期目标是开发一种新的干预策略，以清除单纯疱疹病毒（HSV）的潜伏感染。潜伏期是HSV生物学最迷人的方面，也是HSV疾病最令人烦恼的方面-潜伏期是HSV感染尚未治愈的原因。本文提出的干预策略是：1）通过拮抗病毒microRNA（miRNAs）来破坏潜伏期，我们假设使用与这些miRNAs互补的寡核苷酸来抑制潜伏感染神经元中的“裂解性”病毒基因表达（抗miR）; 2）如果需要，通过拮抗帮助维持潜伏期的宿主功能来进一步破坏潜伏期;和3）使用抗HSV药物阿昔洛韦不可逆地抑制所产生的复制HSV。该项目的R21阶段是三个实验室的合作成果。第一个目的是确定选择的抗miR或影响miRNA或其靶位点的选择的病毒突变（Coen实验室）是否在HSV潜伏期的小鼠和大鼠神经元体外模型中降低裂解基因表达（Leib和Wilson实验室）。将使用基于PCR的方法（Coen和Wilson实验室）测量基因表达。还将在体内小鼠潜伏期模型（Coen实验室）中测试所选突变对裂解基因表达的影响。第二个目的是测试这些抗miR单独或至少与宿主功能的小分子拮抗剂相加组合是否在小鼠和大鼠体外模型中诱导再活化，以及阿昔洛韦是否可以治愈此类模型中的潜伏期。该项目的R33阶段（Coen实验室）专注于在小鼠模型中治愈体内潜伏期的努力。第三个目的是研究来自目的2的抗miR，以及可比较的HSV突变对该模型中基因表达去阻遏的影响。第四个目的是测试这些抗miR和相当的突变单独或与来自Aim 2的小分子至少相加组合是否诱导小鼠模型中的再活化。第五个目的是测试目的4中鉴定的组合与阿昔洛韦一起是否可以在小鼠模型中体内治愈潜伏期。这些研究将伴随血浆和组织中抗miR和化合物浓度的药代动力学测定以及毒性测定。目标是以最小的毒性治愈HSV。

DESCRIPTION (provided by applicant): The long-term objective of this project is to develop a novel intervention strategy to clear latent infections by herpes simplex virus (HSV). Latency is the most fascinating aspect of HSV biology and the most vexing aspect of HSV disease -- latency is why HSV infections have yet to be cured. The intervention strategy proposed here is: 1) Disrupt latency by antagonizing viral microRNAs (miRNAs) that we hypothesize repress "lytic" viral gene expression in latently infected neurons, using oligonucleotides that are complementary to these miRNAs (anti-miRs); 2) If necessary, further disrupt latency by antagonizing host functions that help maintain latency; and 3) Irreversibly inactivate the resultin replicating HSV using the anti-HSV drug acyclovir. The R21 phase of this project is a collaborative effort of three labs. The first aim is to determine whether selected anti-miRs or selected viral mutations affecting miRNAs or their target sites (Coen lab) derepress lytic gene expression in mouse and rat neuron in vitro models of HSV latency (Leib and Wilson labs). Gene expression will be measured using PCR-based methods (Coen and Wilson labs). Selected mutations will also be tested for their effects on lytic gene expression in an in vivo mouse latency model (Coen lab). The second aim is to test whether these anti-miRs, alone or in at least additive combination with small molecule antagonists of host functions, induce reactivation in the mouse and rat in vitro models, and whether acyclovir can cure latency in such models. The R33 phase of this project (Coen lab) focuses on efforts to cure latency in vivo in a mouse model. The third aim investigates anti-miRs from Aim 2, and comparable HSV mutations for effects on derepression of gene expression in this model. The fourth aim is to test whether these anti-miRs and comparable mutations, alone or in at least additive combination with small molecules from Aim 2 induce reactivation in the mouse model. The fifth aim is to test whether combinations identified in Aim 4 together with acyclovir can cure latency in vivo in the mouse model. These studies will be accompanied by pharmacokinetic assays of anti-miR and compound concentrations in plasma and tissues, and assays of toxicities. The goal is to achieve cure of HSV with minimal toxicity.