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来清除单纯疱疹病毒（HSV）的潜伏感染。潜伏期是HSV生物学中最迷人的方面，也是HSV疾病中最令人烦恼的方面——潜伏期是为什么HSV感染尚未被治愈的原因。本文提出的干预策略是：1)利用与这些mirna互补的寡核苷酸（anti- mirna），通过拮抗病毒microRNAs （miRNAs）来破坏潜伏性，我们假设miRNAs在潜伏感染的神经元中抑制“溶解”病毒基因表达；2)如有必要，通过拮抗有助于维持潜伏期的宿主功能进一步破坏潜伏期；3)使用抗HSV药物阿昔洛韦不可逆地灭活复制的HSV。这个项目的R21阶段是三个实验室的合作成果。第一个目的是确定选定的抗mirna或选定的影响mirna或其靶点的病毒突变（Coen实验室）是否在体外HSV潜伏期模型中抑制小鼠和大鼠神经元中的裂解基因表达（Leib和Wilson实验室）。基因表达将使用基于pcr的方法进行测量（Coen和Wilson实验室）。选择的突变也将在体内小鼠潜伏期模型中测试其对裂解基因表达的影响（Coen实验室）。第二个目的是测试这些抗mirs是否单独或至少与宿主功能的小分子拮抗剂加性联合在小鼠和大鼠体外模型中诱导再激活，以及阿昔洛韦是否可以治愈这些模型中的潜伏期。该项目的R33阶段（科恩实验室）专注于在小鼠模型中治疗体内潜伏期的努力。第三个目的是研究来自aim 2和类似HSV突变的抗mir对该模型中基因表达抑制的影响。第四个目标是在小鼠模型中测试这些抗mirs和类似的突变，单独或至少与aim 2的小分子组合是否诱导再激活。第五个目的是在小鼠模型中测试aim 4中鉴定的联合药物与阿昔洛韦是否可以治愈体内潜伏期。这些研究将伴随着血浆和组织中抗mir和化合物浓度的药代动力学分析，以及毒性分析。目标是以最小的毒性治愈单纯疱疹病毒。

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.