RNAi vector deilvery to inhibit JE/WN encephalitis

产品说明：西尼罗河病毒（WN）和日本脑炎病毒（JE）是蚊媒黄病毒，可导致毁灭性的急性神经系统疾病，死亡率高达30%，幸存者永久性神经系统残疾。这些病毒也是潜在的B类生物恐怖主义制剂，目前还没有有效的治疗方法。最近，RNA干扰，其中短RNA分子（siRNA）介导的破坏相应的mRNA分子在序列特异性的方式，已成为一种潜在的抗病毒治疗的工具。在测试了几个基因组序列作为靶标后，我们发现靶向病毒包膜基因中高度保守序列的siRNA可以保护小鼠免受由WNV或JEV诱导的致命性脑炎。由于自然界中存在的许多病毒株即使在保守序列内也可能表现出微小的差异，因此在目的1中，我们将鉴定另外2-3个可以有效抑制JEV和WNV的保守靶序列。我们还将测试用siRNA组合治疗是否会增加保护的幅度和/或广度。此外，为了增强信心，我们还将测试siRNA抑制从不同地理位置分离的多种病毒株的能力。在初步研究中，用慢病毒表达的shRNA或合成siRNA的单次治疗足以提供对6或18小时前诱导的感染的保护。然而，目前使用的方法涉及在与病毒攻击相同的部位颅内施用siRNA，因此siRNA不太可能扩散到足以保护所有脑细胞，这将是在病毒在感染后的稍后时间点广泛传播后治疗有效所需的。因此，为了实现siRNA在临床环境中的治疗潜力，在目标2中，我们将开发用于更好地递送siRNA穿过大脑的方法。因为它将提供最实用的治疗形式，我们将开发siRNA递送的非侵入性方法。为了使脑细胞在静脉内递送后能够摄取，我们将开发涂有转铁蛋白受体抗体的聚乙二醇化免疫脂质体以及使用涂有转铁蛋白的聚合物纳米颗粒。或者，我们将尝试将siRNA递送到脑脊液中。如果这些方法不起作用，我们还将使用瞬时破坏血脑屏障的方法来测试递送功效。将通过测试siRNA预防以及治愈已建立的感染的能力来确定功效。

DESCRIPTION: West Nile (WN) and Japanese encephalitis (JE) viruses are mosquito-borne flaviviruses that cause a devastating acute neurological illness with up to 30% mortality and permanent neurological disabilities in the survivors. These are also potential category B bioterrorism agents and there is no effective treatment for these viruses. Recently RNA interference, where short RNA molecules (siRNA) mediate the destruction of corresponding mRNA molecules in a sequence-specific manner, has emerged as a tool for potential antiviral therapeutics. After testing several genomic sequences as targets, we have found that a siRNA targeting a highly conserved sequence in the viral envelope gene can protect mice from fatal encephalitis induced by either WNV or JEV. Because many strains of viruses exist in nature that may exhibit small differences even within the conserved sequence, in Aim 1, we will identify an additional 2-3 conserved target sequences that can effectively suppress both JEV and WNV. We will also test if treatment with a combination of siRNAs will increase the magnitude and/or breadth of protection. Additionally to enhance confidence, we will also test the siRNAs for their ability to inhibit multiple viral strains isolated from different geographic locations. In preliminary studies, a single treatment with either lentivirally expressed shRNA or synthetic siRNA was sufficient to provide protection from an infection induced 6 or 18 h earlier. However the currently used method involves administration of siRNA intracranially at the same site as viral challenge and thus, the siRNA is unlikely to diffuse enough to protect all brain cells, which would be required for treatment to be effective after the virus has extensively spread at later time points after infection. Thus in order to realize the siRNA treatment potential in a clinical setting, in Aim 2, we will develop methods for better delivery of siRNA across the brain. Because it will provide the most practical form of treatment, we will develop non- invasive methods for siRNA delivery. To enable uptake by brain cells following intravenous delivery, we will develop pegylated immunoliposomes coated with transferrin receptor antibody as well as use polymeric nanoparticles coated with transferrin. Alternatively, we will try siRNA delivery into the cerebrospinal fluid. If these methods do not work, we will also test delivery efficacy using methods that disrupt the blood-brain barrier transiently. Efficacy will be determined by testing the siRNA's ability to prevent as well as to cure an established infection.