RNAi vector deilvery to inhibit JE/WN encephalitis

西尼罗河（WN）和日本脑炎（JE）病毒是蚊媒黄病毒，可导致毁灭性的急性神经系统疾病，幸存者死亡率高达30%，并造成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，对这些病毒没有有效的治疗方法。最近，RNA干扰，即短RNA分子（siRNA）以序列特异性的方式介导相应mRNA分子的破坏，已成为潜在的抗病毒治疗工具。在测试了几个基因组序列作为靶点后，我们发现靶向病毒包膜基因中高度保守序列的siRNA可以保护小鼠免受西尼罗河病毒或乙脑病毒诱导的致命性脑炎。由于自然界中存在许多病毒株，即使在保守序列内也可能表现出微小的差异，因此在Aim 1中，我们将确定另外2-3个可以有效抑制乙脑病毒和西尼罗河病毒的保守靶序列。我们还将测试使用sirna组合治疗是否会增加保护的程度和/或广度。此外，为了增强信心，我们还将测试sirna对从不同地理位置分离的多种病毒株的抑制能力。在初步研究中，用慢病毒表达的shRNA或合成的siRNA进行单次治疗，就足以防止6或18小时前诱导的感染。然而，目前使用的方法涉及在病毒攻击的同一部位颅内给药siRNA，因此，siRNA不太可能扩散到足以保护所有脑细胞的程度，这是在病毒在感染后的较晚时间点广泛传播后治疗有效所必需的。因此，为了在临床环境中实现siRNA的治疗潜力，在Aim 2中，我们将开发更好的siRNA跨脑递送方法。因为它将提供最实用的治疗形式，我们将开发非侵入性的siRNA递送方法。为了使静脉给药后能够被脑细胞吸收，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，并使用涂有转铁蛋白的聚合纳米颗粒。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，我们还将使用暂时破坏血脑屏障的方法来测试递送效果。药效将通过测试siRNA预防和治愈既定感染的能力来确定。

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.