Development of Nanotherapeutics for RSV-induced Lung Disease

描述（由申请人提供）： 该提案的主要目标是研究呼吸道合胞病毒（RSV）非结构蛋白1（NS 1）在RSV感染后通过线粒体系统调节先天免疫和细胞存活的机制，并将这些知识与纳米颗粒技术相结合，以开发预防和治疗RSV感染的新方法。退伍军人通常患有某种形式的慢性肺部疾病，特别容易受到呼吸道病毒感染，如RSV和流感引起的感染。RSV是一种机会性人类呼吸道病原体，可导致婴儿细支气管炎以及免疫功能低下的成人和老年人肺炎，全球每年估计有6400万例感染和166，000例死亡。RSV对老年退伍军人的威胁越来越严重。尽管取得了进展，但RSV诱导的先天免疫反应的确切性质仍不清楚。我们的初步证据表明，NS 1蛋白与线粒体相关联，并在RSV感染的A549上皮细胞中与线粒体抗病毒信号传导（MAVS）蛋白复合，并抑制视黄酸诱导基因-I（RIG-I）和MAVS的信号传导，通过caspase激活和募集结构域（CARD）发生。这会干扰IFN-2的产生，从而抑制先天性免疫应答。NS 1还阻断受感染细胞的程序性细胞死亡（凋亡），并防止线粒体膜电位的丧失，从而促进细胞（和病毒）存活。这些数据导致了RSV利用NS 1减弱RIG-I-MAVS诱导的抗病毒IFN-2产生并调节线粒体功能以增加细胞存活的假设。对这些过程的确切机制的研究有望发现预防或限制RSV感染的新靶点。为了检验这些假设，提出了以下具体目标。在第一个目标中，计划检查NS 1在RSV感染期间减弱RIG-I/MAVS信号传导中的作用。这包括分析NS 1在调节MAVS、LGP 2和RIG-1表达以及在RSV感染期间减弱IFN-2应答中的作用，NS 1蛋白的线粒体定位以及NS 1- MAVS相互作用的结构-功能关系。第二个目标将集中在确定NS 1- MAVS相互作用是否需要防止过早凋亡和内在细胞死亡。这还包括解决NS 1-MAVS复合物是否与抗或促凋亡因子相关并调节它们在线粒体膜上的表达。在第三个目标中，计划开发和测试靶向chlipid（壳聚糖-脂质）纳米颗粒作为RSV诱导的肺部疾病的治疗剂。这些chlipids包封编码LGP 2和NS 1的siRNA的质粒和编码MAVS的肽（称为pLMNS 1），其中每一种单独地显示出显著下调人细胞中的RSV复制。最后，在第四个目的中，提出评估靶向纳米颗粒包封的pLMNS 1用于治疗小鼠模型中的RSV疾病。拟议的研究将由一组优秀的研究人员进行，他们在RSV疾病、细胞凋亡和纳米颗粒技术方面有着良好的记录。这些结果有望发现新的靶点，并启动这些靶点对抗RSV肺病的临床前研究。

DESCRIPTION (provided by applicant): The primary goal of this proposal is to investigate the mechanism whereby respiratory syncytial virus (RSV) nonstructural protein 1 (NS1) modulates innate immunity and cell survival through the mitochondrial system following RSV infection, and to combine this knowledge with nanoparticle technology to develop new ways to prevent and treat RSV infections. Veterans commonly have some form of chronic lung disease and are especially susceptible to respiratory viral infections like those caused by RSV and influenza. RSV is an opportunistic human respiratory pathogen that causes bronchiolitis in infants and pneumonia in immunocompromised adults and the elderly with an estimated 64 million infections and 166,000 deaths annually worldwide. RSV is becoming more and more of a serious threat to aging veterans. Despite progress, the precise nature of the RSV-induced innate immune response remains unclear. Our preliminary evidence shows that the NS1 protein associates with mitochondria and is found in complex with the mitochondrial antiviral signaling (MAVS) protein in RSV-infected A549 epithelial cells and inhibits the signaling of retinoic acid-inducible gene-I (RIG-I) and MAVS occurring via the caspase activation and recruitment domain (CARD). This interferes with IFN-2 production which inhibits the innate immune response. NS1 also blocks programmed cell death (apoptosis) of infected cells and prevents loss of mitochondrial membrane potential, thus promoting cell (and viral) survival. These data have led to the hypothesis that RSV utilizes NS1 to attenuate RIG-I- MAVS-induced antiviral IFN-2 production and to modulate mitochondrial function for increasing cell survival. The study of the precise mechanisms underlying these processes is expected to lead to the discovery of new targets for preventing or limiting RSV infection. To test these hypotheses, the following specific aims are proposed. In the first aim, it is planned to examine the role of NS1 in attenuating RIG-I/MAVS signaling during RSV infection. This includes analysis of the role of NS1 in regulating MAVS, LGP2 and RIG-1 expression and in attenuation of IFN-2 response during RSV infection, mitochondrial localization of NS1 protein and structure-function relationships of NS1- MAVS interaction. The second aim will focus on determining whether the NS1- MAVS interaction is required to prevent premature apoptosis and intrinsic cell death. This also includes addressing whether the NS1-MAVS complex is associated with anti- or pro-apoptotic factors and modulates their expression on the mitochondrial membrane. In the third aim, it is planned to develop and test targeted chlipid (chitosan-lipid) nanoparticles as a therapeutic agent for RSV-induced lung disease. These chlipids encapsulate a plasmid that encodes siRNAs for LGP2 and NS1 and a peptide for MAVS (referred to as pLMNS1), each of which individually has been shown to significantly down- regulate RSV replication in human cells. Lastly, in the fourth aim it is proposed to evaluate targeted nanoparticle-encapsulated pLMNS1 for treating RSV disease in a mouse model. The proposed research will be conducted by an excellent group of investigators with a proven track record in RSV disease, apoptosis and nanoparticle technology. The results are expected to lead to the discovery of novel targets and to the initiation of preclinical studies of these targets against RSV lung disease.