Development of Nanotherapeutics for RSV-induced Lung Disease

描述(由申请人提供)： 这项建议的主要目的是研究呼吸道合胞病毒(RSV)非结构蛋白1(NS1)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，并将这一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合胞病毒和流感引起的呼吸道病毒感染。呼吸道合胞病毒是一种机会性的人类呼吸道病原体，可导致婴儿毛细支气管炎和免疫功能低下的成年人和老年人肺炎，据估计，全球每年有100万人感染呼吸道合胞病毒，166,000人死亡。呼吸道合胞病毒对年迈的退伍军人构成越来越严重的威胁。尽管取得了进展，但RSV诱导的先天性免疫反应的确切性质仍不清楚。我们的初步证据表明，在RSV感染的A549上皮细胞中，NS1蛋白与线粒体结合，并与线粒体抗病毒信号(MAVS)蛋白形成复合体，并通过caspase激活和募集结构域(CARD)抑制维甲酸诱导基因-I(RIG-I)和MAVS的信号转导。这会干扰干扰素-2的产生，而干扰素-2会抑制先天免疫反应。NS1还阻止受感染细胞的程序性细胞死亡(凋亡)，防止线粒体膜电位的丧失，从而促进细胞(和病毒)的生存。这些数据导致了一种假设，即RSV利用NS1来减弱RIG-I-MAVS诱导的抗病毒干扰素-2的产生，并调节线粒体功能以提高细胞存活率。对这些过程背后的精确机制的研究有望导致发现预防或限制RSV感染的新靶点。为了检验这些假设，我们提出了以下具体目标。在第一个目标中，计划研究NS1在RSV感染过程中减弱RIG-I/MAVS信号的作用。这包括分析NS1在调节MAVS、LGP2和RIG-1表达以及在RSV感染过程中减弱干扰素-2应答中的作用、NS1蛋白的线粒体定位以及NS1-MAVS相互作用的结构与功能关系。第二个目标将集中在确定是否需要NS1-MAVS相互作用来防止过早的细胞凋亡和固有的细胞死亡。这也包括解决NS1-MAVS复合体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CHLID(壳聚糖脂)纳米颗粒作为RSV引起的肺部疾病的治疗剂。这些chlipids包裹着一个编码LGP2和NS1的siRNAs和一个MAV的肽(称为pLMNS1)的质粒，每一个单独的肽都被证明能够显著下调RSV在人类细胞中的复制。最后，在第四个目标中，建议评估靶向纳米颗粒包裹的pLMNS1在小鼠模型中治疗RSV疾病的效果。这项拟议的研究将由一群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都有良好的记录。这些结果有望导致发现新的靶点，并启动这些靶点对抗呼吸道合胞病毒肺部疾病的临床前研究。

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.