Development of Nanotherapeutics for RSV-induced Lung Disease

RSV 引起的肺部疾病纳米疗法的开发

• 批准号: 8391625
• 负责人: Shyam S Mohapatra
• 金额: --
• 依托单位: JAMES A. HALEY VA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-10-01 至 2015-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8391625
• 关键词: 3-Dimensional; A549; Address; Adult; Affect; Aging; Antiviral Agents; Apoptosis; Apoptotic; Area; Attenuated; Binding; Biology; Bronchiolitis; CCL4 gene; Caspase; Cell Culture Techniques; Cell Death; Cell Membrane Permeability; Cell Survival; Cells; Cessation of life; Chitosan; Chronic lung disease; Complex; Cyclophosphamide; Data; Development; Dissociation; Doctor of Philosophy; Effectiveness; Elderly; Encapsulated; Epithelial Cells; Fibroblasts; Fluorescein-5-isothiocyanate; Fluorescence Microscopy; Funding; Genes; Genetic; Goals; Grant Review; Host Defense; Hour; Human; Hybrids; Immune; Immune response; Immune system; Immunocompromised Host; In Vitro; Inbred BALB C Mice; Infant; Infection; Influenza; Intercellular adhesion molecule 1; Interferons; Knowledge; Label; Laboratories; Lead; Leadership; Link; Lipids; Lung; Lung Inflammation; Lung diseases; Membrane Potentials; Metabolic; Methods; Mitochondria; Modeling; Molecular Medicine; Motivation; Mucous Membrane; Mus; Natural Immunity; Nature; Nonstructural Protein; Palivizumab; Pathogenesis; Peptide Signal Sequences; Peptides; Physiological; Physiology; Plasmids; Play; Pneumonia; Postdoctoral Fellow; Principal Investigator; Process; Production; Prophylactic treatment; Proteins; Publications; RNA Helicase; RNA Interference; Recombinants; Regulation; Reporting; Research; Research Personnel; Research Project Grants; Respiratory Syncytial Virus Infections; Respiratory Syncytial Virus Vaccines; Respiratory syncytial virus; Role; Safety; Scientist; Signal Pathway; Signal Transduction; Signaling Protein; Small Interfering RNA; Structure-Activity Relationship; System; Technology; Testing; Therapeutic; Therapeutic Agents; Time; Tissue Model; Translations; Treatment Efficacy; Tretinoin; Veterans; Viral; Virus; Virus Diseases; Virus Replication; Work; accomplished suicide; attenuation; base; career; caspase-9; cytochrome c; effective therapy; efficacy testing; fetal; in vivo; mitochondrial membrane; mouse model; nanoparticle; nanotherapeutic; novel; novel therapeutics; pathogen; preclinical study; premature; prevent; professor; programs; promoter; protein structure function; receptor; red fluorescent protein; respiratory; response; skills; targeted delivery; vector control; viral RNA

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.

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 (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，并将这些知识与纳米颗粒技术相结合，开发新方法预防和治疗 RSV 感染。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，如呼吸道合胞病毒和流感引起的感染。 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 6,400 万人感染，166,000 人死亡。 RSV 对老年退伍军人构成越来越严重的威胁。尽管取得了进展，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，NS1 蛋白与线粒体相关，并与 RSV 感染的 A549 上皮细胞中的线粒体抗病毒信号 (MAVS) 蛋白形成复合物，并抑制视黄酸诱导基因 -I (RIG-I) 和MAVS 通过半胱天冬酶激活和招募结构域 (CARD) 发生。这会干扰 IFN-2 的产生，从而抑制先天免疫反应。 NS1 还可以阻止受感染细胞的程序性细胞死亡（细胞凋亡），并防止线粒体膜电位损失，从而促进细胞（和病毒）存活。这些数据得出这样的假设：RSV利用NS1来减弱RIG-I-MAVS诱导的抗病毒IFN-2的产生并调节线粒体功能以增加细胞存活。对这些过程背后的精确机制的研究预计将导致发现预防或限制 RSV 感染的新靶点。为了检验这些假设，提出了以下具体目标。第一个目标是研究 NS1 在 RSV 感染期间减弱 RIG-I/MAVS 信号传导中的作用。这包括分析 NS1 在 RSV 感染期间调节 MAVS、LGP2 和 RIG-1 表达以及减弱 IFN-2 反应中的作用、NS1 蛋白的线粒体定位以及 NS1-MAVS 相互作用的结构功能关系。第二个目标将集中于确定是否需要 NS1-MAVS 相互作用来防止过早凋亡和内在细胞死亡。这还包括解决 NS1-MAVS 复合物是否与抗凋亡因子或促凋亡因子相关并调节它们在线粒体膜上的表达。第三个目标是，计划开发和测试靶向磷脂（壳聚糖-脂质）纳米颗粒作为 RSV 引起的肺部疾病的治疗剂。这些脂质封装了编码 LGP2 和 NS1 的 siRNA 的质粒以及 MAVS 的肽（称为 pLMNS1），其中每一个都已被证明能够显着下调人类细胞中的 RSV 复制。最后，在第四个目标中，建议评估靶向纳米颗粒封装的 pLMNS1 在小鼠模型中治疗 RSV 疾病的效果。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拥有良好的记录。预计这些结果将导致新靶标的发现，并启动这些靶标针对 RSV 肺部疾病的临床前研究。