Targeted Destruction of HIV and HIV-Infected Cells by an Engineered Ribonuclease

通过工程核糖核酸酶靶向破坏 HIV 和 HIV 感染细胞

• 批准号: 7283356
• 负责人: STEWART N LOH
• 金额: $ 23.5万
• 依托单位: UPSTATE MEDICAL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-05-01 至 2009-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7283356
• 关键词: Antiviral Agents; Bacillus amyloliquefaciens ribonuclease; Biological Assay; Capsid; Cell Death; Cell Survival; Cells; Chimeric Proteins; Class; Cleaved cell; Condition; Cytosol; DNA; Dependovirus; Endopeptidases; Endoribonucleases; Engineering; Ensure; Enzyme Inhibitor Drugs; Enzyme Inhibitors; Enzymes; Free Energy; Gene Expression; Gene Transfer; Genetic Materials; Genome; Goals; HIV; HIV Infections; HIV Protease; Human; In Vitro; Infection; Life Cycle Stages; Mediating; Methods; Molecular; Molecular Conformation; Mutation; Outcome Study; Pancreatic ribonuclease; Peptide Hydrolases; Peptides; Principal Investigator; Property; Proteins; RNA; Research; Resistance; Reverse Transcriptase Inhibitors; Ribonucleases; Site; Structure; Tertiary Protein Structure; Testing; Toxin; Transduction Gene; Ubiquitin; Viral; Viral Genes; Virion; Virus; base; cell killing; cellular engineering; cytotoxic; design; design and construction; killings; neuronal cell body; novel; particle; programs; research study; vector; vpr Gene Products

DESCRIPTION (provided by applicant): The goal of this project is to create a new class of potent toxins that selectively target HIV and HIV-infected cells for destruction. The toxin, an engineered bacterial ribonuclease, is catalytically inactive in uninfected cells and does not harm them. The ribonuclease is activated upon cleavage by HIV protease. The method for suppressing and activating catalytic activity is based on a novel forced-unfolding mechanism. Once activated, the enzyme disrupts HIV infection by three independent mechanisms. First, it kills infected cells by degrading cytosolic RNA. Second, it is packaged into the HIV capsid, where it destroys the HIV RNA genome. Third, it is activated and delivered to cells by the HIV particle itself, upon subsequent rounds of infection. These effects are cumulative. The net result is that HIV infection is halted after a single round. By attacking the virus as well as the cells that harbor it, the proposed method is designed to rid HIV completely from the body, especially when used in conjunction with therapies that activate HIV gene expression in latently-infected cells. The first aim describes the design, construction and in vitro testing of two separate designs of the toxin. The proteins are optimized so that ribonuclease activity is high in the protease-cleaved state and undetectable in the uncleaved form. In the second aim, each of the three anti-HIV mechanisms is tested ex vivo. Toxins are delivered into cultured human cells by direct transduction (facilitated by fusion to the cell-penetrating HIV TAT peptide) as well as by a viral gene vector. The cells are then infected with HIV. Cell viability and viral infectivity assays determine the extent to which the toxins selectively kill infected cells and inactivate HIV. A major benefit of this therapy is that the molecules developed here will retain their potency against viral mutation to a much greater degree than existing protease and reverse transcriptase inhibitors. This study creates a new class of molecules that specifically kill HIV and HIV-infected cells, and do not harm healthy cells. These molecules are engineered to remain potent despite viral mutation. This therapy is designed to eliminate the virus as well as diseased cells from the body.

描述（由申请人提供）：该项目的目标是创造一类新型强效毒素，选择性地针对 HIV 和 HIV 感染细胞进行破坏。这种毒素是一种工程细菌核糖核酸酶，在未感染的细胞中没有催化活性，不会伤害它们。核糖核酸酶在被 HIV 蛋白酶切割后被激活。抑制和激活催化活性的方法基于一种新颖的强制展开机制。一旦被激活，这种酶就会通过三种独立的机制破坏艾滋病毒感染。首先，它通过降解胞质 RNA 来杀死受感染的细胞。其次，它被包装到 HIV 衣壳中，破坏 HIV RNA 基因组。第三，在随后的几轮感染中，它被HIV颗粒本身激活并传递到细胞。这些影响是累积的。最终结果是艾滋病毒感染在一轮后停止。通过攻击病毒以及含有病毒的细胞，所提出的方法旨在将艾滋病毒从体内完全清除，特别是当与激活潜伏感染细胞中艾滋病毒基因表达的疗法结合使用时。第一个目标描述了两种独立设计的毒素的设计、构建和体外测试。这些蛋白质经过优化，核糖核酸酶活性在蛋白酶切割状态下较高，而在未切割状态下检测不到。第二个目标是对三种抗艾滋病毒机制中的每一种进行离体测试。毒素通过直接转导（通过与细胞穿透性 HIV TAT 肽融合来促进）以及病毒基因载体传递到培养的人类细胞中。然后这些细胞就会感染艾滋病毒。细胞活力和病毒感染性测定确定毒素选择性杀死受感染细胞并灭活 HIV 的程度。这种疗法的一个主要好处是，这里开发的分子将比现有的蛋白酶和逆转录酶抑制剂更大程度地保留其抗病毒突变的效力。这项研究创造了一种新的分子，可以专门杀死艾滋病毒和艾滋病毒感染的细胞，并且不会伤害健康细胞。尽管病毒发生突变，这些分子仍能保持有效。这种疗法旨在消除体内的病毒和患病细胞。