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）：该项目的目的是创建一类新的有效毒素，这些毒素有选择地靶向HIV和HIV感染的细胞以破坏。毒素是一种工程的细菌核糖核酸酶，在未感染的细胞中催化不活跃，不会损害它们。 HIV蛋白酶在裂解后激活核糖核酸酶。抑制和激活催化活性的方法是基于一种新型的强制性解释机制。一旦激活，该酶通过三种独立机制破坏了HIV感染。首先，它通过降解胞质RNA杀死受感染的细胞。其次，它被包装到HIV Capsid中，在那里破坏了HIV RNA基因组。第三，在随后的感染后，它被HIV颗粒本身激活并传递到细胞中。这些影响是累积的。最终结果是单轮后将HIV感染停止。通过攻击病毒以及藏有其细胞的细胞，该方法旨在完全摆脱身体，尤其是在与疗法一起使用的疗法中，该疗法激活了在潜在受感染的细胞中激活HIV基因的表达时。第一个目的描述了毒素两种单独设计的设计，构建和体外测试。对蛋白质进行了优化，以使蛋白酶切割态中的核糖核酸酶活性很高，并且在未切换的形式中无法检测到。在第二个目标中，三种抗HIV机制中的每一个都经过体内测试。通过直接转导（通过融合到细胞渗透HIV TAT肽的促进）以及通过病毒基因载体将毒素传递到培养的人类细胞中。然后将细胞感染HIV。细胞活力和病毒感染性测定确定毒素选择性地杀死感染细胞和失活的HIV的程度。这种疗法的一个主要好处是，这里开发的分子将保留其对病毒突变的效力，比现有蛋白酶和逆转录酶抑制剂的程度要大得多。这项研究创建了一类新的分子，这些分子专门杀死HIV和感染了HIV的细胞，并且不会损害健康的细胞。尽管病毒突变，这些分子经过设计以保持有效。该疗法旨在消除体内病毒以及病态细胞。