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种新型的强效毒素，选择性地靶向HIV和HIV感染细胞进行破坏。这种毒素是一种工程化的细菌核糖核酸酶，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蛋白酶切割时被激活。抑制和活化催化活性的方法基于新的强制解折叠机理。一旦被激活，这种酶通过三种独立的机制破坏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衣壳中，在那里它破坏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之后就停止了。通过攻击病毒以及携带它的细胞，所提出的方法旨在将HIV完全从体内清除，特别是当与激活潜伏感染细胞中HIV基因表达的疗法结合使用时。第一个目标描述了毒素的两个单独设计的设计、构造和体外测试。对蛋白质进行优化，使得核糖核酸酶活性在蛋白酶切割状态下高，并且在未切割形式下不可检测。在第二个目标中，离体测试三种抗HIV机制中的每一种。毒素通过直接转导（通过与细胞穿透HIV达特肽融合促进）以及通过病毒基因载体递送到培养的人细胞中。然后这些细胞被艾滋病毒感染。细胞活力和病毒感染性测定确定毒素选择性杀死感染细胞和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，比现有的蛋白酶和逆转录酶抑制剂更大的程度。这项研究创造了一类新的分子，专门杀死艾滋病毒和艾滋病毒感染的细胞，而不伤害健康细胞。这些分子经过改造，即使病毒突变也能保持效力。这种疗法的目的是消除病毒以及从身体的病变细胞。