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

描述（由申请人提供）：该项目的目标是创造一类新的强效毒素，选择性地靶向艾滋病毒和感染艾滋病毒的细胞进行破坏。这种毒素是一种经过改造的细菌核糖核酸酶，在未感染的细胞中没有催化活性，不会对细胞造成伤害。这种核糖核酸酶在HIV蛋白酶裂解时被激活。抑制和激活催化活性的方法是基于一种新的强制展开机制。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杀死被感染的细胞。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中。这些影响是累积的。最终的结果是，经过一轮治疗后，艾滋病病毒感染就停止了。通过攻击病毒以及携带病毒的细胞，这种被提出的方法旨在彻底清除体内的艾滋病毒，特别是在与激活潜伏感染细胞中的艾滋病毒基因表达的疗法结合使用时。第一个目标描述了毒素的两种不同设计的设计、构造和体外测试。这些蛋白质经过优化，使核糖核酸酶活性在蛋白酶裂解状态下高，而在非裂解形式下检测不到。在第二个目标中，三种抗hiv机制中的每一种都在体外进行了测试。毒素通过直接转导（通过与穿透细胞的HIV TAT肽融合促进）以及病毒基因载体传递到培养的人类细胞中。然后这些细胞被HIV感染。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和灭活艾滋病毒的程度。这种疗法的一个主要好处是，与现有的蛋白酶和逆转录酶抑制剂相比，这里开发的分子将在更大程度上保持其对抗病毒突变的效力。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。这种疗法旨在消除体内的病毒和患病细胞。

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.