CONSTRUCTION AND OPTIMIZATION OF ANTIHIV RIBOZYMES

描述：（改编自申请人的摘要） 这项拟议的研究项目是开发核酶技术， 这一点是接近现实的治疗。 这将使用 发夹核酶是一种小的反式作用RNA酶， 非常适合细胞RNA失活研究。 以前的工作 阐明了发夹状核酶可以被改造成 有效地切割HIV序列，并开发了一种体外选择 通过该方法，针对给定靶序列的核酶活性 可以优化。 这些发现已被用于产生核酶 靶向强烈抑制HIV-1的保守HIV pol序列 在培养的淋巴细胞中复制。 目前建议的具体目标是：（1）严格界定 抗HIV发夹状核酶抗病毒作用位点和机制， 并评估其在体内的底物选择性;（2）开发和评估 一类新的抗艾滋病毒靶点，使用新的信息定义 发夹状核酶的底物特异性。 预计这一 工作将导致（i）明确的证据表明，工程发夹 核酶通过在细胞膜上的位点特异性切割RNA来抑制HIV的复制。 靶向位点，（ii）探索几种新的策略， 发育、细胞内活化和代谢调节 高活性核酶，以及（iii）开发和快速测试 一些潜在的新的抗HIV治疗剂。

DESCRIPTION: (Adapted from Applicant's Abstract) The overall goal of this proposed research project is to develop ribozyme technology to the point that it is close to therapeutic reality. This will be done using the hairpin ribozyme which is a small trans-acting RNA enzyme that is highly suitable for cellular RNA inactivation studies. Previous work has elucidated rules by which the hairpin ribozyme can be engineered to efficiently cleave HIV sequences and has developed an in vitro selection method through which ribozyme activity against a given target sequence can be optimized. These findings have been used to generate a ribozyme targeted to a conserved HIV pol sequence that strongly inhibits HIV-1 replication in cultured lymphocytes. The Specific Aims of the current proposal are: (1) Rigorously define the site and mechanism of antiviral action of anti-HIV hairpin ribozymes, and assess their substrate selectivity in vivo; (2) Develop and evaluate a novel class of anti-HIV targets, using new information defining substrate specificity of the hairpin ribozyme. It is expected that this work will result in (i) definitive proof that engineered hairpin ribozymes inhibit HIV replication by site- specific RNA cleavage at the targeted site, (ii) the exploration of several novel strategies for the development, intracellular activation, and metabolic regulation of highly active ribozymes, and (iii) the development and rapid testing of a number of potential new anti-HIV therapeutic agents.