MULTITARGET RIBOZYMES AS ANALYTICAL AND THERAPEUTIC AGENTS

多靶点核酶作为分析剂和治疗剂

• 批准号: 2579665
• 负责人: M SCHUBERT
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/2579665
• 关键词: Alphaherpesvirinae; HIV infections; active sites; antiviral agents; beta galactosidase; chemical cleavage; chemical structure function; disease /disorder model; gene expression; gene targeting; genetically modified animals; hippocampus; human immunodeficiency virus 1; laboratory mouse; messenger RNA; molecular cloning; mutant; neurons; phenotype; ribozymes; tissue /cell culture; transfection /expression vector; virus RNA

Ribozymes are catalytic RNAs that can be targeted to specifically cleave selected RNAs. Such target RNAs could be viral RNAs or cellular RNAs. One of the goals of this study was to develop ribozymes against HIV-1 as part of a gene therapy in AIDS patients. Our second goal is to develop ribozymes for specific cellular mRNAs in the CNS. A specific combination of ribozyme target sites together with specific promoters for ribozyme expression could be employed in a reverse genetic approach in transgenic mice. Our plan is to generate specific phenotypes in transgenic mice to cause neuronal dysfunction by multitarget-ribozymes in order to study neuronal function. We have previously developed multitarget-ribozymes that cleave HIV-1 RNA at up to nine different sites, and protect cells from HIV-1 pathogenesis in tissue culture. Our data suggest that multitarget-ribozymes are functional in cell nuclei and they cleave unspliced HIV-1 RNA. In addition, we found that the same multitarget-ribozyme transcript can be translated in the cytoplasm. This strongly suggests that a highly effective negative transdominant mutant HIV-1 Rev protein could be expressed from the same mRNA that also functions as a multitarget-ribozyme. We are currently recloning our HIV-1 multitarget-ribozyme for insertion into a retrovirus vector (MMLV). If the combination of multitarget-ribozyme and transdominant negative HIV-1 Rev protein increases antiviral efficacy against HIV-1, these vector may be included in clinical trials with HIV-1 infected and uninfected twins. For our initial studies of multitarget-ribozyme effectiveness in transgenic mice, we have recently synthesized two octaribozymes, one against the ICP4 protein mRNA of herpes simplex virus and the other against the beta-galactosidase protein mRNA. The efficacy of these ribozymes will initially be tested in tissue culture and subsequently in transgenic mice. The purpose of these studies will be to rule out toxicity of multitarget-ribozymes in vivo. Secondly, these transgenic mice may resist herpes virus neuropathogenesis because of the multitarget-ribozyme. The octaribozyme against the beta-galactosidase will be used to inhibit beta-galactosidase expression in select neurons of the hippocampus of already existing transgenic mice. If multitarget- ribozymes are highly effective in vivo, they could complement gene knock- out experiments for the generation of mouse models for neuronal dysfunction.

核酶是催化 RNA，可以靶向特异性切割 选定的 RNA。 这样的靶RNA可以是病毒RNA或细胞RNA。 这项研究的目标之一是开发针对 HIV-1 的核酶 作为艾滋病患者基因治疗的一部分。 我们的第二个目标是 开发中枢神经系统中特定细胞 mRNA 的核酶。具体的 核酶靶位点与特定启动子的组合 核酶表达可用于反向遗传方法 在转基因小鼠中。 我们的计划是产生特定的表型 转基因小鼠通过多靶点核酶引起神经元功能障碍 以研究神经元功能。我们之前开发过 多靶点核酶，可在多达 9 种不同的位置切割 HIV-1 RNA 位点，并在组织培养中保护细胞免受 HIV-1 发病机制的影响。我们的 数据表明多靶点核酶在细胞核中发挥作用 它们切割未剪接的 HIV-1 RNA。 另外，我们发现同样 多靶点核酶转录物可以在细胞质中翻译。这 强烈表明一种高效的负转显性突变体 HIV-1 Rev 蛋白可以由与 HIV-1 Rev 蛋白相同的 mRNA 表达 作为多靶点核酶发挥作用。我们目前正在重新克隆我们的 HIV-1 用于插入逆转录病毒载体（MMLV）的多靶点核酶。如果 多靶点核酶与转显性阴性 HIV-1 的组合 Rev 蛋白可增加针对 HIV-1 的抗病毒功效，这些载体可能 被纳入针对 HIV-1 感染和未感染双胞胎的临床试验。 对于我们对多靶点核酶有效性的初步研究 转基因小鼠，我们最近合成了两种八核酶，一种 对抗单纯疱疹病毒等的ICP4蛋白mRNA 对抗 β-半乳糖苷酶蛋白 mRNA。这些功效 核酶最初将在组织培养物中进行测试，随后在 转基因小鼠。 这些研究的目的是排除 体内多靶点核酶的毒性。 其次，这些转基因 小鼠可能抵抗疱疹病毒的神经发病机制，因为 多靶点核酶。抗β-半乳糖苷酶的八核酶 将用于抑制选定神经元中的β-半乳糖苷酶表达 现有转基因小鼠的海马体。如果是多目标- 核酶在体内非常有效，它们可以补充基因敲除 进行神经元小鼠模型生成实验 功能障碍。