ENHANCEMENT AND MODULATION OF ANTI-SENSE RNA ACTIVITY

反义 RNA 活性的增强和调节

• 批准号: 3191376
• 负责人: JONATHAN G IZANT
• 金额: $ 24.93万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-04-01 至 1993-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3191376
• 关键词: RNA; RNA binding protein; Retroviridae; Retroviridae disease; Xenopus oocyte; antisense nucleic acid; fusion gene; gene expression; gene therapy; genetic manipulation; genetic transcription; genetically modified animals; infant animal; laboratory mouse; nucleic acid sequence; polymerase chain reaction; posttranscriptional RNA processing; small nuclear RNA; structural genes; tissue /cell culture; transfection; transfer RNA; transforming virus; virus replication

Antisense RNA is a valuable, though often unpredictable, strategy for experimental gene inhibition as well as a potential rational approach to genetic therapeutics. We propose to investigate sophisticated chimeric antisense genes constructed by inserting antisense DNA fragments into structural RNA genes including snRNA and tRNA genes. The novel design of these antisense RNAs exploits the biological activity of both the snRNP and tRNA parent genes and has shown significant antisense inhibition activity in cultured cells and in transgenic mice. We will test enhanced designs of the chimeric genes including improvements in secondary structure, enhancer-like flanking sequences, enzymatic RNA motifs, Ul, and U7 snRNP genes. The details of the molecular activities of the chimeric antisense RNAs will initially be studied in Xenopus oocytes and cultured mouse cells. Transgenic mice containing chimeric antisense genes will be constructed as a model for anti-sense gene therapy because no culture system can adequately mimic the complexity of cell function within intact mammals. The anti-sense inserts will be designed to be complementary to both a readily assayed CAT gene and to oncogenic murine retroviruses. Transgenic mouse lines will be screened for the production of chimeric RNA and then challenged with a target CAT gene (by crossing with a CAT transgenic mouse). We will quantify and characterize the degree of inhibition using the ectopic CAT system and then test the capacity of these constructs to suppress retroviral proliferation and pathogenesis in cultured cells and in whole mice. These studies will provide a critical test of the prospects of antisense RNA gene therapy for the treatment of mammalian retroviruses such as MSV and HIV.

反义RNA是一种有价值的策略，尽管常常不可预测。 实验性基因抑制以及潜在的合理方法 基因疗法。 我们建议研究复杂的嵌合体 将反义DNA片段插入构建的反义基因 结构RNA基因包括snRNA和tRNA基因。 新颖的设计 这些反义 RNA 利用了 snRNP 和 snRNP 的生物活性 tRNA 亲本基因并显示出显着的反义抑制活性 在培养细胞和转基因小鼠中。 我们将测试增强设计 嵌合基因，包括二级结构的改进， 增强子样侧翼序列、酶RNA基序、U1和U7 snRNP 基因。 嵌合反义分子活性的详细信息 RNA 最初将在爪蟾卵母细胞和培养的小鼠细胞中进行研究。 含有嵌合反义基因的转基因小鼠将被构建为 反义基因治疗的模型，因为没有任何培养系统可以 充分模拟完整哺乳动物细胞功能的复杂性。 反义插入片段将被设计为与 易于检测 CAT 基因和致癌鼠逆转录病毒。 转基因 将筛选小鼠品系以生产嵌合RNA，然后 受到目标 CAT 基因的攻击（通过与 CAT 转基因杂交） 老鼠）。 我们将使用以下方法量化和表征抑制程度 异位 CAT 系统，然后测试这些构建体的能力 抑制培养细胞中的逆转录病毒增殖和发病机制 整只老鼠。 这些研究将为前景提供严峻的考验 反义RNA基因疗法治疗哺乳动物逆转录病毒的研究 例如 MSV 和 HIV。