Development of siRNA libraries for antiviral discovery

开发用于抗病毒发现的 siRNA 文库

• 批准号: 6789086
• 负责人: ROGER L KASPAR
• 金额: $ 29.47万
• 依托单位: SOMAGENICS, INC.
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-04-01 至 2005-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6789086
• 关键词: RNA; RNA interference; antiviral agents; biotechnology; cell line; dengue virus; drug discovery /isolation; ganciclovir; gene induction /repression; gene targeting; genetic library; herpes simplex virus 1; high throughput technology; messenger RNA; method development; nucleic acid purification; nucleic acid sequence; polymerase chain reaction; small interfering RNA; thymidine kinase; transfection

The discovery and development of RNA interference and especially the breakthrough of using small interfering RNAs (siRNA) to inhibit gene expression is revolutionizingbiology and opening new avenues for developing novel therapeutics. Despite the current usefulness of the technology, a bottleneck exists for quickly finding effective siRNA target sites in a high throughput-like manner and most researchers currently employ a rational trial-and-error design method. In this proposal, we present a novel cell-based technology to identify effective inhibitors from siRNA libraries (either random or directed). The combination of using a library to find the most effective, robust siRNAs against accessible sites on any given target within a cellular environment, coupled with the inhibitor activity intrinsic to siRNAs, holds great promises in finding effective therapeutics. We have chosen dengue virus, a NIAID Class A threat, as our initial target for developing this technology. Potential target dengue viral sequences will be linked to the HSV thymidine kinase (TK) gene and stably expressed in human cells. Addition of ganciclovir leads to cell killing of any cell expressing the HSV TK protein. Prior to addition of ganciclovir, a proprietary library of siRNAs designed to only target dengue sequences ("directed" library)will be introduced into the cells. Those cells that subsequently survive the killing challenge will be isolated and the rescued siRNAs identified. Among the purified and validated inhibitorsidentified, the most potent anti-dengue siRNAs will be further tested for their anti-dengue viral activity in cell-based and animal models. This approach could potentially be extended to any RNA target of interest, including other viruses and oncogene mRNAs.

RNA干扰的发现和发展，尤其是使用小型干扰RNA（siRNA）抑制基因表达的突破是革命性的生物学，并为开发新的疗法提供了新的途径。尽管该技术目前具有有用性，但仍存在一种以高吞吐量的方式快速找到有效的siRNA目标站点的瓶颈，而大多数研究人员目前采用了合理的试用和纠正设计方法。在此提案中，我们提出了一种基于细胞的新型技术，可确定来自siRNA库（随机或指示）的有效抑制剂。使用 图书馆为在细胞环境中的任何给定靶标上找到最有效，最强大的siRNA，并与siRNA固有的抑制剂活性一起在任何给定目标上找到可访问的位点，在寻找有效的疗法方面具有巨大的承诺。我们选择了NIAID A类威胁的登革热病毒作为开发这项技术的最初目标。潜在靶登革热病毒序列将与HSV胸苷激酶（TK）基因相关，并在人类细胞中稳定表达。添加Ganciclovir会导致任何表达HSV TK蛋白的细胞杀死细胞。在添加Ganciclovir之前，将将仅针对登革热序列的siRNA库（“定向”文库）引入细胞中。那些随后生存的细胞 杀人挑战将被隔离，并确定被救出的siRNA。在纯化和验证的抑制剂识别中，最有效的抗登记siRNA将进一步测试其在基于细胞和动物模型中的抗登记性病毒活性。这种方法可能会扩展到任何感兴趣的RNA靶标，包括其他病毒和癌基mRNA。