Development of siRNA libraries for antiviral discovery

RNA干扰技术的发现和发展，特别是利用小干扰RNA（siRNA）抑制基因表达的突破，正在给生物学带来革命性的变化，并为开发新的治疗药物开辟了新的途径。尽管该技术目前很有用，但以高通量的方式快速找到有效的siRNA靶位点存在瓶颈，大多数研究人员目前采用合理的试错设计方法。在这个建议中，我们提出了一种新的基于细胞的技术，以确定有效的抑制剂从siRNA库（随机或定向）。使用一个 通过构建siRNA文库来寻找针对细胞环境内任何给定靶标上的可接近位点的最有效、最稳健的siRNA，加上siRNA固有的抑制剂活性，在寻找有效治疗剂方面具有很大的希望。我们选择了登革热病毒，一种NIAID A级威胁，作为我们开发这项技术的最初目标。潜在的靶登革病毒序列将与HSV胸苷激酶（TK）基因连接并在人细胞中稳定表达。添加更昔洛韦导致任何表达HSV TK蛋白的细胞的细胞杀伤。在添加更昔洛韦之前，将设计成仅靶向登革热序列的siRNA的专有文库（“定向”文库）引入细胞中。那些随后存活下来的细胞 将分离杀伤激发物并鉴定拯救的siRNA。在已鉴定的纯化和验证的siRNA中，最有效的抗登革热siRNA将在细胞和动物模型中进一步测试其抗登革热病毒活性。这种方法可能扩展到任何感兴趣的RNA靶标，包括其他病毒和致癌基因mRNA。

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.