CCR RNAi Initiative: Validation of siRNAs Against Cancer-Associated Genes

CCR RNAi 计划：针对癌症相关基因验证 siRNA

• 批准号: 7592799
• 负责人: natasha caplen
• 金额: $ 34.91万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 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/7592799
• 关键词: Antineoplastic Agents; Aspartate-Ammonia Ligase; Biological; Biological Assay; Biological Markers; Biological Models; CCR; Cells; Chronic Lymphocytic Leukemia; Clinical effectiveness; Consensus; Data; Development; Drug Interactions; Drug usage; Enzymes; Exhibits; Failure; Gene Expression; Gene Targeting; Genes; Genomics; Growth; Human; Investigation; Lead; Legal patent; Malignant Neoplasms; Manuscripts; Mediating; Molecular; Molecular Target; Nucleic Acids; Pathway interactions; Pharmaceutical Preparations; Phenotype; Process; Protein Complex Subunit; Proteins; Publishing; RNA Interference; Range; Reporting; Research; Single Nucleotide Polymorphism; Small Interfering RNA; Speed; Therapeutic; Transcript; United States Food and Drug Administration; Validation; Variant; Work; asparaginase; concept; design; enzyme activity; gene function; novel

To date we have fully assayed the knockdown mediated by 258 siRNAs corresponding to 129 human genes. Approximately 70% of these siRNAs show a 50% decrease in the steady state levels of the expression of the gene under study. This data was published in 2007 in Nucleic Acids Research. We have conducted extensive study of the reasons why some siRNAs fail to silence and have established that single nucleotide polymorphisms, errors and changes in the consensus transcript and genomic sequences used for the design of siRNAs and differential expression of transcript variants can all contribute to the seeming failure of an siRNA to mediate RNAi. The following describes one example of the impact of this validation process. Using synthetic siRNAS corresponding to the enzyme asparagine synthetase we had previously validated Dr. John Weinsteins group (LMP, CCR) were able to rapidly assess a functional relationship between ASNS expression and the activity of the enzyme drug L-asparaginase (L-ASP). This work has lead to the filing of a patent application and a manuscript describing this work in detail is In Press. This study showed that treatment of cells with siRNAs targeted against ASNS reduces ASNS expression and potentiates the growth inhibitory activity of L-asparaginase (L-ASP), a FDA approved drug used for the treatment of chronic lymphocytic leukemia. This data suggests that L-ASP treatment could be applied to other cancers and suggest that ASNS could be used as a biomarker for the clinical effectiveness of L-ASP. This study presents a paradigm for the use of RNAi analysis to further pharmocogenomic studies and has recently been published in Molecular Cancer Therapeutics. We also investigated the ability of siRNAs to simultaneously decrease the expression of multiple genes. Our concept was to determine if siRNA-mediated RNAi could be used more extensively, than currently reported, for the study of multi subunit protein complexes, proteins exhibiting functional redundancy and for investigation of interactions within biological pathways and networks. This type of analysis could be especially valuable for the development of anti-cancer drugs, as it could assist in identifying molecular target combinations that generate particularly lethal phenotypes. We determined that at least six different gene targets could be simultaneously silenced using well-characterized synthetic siRNAs. This work has been recently published. On-going studies are further investigating factors that may influence the efficacy of synthetic siRNA mediated RNAi.

迄今为止，我们已经全面分析了对应 129 个人类基因的 258 个 siRNA 介导的敲低。这些 siRNA 中大约 70% 显示所研究基因的稳态表达水平降低了 50%。该数据于 2007 年发表在《核酸研究》上。我们对一些 siRNA 无法沉默的原因进行了广泛的研究，并确定用于 siRNA 设计的共有转录本和基因组序列中的单核苷酸多态性、错误和变化以及转录本变体的差异表达都可能导致 siRNA 介导 RNAi 的看似失败。下面描述了这一验证过程的影响的一个示例。使用与天冬酰胺合成酶相对应的合成 siRNA，我们之前已验证 John Weinsteins 博士小组（LMP、CCR）能够快速评估 ASNS 表达与酶药物 L-天冬酰胺酶 (L-ASP) 活性之间的功能关系。这项工作已经提交了专利申请，详细描述这项工作的手稿正在出版。这项研究表明，用针对 ASNS 的 siRNA 处理细胞可减少 ASNS 表达，并增强 L-天冬酰胺酶 (L-ASP) 的生长抑制活性，L-ASP 是 FDA 批准用于治疗慢性淋巴细胞白血病的药物。该数据表明 L-ASP 治疗可应用于其他癌症，并表明 ASNS 可用作 L-ASP 临床有效性的生物标志物。这项研究提出了使用 RNAi 分析进一步进行药物基因组学研究的范例，并于最近发表在《分子癌症治疗》杂志上。我们还研究了 siRNA 同时降低多个基因表达的能力。我们的想法是确定 siRNA 介导的 RNAi 是否可以比目前报道的更广泛地用于研究多亚基蛋白质复合物、表现出功能冗余的蛋白质以及研究生物途径和网络内的相互作用。这种类型的分析对于抗癌药物的开发特别有价值，因为它可以帮助识别产生特别致命表型的分子靶标组合。我们确定使用充分表征的合成 siRNA 可以同时沉默至少 6 个不同的基因靶标。这部作品最近出版了。正在进行的研究正在进一步调查可能影响合成 siRNA 介导的 RNAi 功效的因素。