Improved Delivery Methods for Small Interfering RNAs

改进小干扰 RNA 的递送方法

• 批准号: 7053683
• 负责人: Brian H. Johnston
• 金额: $ 29.74万
• 依托单位: SOMAGENICS, INC.
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-05-26 至 2008-05-25
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7053683
• 关键词: antisense nucleic acid; biotechnology; cell line; chemical conjugate; cholesterol; gene expression; hepatitis C virus; human genetic material tag; laboratory mouse; reporter genes; small interfering RNA; technology /technique development

DESCRIPTION (provided by applicant): Both short interfering RNA (siRNA) and short hairpin RNA (shRNA) are potent gene-silencing agents that have been successfully used in cell culture. However, so far their application in vivo has been limited by the low stability of RNA and poor efficiency of delivery. The attachment of cholesterol groups to antisense oligonucleotides has been shown to improve their delivery, and it was recently demonstrated that cholesterol attached to the 5'-termini of the sense strands of siRNAs improve uptake both in cultured cells and in livers of living mice. In this proposal we develop methods to increase liver delivery and uptake by shRNAs. In contrast to siRNA, which directly goes to RISC complex, shRNA must be first processed by the enzyme Dicer to cleave the hairpin loop. Hence any chemical modifications must not block its ability to act as a substrate for Dicer. All modifications will be tested on a potent shRNA inhibitor of HCV discovered at SomaGenics for improved ability to inhibit HCV-mediated reporter expression both in a human hepatocyte cell line and in a mice liver model, assayed by whole-animal imaging. They will be also tested on a more realistic cell-based model of HCV, the HCV replicon, under SomaGenics' existing agreement with the NIAID antivirial screening program.

描述（由申请人提供）：短干扰RNA（siRNA）和短发夹RNA（shRNA）都是有效的基因沉默剂，已成功用于细胞培养。然而，迄今为止，它们在体内的应用受到RNA稳定性低和递送效率差的限制。胆固醇基团与反义寡核苷酸的附着已被证明可以改善其递送，并且最近证明附着在 siRNA 有义链 5' 末端的胆固醇可改善培养细胞和活小鼠肝脏中的摄取。在本提案中，我们开发了增加 shRNA 肝脏递送和摄取的方法。与直接进入 RISC 复合体的 siRNA 不同，shRNA 必须首先由 Dicer 酶加工以裂解发夹环。因此，任何化学修饰都不能阻止其作为 Dicer 底物的能力。所有修饰都将在 SomaGenics 发现的 HCV 有效 shRNA 抑制剂上进行测试，以提高抑制人肝细胞系和小鼠肝脏模型中 HCV 介导的报告基因表达的能力，并通过整体动物成像进行测定。根据 SomaGenics 与 NIAID 抗病毒筛查计划的现有协议，它们还将在更真实的 HCV 细胞模型（HCV 复制子）上进行测试。