Improved Delivery Methods for Small Interfering RNAs

描述(申请人提供)：短链干扰RNA(SiRNA)和短发夹状RNA(ShRNA)都是有效的基因沉默试剂，已成功用于细胞培养。然而，到目前为止，它们在体内的应用一直受到RNA稳定性低和递送效率低的限制。将胆固醇基团连接到反义寡核苷酸上可以改善其转运，最近的研究表明，连接到siRNAs正义链5‘端的胆固醇可以促进培养细胞和活着的小鼠肝脏的摄取。在这项建议中，我们开发了通过shRNAs增加肝脏递送和摄取的方法。与直接进入RISC复合体的siRNA不同，shRNA必须首先被Dier酶处理，才能切割发夹环。因此，任何化学修饰都不能阻碍其作为底物的能力。所有的修改都将在SomaGenics公司发现的一种有效的丙型肝炎病毒shRNA抑制剂上进行测试，以提高在人类肝细胞系和小鼠肝脏模型中抑制丙型肝炎病毒介导的报告表达的能力，通过全动物成像进行测试。根据SomaGenics与NIAID抗病毒筛查计划的现有协议，他们还将在更现实的丙型肝炎病毒细胞模型--丙型肝炎病毒复制子--上进行测试。

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.