Chemical stabilization of shRNAs for therpeutic use

描述(申请人提供)：小干扰RNAs(SiRNAs)和小发夹RNAs(ShRNAs)是已成功用于细胞培养的有效基因抑制剂。然而，到目前为止，它们在体内的应用一直受到靶向组织递送不足的限制。由于RNA容易被核糖核酸酶降解，特别是在血液中，一些稳定的化学修饰已经针对siRNAs进行了测试。其中一些修饰在保持较高生物活性的同时，显著提高了稳定性。然而，关于shRNA的化学修饰还没有报道，修饰的选择和位置也不是很明显。这是因为，与siRNAs不同的是，shRNA需要酶处理来切割末端环，而任何修饰都需要保持这种活性。虽然众所周知，较长的shRNA是由Dester酶处理的，但一些结果表明，较短的shRNA是由一条不同的但未知的途径处理的。我们已经确定了一种未经修饰的shRNA，它是一种有效的丙型肝炎病毒抑制物。我们建议寻找稳定的化学修饰，在保持高生物活性的同时增加血液中shRNA的半衰期。我们建议合成一组具有不同化学修饰模式的shRNA分子，检测它们在血清中的稳定性，然后在报告系统和丙型肝炎病毒复制子系统中测试它们的活性，这两个系统都在人类细胞中。最成功地将核酸酶抗性与高效性结合在一起的稳定的shRNA将用于抗丙型肝炎病毒药物开发计划。S丙型肝炎病毒感染全球1.75亿人，70%的患者发展为慢性肝病，包括肝硬变和肝细胞癌。没有疫苗，目前的治疗方法往往无效，并有严重的副作用。RNA干扰(RNAi)是一种能够有效抑制基因表达的新兴技术，具有巨大的治疗应用潜力。ShRNA是一类很有前途的RNAi药物，但它们的使用需要化学稳定，以防止它们在体内降解。这项提议将决定如何有效地实现这一稳定。

DESCRIPTION (provided by applicant): Small interfering RNAs (siRNAs) and small hairpin RNAs (shRNAs) are potent gene-inhibiting agents that have been successfully used in cell culture. However, so far their application in vivo has been limited by insufficient delivery to target tissues. Because RNAs are susceptible to degradation by ribonucleases, particularly in the blood, a number of stabilizing chemical modifications have been tested for siRNAs. Some of these modifications have provided a dramatic increase of stability while maintaining high biological activity. However, there are no reports on the chemical modification of shRNA, and the choice and location of modifications is not obvious. This is because, unlike siRNAs, shRNA require enzymatic processing to cleave the terminal loop, and any modifications need to preserve that activity. While it is known that longer shRNAs are processed by enzyme Dicer, some results indicate that short shRNAs are processed by a distinct but unknown pathway. We have identified an unmodified shRNA that is a potent inhibitor of hepatitis C virus. We propose to search for stabilizing chemical modifications that will increase the shRNA half-life in blood while maintaining high biological activity. We propose to synthesize a set of shRNA molecules with various patterns of chemical modification, assay their stability in blood serum, then test their activity in a reporter system and in an HCV replicon system, both in human cells. The stabilized shRNAs that most successfully combine nuclease resistance with high potency will be used in an anti-HCV drug development program. s Hepatitis C virus (HCV) infects 175 million people worldwide, with 70% of patients developing chronic liver disease, including cirrhosis and hepatocellular carcinoma. There is no vaccine and current treatments are often ineffective and have severe side-effects. RNA interference (RNAi) is a newly emerging technology allowing potent inhibition of gene expression, with great potential for therapeutic use. shRNA are a promising class of RNAi drugs, but their use requires chemical stabilization to prevent their degradation in the body. This proposal will determine how to effectively accomplish this stabilization.