Chemical stabilization of shRNAs for therpeutic use

描述（由申请人提供）：小干扰RNA（siRNA）和小发夹RNA（shRNA）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，它们在体内的应用受到不充分递送至靶组织的限制。由于RNA易受核糖核酸酶降解，特别是在血液中，因此已经测试了siRNA的许多稳定化学修饰。这些修饰中的一些提供了稳定性的显著增加，同时保持了高的生物活性。然而，目前还没有关于shRNA化学修饰的报道，修饰的选择和位置也不明显。这是因为，与siRNA不同，shRNA需要酶处理来切割末端环，并且任何修饰都需要保持这种活性。虽然已知较长的shRNA由酶Dicer加工，但一些结果表明短的shRNA由独特但未知的途径加工。我们已经鉴定出一种未经修饰的shRNA，它是丙型肝炎病毒的有效抑制剂。我们建议寻找稳定的化学修饰，增加血液中的shRNA半衰期，同时保持高的生物活性。我们建议合成一组具有各种化学修饰模式的shRNA分子，测定其在血清中的稳定性，然后在人类细胞中的报告系统和HCV复制子系统中测试其活性。最成功地将联合收割机核酸酶抗性与高效力相结合的稳定化的shRNA将用于抗HCV药物开发计划。丙型肝炎病毒（HCV）感染全球1.75亿人，其中70%的患者发展为慢性肝病，包括肝硬化和肝细胞癌。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（RNA interference，RNAi）是近年来发展起来的一种有效抑制基因表达的技术，具有广阔的应用前景。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。这项建议将确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.