Chemical stabilization of shRNAs for therpeutic use

用于治疗用途的 shRNA 的化学稳定性

• 批准号: 7273776
• 负责人: Brian H. Johnston
• 金额: $ 16.56万
• 依托单位: SOMAGENICS, INC.
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-05-15 至 2008-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7273776
• 关键词: Adverse effects; Animals; Antiviral Agents; Biological; Biological Assay; Blood; Cells; Chemicals; Cholesterol; Chronic; Cirrhosis; Class; Cleaved cell; Collaborations; Compatible; Cultured Cells; Data; Development; Dicer Enzyme; Drug Formulations; Emerging Technologies; Encapsulated; Figs - dietary; Gene Expression; Genes; Goals; Half-Life; Hepatitis C virus; Human; Image; In Vitro; Incubated; Lead; Letters; Light; Lipids; Liver; Liver diseases; Location; Luciferases; Modification; Mus; Numbers; Pathway interactions; Patients; Pattern; Pharmaceutical Preparations; Phase; Positioning Attribute; Primary carcinoma of the liver cells; Process; Publishing; Pyrimidine; Pyrimidines; RNA; RNA Interference; RNA-Induced Silencing Complex; Rana; Replicon; Reporter; Reporting; Research; Resistance; Ribonucleases; Route; Safety; Screening procedure; Serum; Site; Small Interfering RNA; Study Section; System; Testing; Therapeutic; Therapeutic Uses; Time; Tissues; Universities; Vaccines; adeno-associated viral vector; anti-hepatitis C; base; density; drug development; human DICER1 protein; in vivo; inhibitor/antagonist; intravenous injection; nanoparticle; nuclease; pressure; prevent; programs; small hairpin RNA; vector

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.

描述（由申请人提供）：小干扰rna （sirna）和小发夹rna （shRNAs）是有效的基因抑制剂，已成功用于细胞培养。然而，到目前为止，它们在体内的应用受到了靶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，因此已经测试了许多稳定sirna的化学修饰。其中一些修饰在保持高生物活性的同时显著增加了稳定性。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，任何修饰都需要保持这种活性。虽然已知较长的shrna是由Dicer酶加工的，但一些结果表明，较短的shrna是通过一种独特但未知的途径加工的。我们已经鉴定出一种未经修饰的shRNA，它是一种有效的丙型肝炎病毒抑制剂。我们建议寻找稳定的化学修饰，以增加shRNA在血液中的半衰期，同时保持较高的生物活性。我们建议合成一组具有不同化学修饰模式的shRNA分子，测定它们在血清中的稳定性，然后在人类细胞的报告系统和HCV复制子系统中测试它们的活性。最成功地将核酸酶抗性与高效结合起来的稳定shrna将用于抗hcv药物开发计划。全世界有1.75亿人感染丙型肝炎病毒（HCV），其中70%的患者发展为慢性肝病，包括肝硬化和肝细胞癌。目前还没有疫苗，目前的治疗方法往往无效，而且有严重的副作用。RNA干扰（RNAi）是一种新兴技术，可以有效抑制基因表达，具有很大的治疗潜力。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。这项建议将决定如何有效地实现这种稳定。