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）：小型干扰RNA（siRNA）和小发夹RNA（shRNA）是成功用于细胞培养的有效基因抑制剂。但是，到目前为止，他们在体内的应用受到了靶向组织的递送不足的限制。由于RNA容易受到核糖核酸酶的降解，尤其是在血液中，因此已经测试了许多siRNA的稳定化学修饰。其中一些修饰为稳定性急剧提高，同时保持了高生物学活性。但是，没有关于ShRNA化学修饰的报道，并且修改的选择和位置并不明显。这是因为与siRNA不同，SHRNA需要酶促加工来裂解末端环，并且任何修改都需要保留该活动。虽然众所周知，较长的shRNA是由酶迪切尔（酶）处理的，但某些结果表明，短shrnas是通过一个独特但未知的途径处理的。我们已经确定了一个未修饰的shRNA，该shRNA是丙型肝炎病毒的有效抑制剂。我们建议寻找稳定化学修饰，这将增加血液中的shRNA半衰期，同时保持高生物学活性。我们建议通过各种化学修饰模式合成一组SHRNA分子，测定它们在血清中的稳定性，然后在人类细胞中测试其在报告基因系统和HCV复制子系统中的活性。抗HCV药物开发计划中，最成功地结合核酸酶耐药性与高效力的稳定shRNA将使用。 S丙型肝炎病毒（HCV）在全球感染了1.75亿人，其中70％的患者患有慢性肝病，包括肝硬化和肝细胞癌。没有疫苗，目前的处理通常无效，并且具有严重的副作用。 RNA干扰（RNAi）是一种新兴的技术，可有效抑制基因表达，具有巨大的治疗使用潜力。 SHRNA是一类有希望的RNAi药物，但是它们的使用需要化学稳定以防止其体内降解。该建议将决定如何有效完成此稳定。