Chemical Stabilization of shRNAs and their development as hepatitis C drugs

描述（由申请人提供）：丙型肝炎病毒（HCV）是一个全球性的健康问题，其治疗和预防仍然是一个重大挑战。现有的治疗方法只是部分有效，而且有严重的副作用。目前尚无疫苗可用。RNA干扰（RNAi）为治疗HCV感染提供了一种新的治疗方法。我们已经确定了一类称为sshrna的shrna，它们具有较小的长度，并且似乎通过与普通shrna或sirna不同的途径起作用。我们进一步发现了针对HCV内部核糖体进入位点（IRES）的非常有效的sshRNA (IC50 < 10 pM)， IRES是病毒RNA的一个高度保守和必需但“不可药物”的部分。在第一阶段，我们测试了大量这些抑制剂的化学修饰版本，以找到能够在不影响其活性的情况下在血清中稳定它们的修饰类型和模式。我们确定了有效修饰的规则，并发现这种修饰消除了未经修饰的sshrna对先天免疫系统的任何刺激，特别是如果它们是钝端。这些研究最近发表在《RNA》杂志上的两篇论文中。在第二阶段，我们将：1)使用第一阶段确定的规则合成我们的替代先导shRNA的化学修饰版本，并验证它们的效力和缺乏免疫刺激特性；2)寻找导联shrna的最佳组合，以减少shrna介导的HCV病毒耐药变异；3)使用小鼠报告细胞模型完成纳米颗粒配方的筛选，以确定提供最佳sshRNA肝脏递送的平台；4)研究铅配制的shRNA的毒性；5)在支持HCV感染的动物模型中验证铅配制shRNA的治疗效果。这项工作完成后，我们将处于非常有利的地位，可以吸引合作伙伴为剩余的IND临床前研究提供资金，提交IND申请，并进行临床试验。

DESCRIPTION (provided by applicant): Hepatitis C virus (HCV) is a worldwide health problem, and its treatment and prevention remain a major challenge. Existing therapies are only partially effective and have serious side effects. No vaccine is currently available. RNA interference (RNAi) offers a novel therapeutic approach for treating HCV infections. We have identified a class of shRNAs, called sshRNAs, which have smaller length and appear to act via a somewhat different pathway than ordinary shRNAs or siRNAs. We have further identified very potent sshRNA (IC50 < 10 pM) targeting the HCV internal ribosome entry site (IRES), a highly conserved and essential but "undruggable" part of the viral RNA. In Phase I, we tested a large number of chemically modified versions of these inhibitors to find types and patterns of modification that would stabilize them in serum without compromising their activity. We identified rules for effective modification and found that such modifications eliminate any stimulation of the innate immune system that can occur with unmodified sshRNAs, particularly if they are blunt-ended. These studies were recently published in two papers in RNA. In Phase II, we will: 1) synthesize chemically modified versions of our alternative lead shRNA using the rules identified in Phase I, and verify their potency and lack of immunostimulatory properties; 2) find optimal combinations of lead shRNAs to minimize shRNA-mediated HCV viral resistant variants; 3) complete screening of nanoparticle formulations to identify the platform that provides the best sshRNA delivery to liver using a mouse reporter model; 4) investigate the toxicity of lead formulated shRNA; and 5) verify the therapeutic efficacy of lead formulated shRNA in an animal model that supports HCV infection. Upon completion of this work, we will be in a very strong position to attract a partner to fund the remaining IND-enabling preclinical studies, file an IND, and proceed with clinical trials.