Chemical Stabilization of shRNAs and their development as hepatitis C drugs

丙型肝炎病毒(丙型肝炎病毒)是一个世界性的健康问题，其治疗和预防仍然是一个重大挑战。现有的治疗方法只有部分有效，而且有严重的副作用。目前还没有疫苗可用。RNA干扰(RNAi)为治疗丙型肝炎病毒感染提供了一种新的治疗方法。我们已经确定了一类称为sshRNAs的shRNAs，它们的长度较小，似乎通过与普通shRNAs或siRNAs略有不同的途径发挥作用。我们进一步确定了针对丙型肝炎病毒内部核糖体进入位点(IRES)的非常有效的sshRNA(IC50和lt；10 PM)，这是病毒RNA的一个高度保守和必要的但“不能下药”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，以找到在不影响其活性的情况下稳定它们在血清中的修饰类型和模式。我们确定了有效修改的规则，发现这种修改消除了未修改的sshRNAs对先天性免疫系统的任何刺激，特别是如果它们是钝端的。这些研究最近在《RNA》杂志上发表了两篇论文。在第二阶段，我们将：1)使用第一阶段确定的规则合成经过化学修饰的替代铅shRNA，并验证其效力和缺乏免疫刺激特性；2)寻找铅shRNA的最佳组合，以最大限度地减少shRNA介导的丙型肝炎病毒耐药变体；3)通过小鼠报告模型对纳米制剂进行全面筛选，以确定提供最佳sshRNA给肝的平台；4)研究铅配方shRNA的毒性；以及5)在支持丙型肝炎感染的动物模型中验证铅配方shRNA的治疗效果。在这项工作完成后，我们将处于非常有利的地位，可以吸引合作伙伴为剩余的启用IND的临床前研究提供资金，提交IND，并继续进行临床试验。

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.