Inhibition of hepatitis C by RNA-based therapeutics

描述(申请人提供)：丙型肝炎病毒(丙型肝炎病毒)感染的治疗和预防仍然是控制这一世界性健康问题的主要挑战；现有的治疗方法只有部分有效，目前还没有疫苗可用。RNA干扰为治疗丙型肝炎病毒感染提供了一种新的治疗方法的潜力。为此，我们正在开发针对丙型肝炎病毒基因组保守的内部核糖体进入位点(IRES)元件的小发夹干扰RNA(ShRNAs)和修饰的siRNAs。第一阶段实验利用了一个报告基因质粒，其中萤火虫荧光素酶(Fluc)的表达依赖于丙型肝炎病毒IRES。直接转染丙型肝炎病毒IRES shRNAs，或从PolIll启动子载体表达的shRNAs，有效地阻断了人肝细胞和小鼠模型中丙型肝炎病毒IRES介导的flc的表达，在该模型中，核酸通过尾静脉流体动力传递到肝细胞中。体内生物发光成像显示，在小鼠模型中，HCVwt shRNA对依赖于丙型肝炎病毒IRES的报告基因的表达抑制高达99%；双突变或无关的shRNAs几乎或没有影响。第二阶段在第一阶段成果的基础上，通过1)衍生化siRNAs以促进肝脏靶向和摄取，以及2)制备将shRNAs特异性地运送到肝脏的腺相关病毒(AAV)血清型，以开发高效的向肝脏递送抑制剂的方法。将对shRNA、siRNAs(未修饰和修饰)和AAV递送的shRNAs进行并列实验，以确定阻止丙型肝炎病毒IRES依赖的基因表达的能力。由于丙型肝炎病毒不会感染组织培养细胞，因此在进行大规模临床前动物研究之前，将在稳定表达丙型肝炎病毒复制子的人肝细胞中测试铅抑制剂。使用单光子发射计算机断层扫描(SPECT)可以进行非侵入性成像，从而优化了丙型肝炎病毒IRES依赖的生长抑素受体2报告基因在小鼠和大型动物(如猴子)中的递送和si/shRNA靶向表达。在研究丙型肝炎病毒感染动物模型(包括慢性感染丙型肝炎病毒的非人灵长类动物和嵌合的KMT小鼠模型)中的抑制剂之前，将在兔子和猴子身上进行药代动力学和毒理学实验。

DESCRIPTION (provided by applicant): Treatment and prevention of hepatitis C virus (HCV) infections remains a major challenge for controlling this worldwide health problem; existing therapies are only partially effective and no vaccine is currently available. RNA interference offers the potential of a novel therapeutic approach for treating HCV infections. Toward this end, we are developing small hairpin interfering RNAs (shRNAs) and modified siRNAs targeting the conserved internal ribosome entry site (IRES) element of the HCV genome. Phase I experiments utilized a reporter gene plasmid in which firefly luciferase (fLuc) expression is dependent on the HCV IRES. Direct transfection of HCV IRES shRNAs, or alternatively shRNAs expressed from pol Ill-promoter vectors, efficiently blocked HCV IRES-mediated fLuc expression in human hepatocytes and a mouse model where nucleic acids were delivered to cells in the liver by hydrodynamic transfection via the tail vein. In vivo bioluminescent imaging revealed that HCVwt shRNA inhibited HCV IRES-dependent reporter gene expression up to 99% in the mouse model; doubly mutated or irrelevant shRNAs had little or no effect. Phase II builds on the Phase I accomplishments to develop efficient methods for inhibitor delivery to liver by 1) derivatizing siRNAs to facilitate liver targeting and uptake and 2) preparation of adeno-associated virus (AAV) serotypes that deliver shRNAs specifically to the liver. Side-by-side experiments with shRNA, siRNAs (unmodified and modified) and AAV-delivered shRNAs will be performed to determine ability to block HCV IRES-dependent gene expression. As HCV does not infect tissue culture cells, lead inhibitors will be tested in human hepatocytes stably expressing HCV replicons prior to engaging in large scale pre-clinical animal studies. The use of single photo emission computed tomography (SPECT) allows non-invasive imaging and hence optimization of delivery and si/shRNA targeting of HCV IRES-dependent somatostatin receptor-2 reporter expression in mice as well as large animals such as monkeys. Pharmacokinetic and toxicology experiments will be done in rabbits and monkeys prior to investigating the inhibitors in HCV-infected animal models, including non-human primates chronically infected with HCV and the chimeric KMT mouse model.