Inhibition of hepatitis C by RNA-based therapeutics

描述（由申请人提供）：治疗和预防丙型肝炎病毒（HCV）感染仍然是控制这一全球卫生问题的主要挑战；现有的治疗方法仅部分有效，目前也没有疫苗。RNA干扰为治疗HCV感染提供了一种新的治疗方法。为此，我们正在开发针对HCV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 （shrna）和修饰sirna。一期实验利用报告基因质粒，其中萤火虫荧光素酶（fLuc）的表达依赖于HCV IRES。直接转染HCV IRES shrna，或通过pol - ill启动子载体表达shrna，在人肝细胞和小鼠模型中有效阻断HCV IRES介导的fLuc表达，其中核酸通过尾静脉通过流体动力学转染传递到肝脏细胞。体内生物发光成像显示，HCVwt shRNA在小鼠模型中抑制HCV ires依赖性报告基因表达达99%；双重突变或不相关的shrna影响很小或没有影响。II期以I期成果为基础，开发有效的方法将抑制剂递送到肝脏：1)衍生sirna以促进肝脏靶向和摄取；2)制备腺相关病毒（AAV）血清型，将shrna特异性递送到肝脏。将对shRNA、sirna（未修饰和修饰）和aav递送的shRNA进行并行实验，以确定阻断HCV ires依赖性基因表达的能力。由于HCV不会感染组织培养细胞，在进行大规模临床前动物研究之前，将在稳定表达HCV复制子的人肝细胞中测试铅抑制剂。单光发射计算机断层扫描（SPECT）的使用允许非侵入性成像，从而优化递送和si/shRNA靶向HCV ires依赖性生长抑素受体-2报告基因在小鼠和猴子等大型动物中的表达。在研究HCV感染动物模型（包括慢性HCV感染的非人灵长类动物模型和嵌合KMT小鼠模型）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.