Tacere Therapeutics, San Jose, California, USA.
Mol Ther. 2012 Sep;20(9):1737-1749. doi: 10.1038/mt.2012.119. Epub 2012 Jun 26.
The hepatitis C virus (HCV) chronically infects 2% of the world population and effective treatment is limited by long duration and significant side-effects. Here, we describe a novel drug, intended as a "single-shot " therapy, which expresses three short hairpin RNAs (shRNAs) that simultaneously target multiple conserved regions of the HCV genome as confirmed in vitro by knockdown of an HCV replicon system. Using a recombinant adeno-associated virus (AAV) serotype 8 vector for delivery, comprehensive transduction of hepatocytes was achieved in vivo in a nonhuman primate (NHP) model following a single intravenous injection. However, dose ranging studies performed in 13 NHP resulted in high-expression levels of shRNA from wild-type (wt) Pol III promoters and dose-dependent hepatocellular toxicity, the first demonstration of shRNA-related toxicity in primates, establishing that the hepatotoxicity arises from highly conserved features of the RNA interference (RNAi) pathway. In the second generation drug, each promoter was re-engineered to reduce shRNA transcription to levels that circumvent toxicity but still inhibit replicon activity. In vivo testing of this modified construct in 18 NHPs showed conservation of hepatocyte transduction but complete elimination of hepatotoxicity, even with sustained shRNA expression for 50 days. These data support progression to a clinical study for treatment of HCV infection.
丙型肝炎病毒（HCV）慢性感染全球人口的 2%，有效的治疗方法受到治疗周期长和严重副作用的限制。在这里，我们描述了一种新型药物，旨在作为一种“单次给药”疗法，该药物表达了三种短发夹 RNA（shRNA），可通过体外 HCV 复制子系统的敲低来同时靶向 HCV 基因组的多个保守区域。使用重组腺相关病毒（AAV）血清型 8 载体进行递送，在单次静脉注射后，在非人类灵长类动物（NHP）模型中实现了对肝细胞的全面转导。然而，在 13 只 NHP 中进行的剂量范围研究导致了来自野生型（wt）Pol III 启动子的 shRNA 的高表达水平和剂量依赖性肝细胞毒性，这是首次在灵长类动物中证明 shRNA 相关毒性，表明这种肝毒性来自 RNA 干扰（RNAi）途径的高度保守特征。在第二代药物中，每个启动子都经过重新设计，以降低 shRNA 的转录水平，从而避免毒性，但仍能抑制复制子活性。在 18 只 NHP 中对该改良构建体进行的体内测试表明，肝细胞转导得以保留，但即使在 50 天内持续表达 shRNA，也完全消除了肝毒性。这些数据支持进行 HCV 感染治疗的临床研究。
