Gene therapy of Hepatitis C using monkey by siRNA

1)我们通过对siRNA寡核苷酸进行2' o甲基化、硫代化等化学修饰，成功地阻止了siRNA/阳离子脂质体静脉给药后干扰素的诱导，使剂量增加到20mg/kg。利用这种经过化学修饰的siRNA，对接种GBV-B的绢毛猴进行处理。然而，病毒滴度没有下降，尽管转氨酶有下降的趋势。2)作为一种新的siRNA载体，我们利用维生素E作为siRNA在体内的载体分子，具有通往大多数器官的生理转运途径。a-生育酚在27/29 mer siRNA的5'端与反义链（Toc-siRNA）共价结合。27/29分子量的Toc-siRNA被设计成在细胞质中被Dicer切割，在释放a-生育酚后产生21/21分子量成熟的siRNA。Toc-siRNA中a-生育酚的抗氧化活性被破坏。通过这种新的给药策略，全身和静脉注射临床相关剂量（2mg /kg）靶向载脂蛋白B（apoB）的Toc-siRNA可使肝脏内源性apoB mRNA减少约80%。通过观察肝脏脂滴积聚作为一种表型，证实了载脂蛋白ob的下调。在生化和病理分析中没有观察到干扰素的诱导和其他明显的副作用。这些发现证明了a-生育酚结合sirna在治疗疾病方面的治疗潜力。

1) We succeeded in preventing induction of interferon after intravenous administration of siRNA/cationic liposome by chemical modifications including 2'O-methylation, phosphorothionate to siRNA oligonucleotides, resulting in increase the dose up to 20mg/kg. With this new siRNA with chemical modifications, GBV-B inoculated tamarins were treated. However, viral titres were not decreased, although transaminases tended to be decreased.2)As a new siRNA vector, we use the vitamin E as a carrier molecule of siRNA in vivo which has -a physiological transport pathway to most of organs. The a-tocopherol was covalently bound to anti-sense strand of 27/29 mer siRNA at the 5' end (Toc-siRNA). The 27/29 mer Toc-siRNA was designed to be cleaved by Dicer in the cytosol and to produce 21/21 mer maturated siRNA after releasing a-tocopherol. The anti-oxidant activity of a-tocopherol in Toc-siRNA was abolished. With this new delivery strategy, systemic and intravenous injection of clinically relevant dose (2 mg/kg) of Toc-siRNA targeting apolipoprotein B(apoB) can reduce endogenous apoB mRNA in the liver by about 80%. The down-regulation of apoB was confirmed by the observation of lipid droplets accumulation in the liver as a phenotype. Neither induction of interferons nor other overt side effects were observed in biochemical and pathological analysis. These findings demonstrate the therapeutic potential of a-tocopherol-bound siRNAs for the treatment of disease.