Gene therapy of Hepatitis C using monkey by siRNA

1)通过2‘O-甲基化、硫代磷酸等化学修饰，成功地阻止了静脉注射siRNA/阳离子脂质体后干扰素的诱导，使剂量增加到20 mg/kg。用这种经过化学修饰的新的siRNA处理了GBV-B接种的罗望子素。虽然转氨酶有下降的趋势，但病毒滴度并没有下降。2)作为一种新的siRNA载体，我们使用维生素E作为体内siRNA的载体分子，它有一条生理转运途径到达大部分器官。α-生育酚在5‘端与27/29聚体siRNA的反义链(Toc-siRNA)共价结合。27/29聚体Toc-siRNA被设计为在胞浆中被DICER切割，并在释放α-生育酚后产生21/21聚体成熟的siRNA。Toc-siRNA中的α-生育酚的抗氧化活性被取消。在这种新的递送策略下，全身和静脉注射临床相关剂量(2 mg/kg)的针对载脂蛋白B(ApoB)的Toc-siRNA可以使肝脏内源性apoB mRNA减少约80%。通过观察脂滴在肝脏中的积聚，证实了apoB的下调。生化和病理分析均未发现干扰素的诱导和其他明显的副作用。这些发现证明了α-生育酚结合的siRNAs在疾病治疗中的治疗潜力。

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.