Gene therapy of Hepatitis C using monkey by siRNA

1)我们成功地通过化学修饰（包括2 'O-甲基化、硫代磷酸酯化到siRNA寡核苷酸，导致剂量增加至20 mg/kg）来防止静脉内施用siRNA/阳离子脂质体后干扰素的诱导。用这种具有化学修饰的新siRNA处理GBV-B接种的绢毛猴。2）作为一种新的siRNA载体，我们使用维生素E作为siRNA的载体分子，它具有一个生理性的转运途径，可以到达大多数器官。α-生育酚在5'端共价结合至27/29 mer siRNA的反义链（Toc-siRNA）。27/29 mer Toc-siRNA被设计成在胞质溶胶中被Dicer切割，并在释放α-生育酚后产生21/21 mer成熟的siRNA。Toc-siRNA中α-生育酚的抗氧化活性被消除。通过这种新的递送策略，全身和静脉内注射临床相关剂量（2 mg/kg）的靶向载脂蛋白B（apo B）的Toc-siRNA可使肝脏中的内源性apo B mRNA减少约80%。通过观察肝脏中的脂滴积聚作为表型证实了apoB的下调。在生物化学和病理学分析中均未观察到干扰素的诱导作用和其他明显的副作用。这些发现证明了α-生育酚结合的siRNA用于治疗疾病的治疗潜力。

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.