Gene therapy of Hepatitis C using monkey by siRNA

我们合成了针对GBV-B基因组序列的siRNA寡核苷酸，它与对应的丙型肝炎病毒基因组序列只有两个核苷酸的差异。该位点选自GBV-B/丙型肝炎病毒基因组中最保守的5‘非翻译区，使siRNA对病毒的突变具有抗性。为了将siRNA运送到肝脏，我们开发了一种阳离子脂质体LIC-101，它由新的阳离子脂类类似物组成。用LIC-101配制的针对GBV-B的1.0、2.5和5.0 mg/kg的siRNA从恒河猴的隐静脉缓慢注射，连续3天。第二天，将GBV-B病毒颗粒直接注射到肝脏。我们观察到GBV-B siRNA/LIC101对GBV-B病毒的复制有显著的抑制作用，5.0 mg/kg的siRNA/LIC-101在接种病毒后4个月以上完全抑制了GBV-B的复制，即使只注射了3天。没想到，对照siRNA/LIC-101还能延缓病毒复制。由于对照小干扰RNA/LIC-101诱导了血清干扰素α和γ，因此，对照小干扰RNA/LIC-101对病毒复制的延迟可能是由于诱导的干扰素的抗病毒作用所致。相反，GBV-B siRNA/LIC-101产生的干扰素明显少于对照siRNA/LIC-101。因此，我们认为GBV-B siRNA/LIC-101的抑制作用包括siRNA和干扰素的作用。由于脂质载体合成的siRNA是通过位于内膜的Toll样受体识别的，并以序列依赖的方式激活免疫系统。综上所述，我们成功地利用siRNA抑制了GBV-B在猴肝脏中的复制。我们正在尝试抑制干扰素反应的新方法，以避免副作用。

We synthesized the siRNA oligonucleotides targeting to the of GBV-B genome which is different from that of the corresponding sequence of HCV genome by only two nucleotides. This site was selected from 5' untranslated region, the most conservative region in the GBV-B/HCV genome, making the siRNA resistant to the mutation of the virus. For the delivery of the siRNA to the liver, we have developed a cationic liposome, LIC-101, which consists of new novel cationic lipid analogue.The five marmosets were used in this study. The 1.0, 2.5 and 5.0mg/kg of siRNA to GBV-B formulated by LIC-101 were slowly administered from the saphenous vein of the marmosets for three days. On the second day, GBV-B virus particles were directly injected to the liver. We could observe the dramatic and dose-dependent effect of the GBV-B siRNA/LIC101 to inhibit the replication GBV-B virus ; The 5.0mg/kg siRNA/LIC-101 completely suppressed the replication of GBV-B for more than 4 months after inoculation of the virus, even though the siRNA was injected for only three days. Unexpectedly, the control siRNA/LIC-101also can delay the virus replication. Since the control siRNA/LIC-101 induced the serum interferons α and γ, this delay of virus replication by the control siRNA/LIC-101 on the viral replication should be caused by an anti-viral effect of the induced interferons. In contrast, GBV-B siRNA/LIC-101 induced much less interferons than the control siRNA/LIC-101. Therefore, inhibitory effect of GBV-B siRNA/LIC-101 was considered to include those by both siRNA and interferons. Since synthetic siRNA formulated with lipid vectors is recognized through toll-like receptors located in the endosomal membrane and activates immune system in a sequence dependent manner.In conclusion, we succeeded to inhibit replication of GBV-B in liver of monkey with siRNA. We are trying the new method to suppress interferon response to escape the side effect.