Gene therapy of Hepatitis C using monkey by siRNA

我们合成了针对GBV-B基因组的siRNA寡核苷酸，该siRNA寡核苷酸与HCV基因组的相应序列仅相差两个核苷酸。该位点选自GBV-B/HCV基因组中最保守的5'非翻译区，使siRNA能够抵抗病毒的突变。为了将siRNA递送到肝脏，我们开发了一种阳离子脂质体，LIC-101，它由一种新的阳离子脂质类似物组成。这5只狨猴被用于这项研究。用li -101配制的siRNA 1.0、2.5和5.0mg/kg /kg从狨猴的隐静脉中缓慢给药3天。第二天，将GBV-B病毒颗粒直接注射到肝脏。我们可以观察到GBV-B siRNA/LIC101抑制GBV-B病毒复制的显著剂量依赖性效应；5.0mg/kg siRNA/LIC-101在接种病毒后完全抑制GBV-B的复制超过4个月，即使siRNA仅注射3天。出乎意料的是，对照siRNA/ lic -101也能延缓病毒的复制。由于对照siRNA/LIC-101诱导了血清干扰素α和γ，因此对照siRNA/LIC-101对病毒复制的延迟应该是由诱导干扰素的抗病毒作用引起的。相比之下，GBV-B siRNA/LIC-101诱导的干扰素比对照siRNA/LIC-101少得多。因此，我们认为GBV-B siRNA/LIC-101的抑制作用包括siRNA和干扰素的抑制作用。由于脂质载体合成的siRNA通过位于内体膜上的toll样受体识别，并以序列依赖的方式激活免疫系统。综上所述，我们成功地用siRNA抑制了GBV-B在猴子肝脏中的复制。我们正在尝试新的方法来抑制干扰素的反应，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.