由野生型HBV乙型肝炎病毒抗原的生产和复制的抑制依赖性复

StephenW

Antiviral Res. 2016 Aug 30. pii: S0166-3542(16)30125-5. doi: 10.1016/j.antiviral.2016.08.007. [Epub ahead of print]
Suppression of hepatitis B virus antigen production and replication by wild-type HBV dependently replicating HBV shRNA vectors in vitro and in vivo.Li B1, Sun S2, Li M3, Cheng X4, Li H4, Kang F4, Kang J4, Dörnbrack K5, Nassal M6, Sun D7.
Author information

• 1Chinese PLA Medical School, Chinese PLA General Hospital, 100853, Beijing, PR China; The Liver Disease Diagnosis and Treatment Center of PLA, Bethune International Peace Hospital, Shijiazhuang, 050082, PR China.
• 2The Liver Disease Diagnosis and Treatment Center of PLA, Bethune International Peace Hospital, Shijiazhuang, 050082, PR China; Troop 66220 of PLA, Xingtai, Hebei Province, 054000, PR China.
• 3The Liver Disease Diagnosis and Treatment Center of PLA, Bethune International Peace Hospital, Shijiazhuang, 050082, PR China; The Fourth Department of the Fifth Hospital, Shijiazhuang City, PR China.
• 4The Liver Disease Diagnosis and Treatment Center of PLA, Bethune International Peace Hospital, Shijiazhuang, 050082, PR China.
• 5Internal Medicine II/Molecular Biology, University Hospital Freiburg, D-79106, Freiburg, Germany.
• 6Internal Medicine II/Molecular Biology, University Hospital Freiburg, D-79106, Freiburg, Germany. Electronic address: [email protected].
• 7The Liver Disease Diagnosis and Treatment Center of PLA, Bethune International Peace Hospital, Shijiazhuang, 050082, PR China. Electronic address: [email protected].
  

AbstractChronic infection with hepatitis B virus (HBV), a small DNA virus that replicates by reverse transcription of a pregenomic (pg) RNA precursor, greatly increases the risk for terminal liver disease. RNA interference (RNAi) based therapy approaches have shown potential to overcome the limited efficacy of current treatments. However, synthetic siRNAs as well as small hairpin (sh) RNAs expressed from non-integrating vectors require repeated applications; integrating vectors suffer from safety concerns. We pursue a new concept by which HBV itself is engineered into a conditionally replicating, wild-type HBV dependent anti-HBV shRNA vector. Beyond sharing HBV's hepatocyte tropism, such a vector would be self-renewing, but only as long as wild-type HBV is present. Here, we realized several important aspects of this concept. We identified two distinct regions in the 3.2 kb HBV genome which tolerate replacement by shRNA expression cassettes without compromising reverse transcription when complemented in vitro by HBV helper constructs or by wild-type HBV; a representative HBV shRNA vector was infectious in cell culture. The vector-encoded shRNAs were active, including on HBV as target. A dual anti-HBV shRNA vector delivered into HBV transgenic mice, which are not susceptible to HBV infection, by a chimeric adenovirus-HBV shuttle reduced serum hepatitis B surface antigen (HBsAg) up to ∼4-fold, and virus particles up to ∼20-fold. Importantly, a fraction of the circulating particles contained vector-derived DNA, indicating successful complementation in vivo. These data encourage further investigations to prove antiviral efficacy and the predicted self-limiting vector spread in a small animal HBV infection model.
Copyright © 2016. Published by Elsevier B.V.


KEYWORDS: Chronic hepatitis B; HBV transgenic mice; HBV vector; Hepatitis B virus; RNAi; shRNA delivery

PMID:27591142DOI:10.1016/j.antiviral.2016.08.007

StephenW

抗病毒水库。 2016年30月PII：S0166-3542（16）30125-5。 DOI：10.1016 / j.antiviral.2016.08.007。 [打印EPUB提前]
由野生型HBV乙型肝炎病毒抗原的生产和复制的抑制依赖性复制在体外和体内的HBV的shRNA的载体。
李B1，孙S2，李M3，程X4，李H4，康F4，康J4，DörnbrackK5，Nassal M6，孙D7。
作者信息

    1.汉语解放军医学院，中国解放军总医院，100853中国北京;肝病诊断​​和PLA的治疗中心，白求恩国际和平医院，石家庄，050082，中国公关。
    2The肝脏疾病的诊断和解放军白求恩国际和平医院，石家庄，050082的治疗中心，中国; PLA，邢台，河北省，054000，PR中国的部队66220。
    3The肝脏疾病的诊断和解放军白求恩国际和平医院，石家庄，050082的治疗中心，中国;第五医院的四系，石家庄市，中国公关。
    4The肝脏疾病诊断以及中国解放军治疗中心，白求恩国际和平医院，石家庄，050082。
    5Internal医药II /分子生物学，大学医​​院弗赖堡，D-79106，德国弗赖堡。
    6Internal医药II /分子生物学，大学医​​院弗赖堡，D-79106，德国弗赖堡。电子地址：[email protected]。
    。第七条肝病诊断以及中国解放军治疗中心，白求恩国际和平医院，石家庄，050082。电子地址：[email protected]。

抽象

乙型肝炎病毒（HBV），这由一个前基因组（PG）的RNA前体的反转录复制的小DNA病毒，慢性感染大大增加了终端肝病的风险。 RNA干扰（RNAi）的基础治疗方法已经表明潜在克服目前的治疗的疗效有限。然而，从非整合载体表达的合成的siRNA以及小发夹（SH）的RNA需要反复应用;整合型载体的安全问题受到影响。我们追求一个新的概念由乙肝病毒本身被设计成一个条件复制，野生型HBV相关的抗乙肝病毒shRNA表达载体。超出共享的HBV的肝向性，这样的载体是自我更新，但只要仅与野生型HBV的存在。在这里，我们实现了这个概念的几个重要方面。我们确定了3.2 kb的HBV基因组两个不同的区域其容忍的shRNA表达盒更换无需通过HBV辅助构建或野生型HBV体外补充时牺牲逆转录;具有代表性的乙肝病毒shRNA表达载体在细胞培养感染。矢量编码的shRNA活跃，包括乙肝病毒作为目标。输送到HBV转基因小鼠，其不容易受到HBV感染，通过嵌合腺病毒 - HBV穿梭降低血清乙肝表面抗原（HBsAg）最多~4倍，和病毒颗粒高达〜双抗HBV shRNA表达载体20倍。重要的是，在循环颗粒的部分含有载体来源的DNA，这表明在体内成功互补。这些数据鼓励进一步研究证明抗病毒疗效，并在小动物HBV感染模型预测的自限性载体传播。

版权所有©2016年出版由Elsevier B.V.
关键词：

慢性乙型肝炎; HBV转基因小鼠;乙肝病毒载体;乙型肝炎病毒; RNA干扰;交货的shRNA

结论：
    27591142
DOI：
    10.1016 / j.antiviral.2016.08.007