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Inhibition of hepatitis B virus replication via HBV DNA cleavage by Cas9 from Staphylococcus aureus
Antiviral Research ( IF 4.5 ) Pub Date : 2018-02-16 , DOI: 10.1016/j.antiviral.2018.02.011
Yu Liu , Miaoxian Zhao , Mingxing Gong , Ying Xu , Cantao Xie , Haohui Deng , Xueying Li , Hongkai Wu , Zhanhui Wang

Chronic hepatitis B virus (HBV) infection is difficult to cure due to the presence of covalently closed circular DNA (cccDNA). Accumulating evidence indicates that the CRISPR/Cas9 system effectively disrupts HBV genome, including cccDNA, in vitro and in vivo. However, efficient delivery of CRISPR/Cas9 system to the liver or hepatocytes using an adeno-associated virus (AAV) vector remains challenging due to the large size of Cas9 from Streptococcus pyogenes (Sp). The recently identified Cas9 protein from Staphylococcus aureus (Sa) is smaller than SpCas9 and thus is able to be packaged into the AAV vector. To examine the efficacy of SaCas9 system on HBV genome destruction, we designed 5 guide RNAs (gRNAs) that targeted different HBV genotypes, 3 of which were shown to be effective. The SaCas9 system significantly reduced HBV antigen expression, as well as pgRNA and cccDNA levels, in Huh7, HepG2.2.15 and HepAD38 cells. The dual expression of gRNAs/SaCas9 in these cell lines resulted in more efficient HBV genome cleavage. In the mouse model, hydrodynamic injection of gRNA/SaCas9 plasmids resulted in significantly lower levels of HBV protein expression. We also delivered the SaCas9 system into mice with persistent HBV replication using an AAV vector. Both the AAV vector and the mRNA of Cas9 could be detected in the C3H mouse liver cells. Decreased hepatitis B surface antigen (HBsAg), HBV DNA and pgRNA levels were observed when a higher titer of AAV was injected, although this decrease was not significantly different from the control. In summary, the SaCas9 system accurately and efficiently targeted the HBV genome and inhibited HBV replication both in vitro and in vivo. The system was delivered by an AAV vector and maybe used as a novel therapeutic strategy against chronic HBV infection.



中文翻译:

金黄色葡萄球菌Cas9通过HBV DNA切割抑制乙型肝炎病毒复制

由于存在共价闭合的环状DNA(cccDNA),慢性乙型肝炎病毒(HBV)感染难以治愈。越来越多的证据表明,CRISPR / Cas9系统有效地破坏HBV基因组,包括cccDNA的,在体外体内。然而,由于化脓性链球菌(Sp)产生的Cas9较大,使用腺相关病毒(AAV)载体将CRISPR / Cas9系统有效递送至肝脏或肝细胞仍然具有挑战性。最近鉴定的金黄色葡萄球菌Cas9蛋白(Sa)比SpCas9小,因此可以包装到AAV向量中。为了检查SaCas9系统对HBV基因组破坏的功效,我们设计了5种针对不同HBV基因型的指导RNA(gRNA),其中3种被证明是有效的。SaCas9系统显着降低了Huh7,HepG2.2.15和HepAD38细胞中的HBV抗原表达以及pgRNA和cccDNA水平。gRNA / SaCas9在这些细胞系中的双重表达导致更有效的HBV基因组切割。在小鼠模型中,水动力注射gRNA / SaCas9质粒导致HBV蛋白表达水平明显降低。我们还使用AAV载体将SaCas9系统交付给具有持久性HBV复制的小鼠。在C3H小鼠肝细胞中都可以检测到AAV载体和Cas9的mRNA。当注射更高滴度的AAV时,观察到乙型肝炎表面抗原(HBsAg),HBV DNA和pgRNA水平降低,尽管这种降低与对照组无显着差异。综上所述,SaCas9系统可准确有效地靶向HBV基因组并抑制HBV复制。体外体内。该系统由AAV载体提供,可以用作针对慢性HBV感染的新型治疗策略。

更新日期:2018-02-16
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