通过HBV DNA切割由金黄色葡萄球菌的Cas9抑制乙型肝炎病毒复制

StephenW

Antiviral Res. 2018 Feb 16. pii: S0166-3542(17)30675-7. doi: 10.1016/j.antiviral.2018.02.011. [Epub ahead of print]
Inhibition of hepatitis B virus replication via HBV DNA cleavage by Cas9 from Staphylococcus aureus.Liu Y1, Zhao M1, Gong M1, Xu Y1, Xie C1, Deng H1, Li X1, Wu H2, Wang Z3.
Author information
1State Key Laboratory of Organ Failure Research, Guangdong Provincial Key Laboratory of Viral Hepatitis Research, Department of Infectious Diseases and Hepatology Unit, Nanfang Hospital, Southern Medical University, Guangzhou, China.2State Key Laboratory of Respiratory Disease, The First Affiliated Hospital of Guangzhou Medical University, Guangzhou Medical University, Guangzhou, China.3State Key Laboratory of Organ Failure Research, Guangdong Provincial Key Laboratory of Viral Hepatitis Research, Department of Infectious Diseases and Hepatology Unit, Nanfang Hospital, Southern Medical University, Guangzhou, China. Electronic address: [email protected].

AbstractChronic 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.


KEYWORDS: DNA targeting; Gene therapy; Hepatitis B virus; SaCas9 system

PMID:29458131DOI:10.1016/j.antiviral.2018.02.011

StephenW

抗病毒研究。 2018年2月16日。pii：S0166-3542（17）30675-7。 doi：10.1016 / j.antiviral.2018.02.011。 [电子版提前打印]
通过HBV DNA切割由金黄色葡萄球菌的Cas9抑制乙型肝炎病毒复制。
Liu Y1，Zhao M1，Gong M1，Xu Y1，Xie C1，Deng H1，Li X1，Wu H2，Wang Z3。
作者信息

1
    广东省南方医科大学南方医院传染病肝病研究室广东省病毒性肝炎研究重点实验室器官衰竭研究国家重点实验室。
2
    呼吸疾病国家重点实验室，广州医科大学附属第一医院，广州医科大学，广州。
3
    广东省南方医科大学南方医院传染病肝病研究室广东省病毒性肝炎研究重点实验室器官衰竭研究国家重点实验室。电子地址：[email protected]。

抽象

由于共价闭合环状DNA（cccDNA）的存在，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系统在体外和体内有效破坏HBV基因组，包括cccDNA。然而，由于来自化脓性链球菌（Sp）的大量Cas9，使用腺伴随病毒（AAV）载体将CRISPR / Cas9系统有效递送至肝或肝细胞仍然具有挑战性。最近发现的来自金黄色葡萄球菌（Sa）的Cas9蛋白小于SpCas9，因此能够包装入AAV载体中。为了检验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基因型的指导性RNA（gRNA），其中3种被证明是有效的。 SaCas9系统在Huh7，HepG2.2.15和HepAD38细胞中显着降低HBV抗原表达以及pgRNA和cccDNA水平。在这些细胞系中gRNA / SaCas9的双重表达导致更有效的HBV基因组切割。在小鼠模型中，gRNA / SaCas9质粒的水动力注射导致显着较低水平的HBV蛋白表达。我们还使用AAV载体将SaCas9系统交付给持续HBV复制的小鼠。在C3H小鼠肝细胞中可以检测到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）降低，HBV DNA和pgRNA水平降低，尽管这种降低与对照没有显着差异。总之，SaCas9系统准确有效地靶向HBV基因组并在体外和体内抑制HBV复制。该系统由AAV载体递送，并可用作针对慢性HBV感染的新型治疗策略。
关键词：

DNA靶向;基因治疗;乙型肝炎病毒; SaCas9系统

结论：
    29458131
DOI：
    10.1016 / j.antiviral.2018.02.011

antiHBVren

怎么感觉像以毒攻毒呢，还不是西医的精准治疗