scholarly journals Adenovirus Vectors Expressing Eight Multiplex Guide RNAs of CRISPR/Cas9 Efficiently Disrupted Diverse Hepatitis B Virus Gene Derived from Heterogeneous Patient

2021 ◽  
Vol 22 (19) ◽  
pp. 10570
Author(s):  
Yuya Kato ◽  
Hirotaka Tabata ◽  
Kumiko Sato ◽  
Mariko Nakamura ◽  
Izumu Saito ◽  
...  

Hepatitis B virus (HBV) chronically infects more than 240 million people worldwide, causing chronic hepatitis, cirrhosis, and hepatocellular carcinoma (HCC). Genome editing using CRISPR/Cas9 could provide new therapies because it can directly disrupt HBV genomes. However, because HBV genome sequences are highly diverse, the identical target sequence of guide RNA (gRNA), 20 nucleotides in length, is not necessarily present intact in the target HBV DNA in heterogeneous patients. Consequently, possible genome-editing drugs would be effective only for limited numbers of patients. Here, we show that an adenovirus vector (AdV) bearing eight multiplex gRNA expression units could be constructed in one step and amplified to a level sufficient for in vivo study with lack of deletion. Using this AdV, HBV X gene integrated in HepG2 cell chromosome derived from a heterogeneous patient was cleaved at multiple sites and disrupted. Indeed, four targets out of eight could not be cleaved due to sequence mismatches, but the remaining four targets were cleaved, producing irreversible deletions. Accordingly, the diverse X gene was disrupted at more than 90% efficiency. AdV containing eight multiplex gRNA units not only offers multiple knockouts of genes, but could also solve the problems of heterogeneous targets and escape mutants in genome-editing therapy.

1994 ◽  
Vol 21 (1) ◽  
pp. 103-109 ◽  
Author(s):  
Clara Balsano ◽  
Olivier Billet ◽  
Myriam Bennoun ◽  
Catherine Cavard ◽  
Alain Zider ◽  
...  

1999 ◽  
Vol 73 (12) ◽  
pp. 10399-10405 ◽  
Author(s):  
Kurt Reifenberg ◽  
Heike Wilts ◽  
Jürgen Löhler ◽  
Petra Nusser ◽  
Ralph Hanano ◽  
...  

ABSTRACT The function of the X protein in the life cycle of mammalian hepadnaviruses is unclear. Based on tissue culture experiments it has been suggested that this protein represents a transcriptional transactivator which might be essential for the expression of the viral core gene. Here we have examined whether the activity of the human hepatitis B virus (HBV) core gene in vivo depends on X coexpression. To this end we compared core gene expression between four lineages of transgenic mice carrying the HBV core gene in cisarrangement with the X gene (cex lineage) and six lineages containing a modified construct in which the start codon of the X gene had been deleted (ce lineage). Whereas all cex lineages consistently exhibited a high-level hepatic core gene expression, the liver-specific core gene expression pattern of the ce lineages was heterogenous with four lineages virtually not expressing the core gene. This defect was due to a strongly reduced transcription since no core mRNA could be detected by Northern blotting. To test whether core gene expression could be restored by providing an intact X gene in trans, we crossbred mice of two lines which expressed no core mRNA or core protein with transgenic mice expressing the X-gene product under the transcriptional regulation of the liver-specific major-urinary-protein promoter/enhancer (MUP-X mice). The introduction of the MUP-X transgene induced core mRNA expression and core protein biosynthesis in the livers of the double-transgenic mice. This demonstrates that the X-gene product has the capacity to transactivate HBV core gene expression in vivo.


Author(s):  
Clara Balsano ◽  
O. Billet ◽  
Myriam Bennoun ◽  
Catherine Cavard ◽  
A. Zider ◽  
...  

2021 ◽  
Vol 12 ◽  
Author(s):  
Kun Yan ◽  
Jiangpeng Feng ◽  
Xing Liu ◽  
Hongyun Wang ◽  
Qiaohong Li ◽  
...  

Curative therapies for chronic hepatitis B virus (HBV) infection remain a distant goal, and the persistence of stable covalently closed circular DNA (cccDNA) during HBV replication is a key barrier that is hard to break through using the drugs currently approved for HBV treatment. Due to the accuracy, efficiency, and cost-effectiveness of genome editing, CRISPR/Cas technologies are being widely used for gene therapy and in antiviral strategies. Although CRISPR/Cas could possibly clear cccDNA, ensuring its safety is requirement for application. In our study, we analyzed the liver specificity of several promoters and constructed candidate promoters in the CRISPR/Staphylococcus aureus Cas9 (SaCas9) system combined with hepatotropic AAV8 (whereby AAV refers to adeno-associated virus) to verify the efficacy against HBV. The results revealed that the reconstructed CRISPR/SaCas9 system in which the original promoter replaced with a liver-specific promoter could still inhibit HBV replication both in vitro and in vivo. Three functional guide RNAs (gRNAs), T2, T3, and T6, which target the conserved regions of different HBV genotypes, demonstrated consistently better anti-HBV effects with different liver-specific promoters. Moreover, the three gRNAs inhibited the replication of HBV genotypes A, B, and C to varying degrees. Under the action of the EnhII-Pa1AT promoter and AAV8, the expression of SaCas9 was further decreased in other organs or tissues in comparison to liver. These results are helpful for clinical applications in liver by ensuring the effects of the CRISPR/Cas9 system remain restricted to liver and, thereby, reducing the probability of undesired and harmful effects through nonspecific targeting in other organs.


2015 ◽  
Vol 41 (08) ◽  
Author(s):  
C Klein ◽  
CT Bock ◽  
H Wedemeyer ◽  
T Wüstefeld ◽  
S Locarnini ◽  
...  

2012 ◽  
Vol 23 (3) ◽  
pp. 278-285 ◽  
Author(s):  
Feng-Jun Liu ◽  
En-Qiang Chen ◽  
Qiao-Ling Zhou ◽  
Tao-You Zhou ◽  
Cong Liu ◽  
...  

1997 ◽  
Vol 94 (15) ◽  
pp. 8162-8167 ◽  
Author(s):  
P. Chirillo ◽  
S. Pagano ◽  
G. Natoli ◽  
P. L. Puri ◽  
V. L. Burgio ◽  
...  

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