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394
Gene Editing By CRISPR/Cas Targeting Cccdna
Exerts Strong Anti-Viral Effect in Combination
with Nucleoside Analogue.
Kazuhiro Murai, Takahiro Kodama, Hayato Hikita, Makoto
Fukuoka, Keisuke Fukutomi, Takuo Yamai, Tasuku Nakabori,
Ryoko Yamada, Ryotaro Sakamori, Tomohide Tatsumi and
Tetsuo Takehara, Department of Gastroenterology and
Hepatology, Osaka University, Graduate School of Medicine
Background: Hepatitis B virus (HBV) forms covalently closed
circular DNA (ccc DNA) in infected hepatocyte nucleus. Since
existing anti-HBV therapies have little effect on cccDNA,
it is difficult to completely eliminate HBV. In this study, we
investigated therapeutic potential of Clustered Regularly
Interspaced Short Palindromic Repeat (CRISPR)/Cas
targeting HBV genome. Methods: We used Streptococcus
pneumoniae Cas9 (SpCas9) and Staphylococcus aureus
Cas9 (SaCas9) as the CRISPR/Cas system. We designed
each set of multiple guide RNAs targeting HBV genome
(HBV-gRNA) and control gRNAs not targeting HBV or human
genome (Cont-gRNA) for SpCas9 and SaCas9, respectively,
since they recognize different proto-spacer adjacent motif
(PAM) sequence. We used lentivirus and adeno-associated
virus for transduction of SpCas9 (LV-SpCas9) and SaCas9
(AAV-SaCas9), respectively, into HBV genome integrated
cell line (HepG2.2.15) or HBV susceptible cell line (HepG2-
hNTCP-C4) upon infection of HBV inoculum produced from
HepAD38.7. We examined the effect of CRISPR/Cas therapy
on HBV replication and intracellular cccDNA levels. Indel
formation in HBV genome was also assessed using Surveyor
assay. Results: We first treatedHepG2.2.15 by CRISPR/
Cas system.Twenty days aftertransduction of LV-SpCas9
containing HBV-gRNA into HepG2.2.15, indel formation was
observed in all the regions of HBV genome targeted by 3
HBV-gRNAs. Transduction of LV-SpCas9 containing HBVgRNAs
significantly decreased the levels of HBV DNA, HBs
antigen and HBe antigen in the cell culture supernatant and
intracellular pgRNA levels in HepG2.2.15 cells compared to
those transduced with LV-SpCas9 containing Cont-gRNAs.
The similar anti-viral effects were also observed in HepG2.2.15
cells when transduced with AAV-SaCas9 containing HBVgRNAs.
Next, we treated HBV-infected HepG2-hNTCP-C4 by
CRISPR/Cas system. Transduction of LV-SpCas9 targeting
HBV-gRNAs significantly reduced the expression level of
cccDNA compared to those transduced with LV-SpCas9
containing Cont-gRNAs. Furthermore, combination therapy
using entecavir (ETV), a nucleoside analogue, and LV-SpCas9
targeting HBV-gRNAs further reduced the expression level
of cccDNA in HBV-infected HepG2-hNTCP-C4 compared
to those reduced by CRISPR/Cas or ETV monotherapy.
Conclusion: CRISPR/Cas targeting HBV genome reduces
HBV replication and cccDNA levels, and thus could be a
potential anti-viral therapy toward HBV. |
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发表于 2018-10-15 08:40
