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82
NOVEL ANTI-HBV THERAPIES USING CRISPR/Cas9
TARGETING HBV GENOME STRONGLY SUPPRESS HBV.
Kazuhiro Murai, Takahiro Kodama, Hayato Hikita, Akiyoshi
Shimoda, Makoto Fukuoka, Keisuke Fukutomi, Yuki
Tahata, Yuki Makino, 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
Current anti-HBV therapies have little effect on cccDNA and
fail to eliminate HBV completely HBV uses host cellular DNA
repair machinery for cccDNA biosynthesis We investigated
efficacy of novel therapeutics targeting the HBV genome
using Clustered Regularly Interspaced Short Palindromic
Repeat (CRISPR)/Cas9 in combination with inhibitors of DNA
repair pathway Methods: We designed gRNA targeting HBV
genome (HBV-gRNA) and control gRNAs not targeting HBV
or human genome (Cont-gRNA) We lentivirally transduced
tandem Cas9 and HBV-gRNA (or Cont-gRNA) expressing
vector (HBV-gRNA/Cas9 or Cont-gRNA/Cas9) into HBV
genome-integrated cells (HepG2 2 15) or primary human
hepatocytes (PHHs) 19 days after HBV infection HBV
susceptible cells with inducible Cas9 expression (HepG2-
hNTCP-iCas9) were lentivirally transduced with single HBVgRNA
or Cont-gRNA vector Two days after HBV infection,
Cas9 expression was induced by doxycycline (DOX)
administration We examined the anti-viral effect of CRISPR
therapy by HBV-gRNA compared to Cont-gRNA either alone
or in combination with inhibition of DNA repair pathways
Results: Lentiviral transduction of HBV-gRNA/Cas9 induced
indel formation at the region of HBV genome targeted by HBVgRNA
in HepG2 2 15 cells HBV-gRNA/Cas9 transduction
significantly reduced the levels of HBV DNA and HBs antigen
in the supernatant and intracellular pregenomic RNA (pgRNA)
levels in HepG2 2 15 cells compared to Cont-gRNA/Cas9
Similarly, HBV-gRNA/Cas9 transduction reduced the levels
of HBV DNA and HBs antigen in the supernatant, and levels
of intracellular pgRNA and cccDNA in PHHs HBV-gRNA
transduction followed by DOX-induced Cas9 expression
also reduced the levels of HBV DNA and HBs antigen in the
supernatant, and levels of intracellular pgRNA and cccDNA in
HepG2-hNTCP-iCas9 cells While siRNA-mediated inhibition
of BRCA1 did not affect anti-HBV effect of CRISPR therapy,
siRNA-mediated inhibition of PARP1 or PARP1 inhibitor
olaparib significantly augmented the reduction of intracellular
pgRNA upon CRISPR therapy Conclusion: Efficacy of anti-
HBV therapy targeting HBV genome by CRISPR/Cas9 may
be enhanced by modulating host DNA repair pathways.
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发表于 2020-10-19 12:39