studies

HEPATOLOGY, VOLUME 62, NUMBER 1 (SUPPL) AASLD ABSTRACTS 1003A
HBV core-related antigen level. In the human hepatocytes,
HDI decreased HBs antigen level by 70%, HBV DNA level
by 90% and 3.5kb HBV RNA level by 50% in the cell. HDI
increased HBs antigen level in the HepG2 cells transfected
with the entire HBV genome and also increased 3.5 kb HBV
RNA level by 3~4-folds. HDI increased HBV DNA associated
with acetylated histone H4 as well as acetylation of histone H4
in the HepG2.2.15 cells. Furthermore, histone acetyltransferase
inhibitor garcinol partially inhibited the increase in 3.5kb
HBV RNA level by HDI indicating the possible role of histone
acetylation on the regulation of HBV replication. Conclusion:
HDI increases HBV replication but decreases HBs antigen production
in hepatoma cell line with HBV DNA integration. In
non-tumor hepatocytes infected with HBV, HDI inhibits HBV
production as well as HBV replication. Epigenetic mechanism
may be involved in the regulation of HBV replication at multisteps
in human hepatocytes.
Disclosures:
Tatsuo Kanda - Grant/Research Support: MSD K.K.; Speaking and Teaching:
AJINOMOTO PHARMACEUTICALS CO., LTD, CHUGAI PHARMACEUTICAL
CO., LTD, GlaxoSmithKlein, BMS, Jansen, Daiichisankyo; Stock Shareholder:
Mitsubishi Tanabe Pharma
Osamu Yokosuka - Grant/Research Support: Chugai, Taiho, Bristol Myers,
Takeda
The following authors have nothing to disclose: Shingo Nakamoto, Shuang Wu,
Yuki Haga, Reina Sasaki, Masato Nakamura, Hiroshi Shirasawa
1629
CRISPR/Cas9 “double”-nickase mediated inactivation of
hepatitis B virus replication
Madina Karimova 2 , Niklas Beschorner 2 , Werner Dammermann 1 ,
Jan Chemnitz 2 , Daniela S. Indenbirken 2 , Adam Grundhoff 2,3 , Stefan
Lueth 1 , Frank Buchholz 4 , Schulze zur Wiesch Julian 1,3 , Joachim
Hauber 2,3 ; 1 1. Department of Medicine, University Medical Center
Hamburg-Eppendorf, Hamburg, Germany; 2 Heinrich Pette Institute
– Leibniz Institute for Experimental Virology, Hamburg, Germany;
3 German Center for Infection Research (DZIF), partner site Hamburg,
Hamburg, Germany; 4 Department of Medical Systems Biology,
University Hospital and Medical Faculty Carl Gustav Carus,
TU Dresden, Dresden, Germany
Background: Current antiviral therapies cannot cure hepatitis B
virus (HBV) infection, since successful HBV eradication would
require the inactivation of the viral genome, which primarily
persists in host cells as episomal covalently closed circular
DNA (cccDNA) and, to a lesser extent, as chromosomally integrated
sequences. However, novel designer-enzymes, such as
the CRISPR/Cas9 RNA-guided nuclease system, provide the
technology for the development of advanced therapy strategies
that directly attack the HBV genome. Methods: We report here
the identification of cross-genotype conserved HBV sequences
in the HbS and HbX region of the HBV genome that are specifically
and effectively inactivated by a Cas9 double-nickase
approach. Pairs of appropriately spaced Cas9 nickase
mutants introduced two single-strand breaks on the opposite
DNA strands that were subject to NHEJ repair in order to avoid
off-target mutations by improving specificity by up to 1,500-fold
relative to the wild-type Cas9 enzyme. Results: We show that
this approach equally inactivated episomal cccDNA as well as
chromosomally integrated HBV target sites in reporter cell lines
as well as in chronically infected hepatoma cell lines. Analysis
of Cas9n activity on ORF S and X target sites by next generation
sequencing revealed efficient editing of cccDNA molecules
targeted by either gRNA in HBV-infected HepG2.2.15,
HepG2-H1.3 and HepG2-NTCP cells. The efficiency of X-specific
sgRNAs was particularly high, with approximately 90%
of all amplicons reads showing clearly discernible indel signatures.
Conclusion: Our data support the feasibility of using
the CRISPR/Cas9 nickase system for novel therapy strategies
aiming to provide a cure for HBV infection.
(A) Depiction of the HBV genome with location of amplicons
spanning the S- and X-specific sgRNA target regions. The two
amplicons are shown as dark gray boxes labeled S- and X-amplicon,
respectively. Arrows shown at the top indicate the genomic
location of gRNA target sequences.
Disclosures:
Stefan Lueth - Advisory Committees or Review Panels: BMS, Gilead, MSD, Janssen
The following authors have nothing to disclose: Madina Karimova, Niklas Beschorner,
Werner Dammermann, Jan Chemnitz, Daniela S. Indenbirken, Adam
Grundhoff, Frank Buchholz, Schulze zur Wiesch Julian, Joachim Hauber
1630
GORASP2 regulates HCV and HBV through RNA and/or
DNA replication and virion trafficking
Dachuan Cai 1,2 , Jian Hong 1 , Xiao Liu 1 , Sae Hwan Lee 1 , Dahlene
Fusco 1 , Esperance A. Schaefer 1 , Cynthia Brisac 1 , Anna Lidofsky 1 ,
Wenyu Lin 1 , Raymond T. Chung 1 ; 1 Gastrointestinal Unit, Massachusetts
General Hospital, Harvard Medical School, Boston, MA;
2 Department for infectious diseases, The second affiliated hospital
of Chongqing medical university, Chongqing, China
Background/Aims: Hepatitis B virus (HBV) and HCV constitute
major causes of blood transmitted hepatitis. Chronic HCV
and HBV infection induce liver cirrhosis and hepatocellular
carcinoma (HCC). However, the mechanisms by which these
infections alter host immunity are not well understood. In a
previous RNA-Seq analysis, we identified Golgi reassembly
stacking protein 2 (GORASP2) as a host factor that participates
in IFN-a regulation of HCV replication (Lin et al, J Hepatology
2015, 62:1024). GORASP2 is involved in establishing the
Golgi apparatus structure. We hypothesized that GORASP2
protein regulates HCV and HBV through HCV RNA replication,
HBV DNA and pregenomic RNA replication, and HCV/
HBV virion trafficking. Methods: We performed siRNA knockdown
or overexpression of GORASP2 in JFH1 HCV-infected
Huh7.5.1 cells or HepG2.2.15 HBV replicon cells with or
without IFN treatment. Selected gene mRNA variants and their
proteins, together with HBV replication in cells and HBV virions
in supernatant, were monitored by qRT-PCR and Western blot.
HBV replication was assessed by measuring HBV X and S protein
levels, HBV DNA levels and pregenomic RNA in cells, and
supernatant virus DNA. Results: We found that GORASP2 has
two isoforms based on RNA-Seq exon splicing bioinformatics
analysis. GORASP2 V2 includes exon #2 while V1 does not.
We found that siRNA knockdown of GORASP2 increased HCV
RNA and protein levels in JFH1-infected Huh7.5.1 cells and
HBV levels in DNA and pregenomic RNA in HepG2.2.15 cells.
GORASP2 mRNA and protein expression were not affected by
IFN treatment, indicating that GORASP2 expression is independent
of direct IFN regulation. We found that GORASP2
V1 overexpression reduced HCV RNA and protein levels in
JFH1-infected cells and HBV DNA and pregenomic RNA levels
in HepG2.2.15 cells. In contrast, overexpression of GORASP2
V2 had no significant effect on HCV RNA or protein levels
in JFH1 cells. Golgi phosphoprotein 3 (GOLPH3) has been