studies

1002A AASLD ABSTRACTS HEPATOLOGY, October, 2015
NUCs. Proliferating NK cells with antiviral capacity increased
significantly, and HBsAg significantly decreased, when NUC
treatment was preceded by Peg-IFNα compared to de-novo
NUCs. Interestingly, the frequency of antiviral NK cells, in
those patients exposed to Peg-IFNα, without further therapy,
reduced to baseline, 9-12 months post treatment cessation.
In addition, increased IFNγ production and cytotoxicity, but
reduced TRAIL expression, were noted in those patients (9/12)
with HBsAg decline on sequential NUCs. CONSLUSIONS:
The potent and cumulative expansion of activated NK cells
with antiviral potential induced by Peg-IFNα is maintained on
sequential NUCs. Patients receiving sequential NUCs achieved
a greater decline in HBsAg than those treated with de novo
NUCs, particularly those with TRAIL low IFNγ/CD107 hi NK cells.
TRAIL expression in this setting may be pathogenic, in line with
our previous data showing that TRAIL+ NK cells can delete
antiviral T cells (Peppa et al, JEM 2013), suggesting the TRAIL
pathway as a potential future therapeutic target. These findings
provide mechanistic insights to support further consideration of
sequential Peg-IFNα/NUC therapy to enhance HBsAg reduction,
and achieve sustained treatment responses.
Disclosures:
Ivana Carey - Grant/Research Support: Gilead, Roche; Speaking and Teaching:
BMS
Graham R. Foster - Advisory Committees or Review Panels: GlaxoSmithKline,
Novartis, Boehringer Ingelheim, Tibotec, Chughai, Gilead, Janssen, Idenix,
GlaxoSmithKline, Novartis, Roche, Tibotec, Chughai, Gilead, Merck, Janssen,
Idenix, BMS; Board Membership: Boehringer Ingelheim; Grant/Research Support:
Chughai, Roche, Chughai; Speaking and Teaching: Roche, Gilead, Tibotec,
Merck, BMS, Boehringer Ingelheim, Gilead, Janssen
Patrick T. Kennedy - Grant/Research Support: Roche, Gilead; Speaking and
Teaching: BMS, Roche, Gilead
The following authors have nothing to disclose: Upkar S. Gill, Dimitra Peppa,
Lorenzo Micco, Harsimran D. Singh, Mala K. Maini
1627
Analysis of the effect on HBV life cycle by HBV genome
editing using two different CRISPR/Cas9 systems
Hiromi Abe, Keiichi Masaki, Tetsushi Sakuma, Masataka Tsuge,
Nobuhiko Hiraga, Michio Imamura, C. Nelson Hayes, Hiroshi
Aikata, Takashi Yamamoto, Kazuaki Chayama; Hiroshima University,
Hiroshima, Japan
Background and aim: Interferon and nucleoside analogue therapy
effectively controls HBV replication in chronically infected
patients. However, it is still difficult to eradicate HBV completely
because covalently closed circular DNA (cccDNA) stably
remains in the nucleus of hepatocytes. Recently, a new
genome editing system, Cas9-nickase, which has mutagenic
Cas9 activity, has been developed to target specific regions
of double stranded DNA sequences with less off target effects
compared with the original CRISPR/cas9. Using this approach
we developed an anti-HBV system that targets three different
portion of the HBV genome using only one plasmid. The aim
of this study is to investigate the efficacy of these two CRISPR/
Cas9 systems. Method: Two CRISPR/cas9 plasmids, nuclease
and nickase types, were constructed to target three regions
of the HBV genome: HBs, polymerase, and core. The original
CRISPR-nuclease digests the HBV genome at these three
regions using three target guide RNAs whereas the nickase
type enzyme nicked both strands of the target site DNA using
three pairs of guide RNAs transcribed from one plasmid.
HepG2 cells grown in 6 well-plates in DMEM containing 10%
FBS were co-transfected with 1.4×HBV genome and one of
the two CRISPR-Cas9 encoding plasmids in a 1:2 ratio. Three
days after co-transfection, cells and culture medium were harvested
to evaluate the efficacy of the genome editing. Core
associated HBV DNA was measured by qPCR after immunoprecipitation
with anti-HBc antibody. The CRISPR/Cas9 plasmid
was introduced into Baculovirus infection system. 14 days after
infection, we measured HBsAg, eAg, and HBV DNA in the
supernatant and intracellular HBV DNA and core-associated
HBV DNA. Results: More than 78-95% of double stranded
DNA of HBs, core and polymerase cleavage by both nuclease
and nickase activity of CRISPR/Cas9 were confirmed by reporter-based
assay. When we co-transfected 1.4×HBV genome
and each CRISPR/Cas9-encoding plasmid, both HBsAg and
eAg levels declined significantly in comparison with control
plasmids in both types of CRISPR/Cas9 systems. Nuclease type
Cas9 more efficiently reduced both HBsAg and eAg levels than
nuclease type to less than 10% of control. Cas9-nickase also
reduced HBsAg and eAg to approximately 10% of control cells
(P