studies

HEPATOLOGY, VOLUME 62, NUMBER 1 (SUPPL) AASLD ABSTRACTS 223A
enrich cccDNA, was measured by qPCR. Pre-core/core RNA
(C probe) and total HBV RNA (Total probe) were measured
by RT-qPCR. The Guide for the Care and Use of Laboratory
Animals was strictly adhered to. Results: During NUC lead-in,
total liver HBV DNA decreased 1.1-2.5 log 10
in HBeAg+ but
not appreciably in HBeAg- chimps. cccDNA in HBeAg+ chimps
decreased 0.7 ± 0.6 log 10
. Following addition of ARC-520 in
HBeAg+, total liver DNA decreased from baseline by 1.5–2.9
log 10
and cccDNA by 1.4 ± 0.7 log 10
, the degree of reduction
generally correlating with duration of treatment. Neither
total HBV DNA nor cccDNA levels changed remarkably in
HBeAg- during the study, which at baseline had 2-4 orders of
magnitude less cccDNA than HBeAg+ chimps. HBeAg- chimps
had 50-fold more DNase-sensitive HBV DNA, possibly indicating
the majority is integrated DNA rather than cccDNA.
HBV RNA was not reduced by NUCs, but with addition of
ARC-520 RNA reductions tracked qHBsAg reductions. In
HBeAg+, Total probe detected 1-2x as many transcripts as
the C probe, suggesting similar levels of core/pre-core and
S transcripts. In HBeAg-, the Total probe detected 37x more
transcripts than the C probe, supporting a greater proportion of
HBsAg transcripts being produced from integrated HBV DNA
in HBeAg- chimps. Integration between DR1 and DR2 would
result in HBV RNA lacking ARC-520 target sites, consistent
with greater HBsAg reduction in HBeAg+ (1.7 ± 0.5 log 10
)
than HBeAg- chimps (0.7 ± 0.1 log 10
). Administration of siRNA
targeted to integrant-produced transcripts resulted in HBsAg
reductions up to 2.3 log 10
beyond those produced by ARC-520
in HBeAg-. Conclusions: 1) ARC-520 reduced total liver DNA
and cccDNA beyond levels achieved in HBeAg+ with NUCs
during lead-in; 2) ARC-520 but not NUCs reduced HBV RNA
and antigens; 3) integrated HBV DNA may be important in
maintaining HBsAg in chronic HBV, especially in HBeAg-. This
finding, if confirmed, has important implications for development
of new HBV therapies.
Disclosures:
Christine I. Wooddell - Employment: Arrowhead Research Corporation
Ryan M. Peterson - Employment: Arrowhead Research Corporation
Julia O. Hegge - Employment: Arrowhead Research Corp
Robert Gish - Advisory Committees or Review Panels: Gilead, AbbVie, Arrowhead;
Consulting: Eiger, Isis, Genentech; Speaking and Teaching: Gilead, Abb-
Vie; Stock Shareholder: Arrowhead
Stephen Locarnini - Consulting: Gilead, Arrowhead; Employment: Melbourne
Health
Robert E. Lanford - Grant/Research Support: Arrowhead Research
David L. Lewis - Employment: Arrowhead Research Corporation
The following authors have nothing to disclose: Deborah Chavez, Jason E. Goetzmann,
Bernadette Guerra, Helen Lee, Christopher R. Anzalone
33
Inhibition of Hepatitis B Virus Replication by the HBV
Core Inhibitor NVR 3-778
Angela Lam, Suping Ren, Robert Vogel, Christine Espiritu, Mollie
Kelly, Vincent Lau, George D. Hartman, Lalo Flores, Klaus Klumpp;
Novira Therapeutics Inc., Doyleston, PA
Background: Hepatitis B virus (HBV) chronically infects more
than 350 million people worldwide, and is a major cause of
severe liver disease including cirrhosis and hepatocellular carcinoma.
NVR 3-778 represents a new class of HBV core inhibitors
and is currently in clinical development for the treatment of
chronic hepatitis B. We present the preclinical characterization
of NVR 3-778 and its antiviral properties in cells expressing
HBV. Methods: HBV core assembly was examined using fluorescence
quenching and electron microscopy. HBV replication
inhibition was measured as reduction of secreted HBV DNA in
HBV producing HepG2.2.15 cells. The production, encapsidation
and secretion of HBV RNA was determined using Northern
blot and Quantigene assays. Antiviral activity of NVR 3-778
against nucleoside resistant HBV variants was determined by
cell based phenotyping. HBV broad spectrum antiviral activity
was examined by testing NVR 3-778 across all HBV genotypes
(A-H). Results: NVR 3-778 inhibited HBV replication in
HepG2.2.15 cells and could fully block the production of HBV
DNA.. The compound could effectively induce mis-assembly of
recombinant HBV core protein and electron microscopy indicated
the formation of capsid-like particles. In HBV expressing
cells, NVR 3-778 blocked the encapsidation of HBV pgRNA,
HBV DNA formation, and the release of HBV DNA and RNA
containing particles. NVR 3-778 was similarly active against
wild-type and HBV nucleos(t)ide resistant variants with defined
amino acid changes within the reverse transcriptase protein:
rtL180M/M204V, rtN236T, rtA181V, rtA181V/N236T, and
rtL180M/M204V/N236T. Furthermore, in transfection assays
NVR 3-778 inhibited HBV replication across representative
HBV strains from genotypes A to H (EC 50
values from 0.20 to
0.58 mM). Conclusions: Preclinical antiviral profiling studies
showed that the antiviral HBV replication inhibitor NVR 3-778
could induce mis-assembly of core protein into capsid-like particles
in vitro. Treatment of HBV producing cells with NVR 3-778
was associated with inhibition of pgRNA encapsidation. Consequently,
the downstream processes of HBV DNA production
and secretion of infectious HBV DNA-containing virions (Dane
particles) were inhibited. In addition, NVR 3-778 also blocked
the secretion of HBV RNA-containing particles. NVR 3-778 was
active against HBV variants resistant to nucleos(t)ide analogs
and was broadly active across all HBV genotypes. These results
support the clinical development of NVR 3-778 as a potential
new therapy for chronic hepatitis B patients alone or in combination
with nucleoside analogs or other antiviral agents.
Disclosures:
Angela Lam - Employment: Novira Therapeutics, Merck & Co
Christine Espiritu - Employment: Novira Therapeutics
Mollie Kelly - Independent Contractor: Novira Therapeutics
George D. Hartman - Management Position: Novira Therapeutics
Klaus Klumpp - Board Membership: Riboscience LLC; Employment: Novira Therapeutics
Inc
The following authors have nothing to disclose: Suping Ren, Robert Vogel, Vincent
Lau, Lalo Flores
34
Dual-gRNAs and gRNA-microRNA (miRNA)-gRNA ternary
cassette combined CRISPR/Cas9 system and RNAi
approach promotes the clearance of HBV cccDNA
Jie Wang, Fengmin Lu; Peking University Health Science Center,
Beijing, China
Purpose: To investigate the potential use of CRISPR/Cas9
approach for eradicating hepatitis B virus (HBV) infection, we
designed and screened for the most effective gRNAs against
HBV of genotypes A-D, and developed a novel gRNA-microRNA
(miRNA)-gRNA ternary cassette. Methods: A total of
15 gRNAs against HBV of genotypes A-D were designed. 11
combinations of two above gRNAs (dual-gRNAs) covering the
different regions of HBV genome were chosen. Based on the
efficiency of dual-gRNAs in suppressing HBV replication, a
novel gRNA-miRNA-gRNA ternary cassette driven by a single
U6 promoter was designed by integrating an anti-HBV primiR31
mimic between two HBV-specific gRNAs, in which two
gRNAs could be released through Drosha/DGCR8 processing.
The HBV replication was examined by the measurement
of HBV surface antigen (HBsAg) and e antigen (HBeAg) in
the culture supernatant. The destruction of HBV-expressing vec-