studies

1214A AASLD ABSTRACTS HEPATOLOGY, October, 2015
mechanism triggered by introduction of small interfering RNA
(siRNA). Tekmira’s clinically-validated lipid nanoparticle (LNP)
platform enables effective delivery of siRNAs in vivo and in
vitro. HDV-targeted siRNA combined with LNP technology may
thus provide a novel opportunity to alleviate HDV pathogenesis.
There is limited data regarding which of the HDV RNA
species are susceptible to gene silencing. siRNAs targeting
positive or negative strand HDV RNAs were designed and
delivered to the Huh7-D12 stable HDV-expressing cell line
using LNP technology. HDV RNAs were quantified using a
branched DNA assay and nuclear/cytoplasmic fractionation,
and HDAg protein was evaluated by ELISA. Rapid inhibitory
effects were detected 1 day after LNP treatment, with HDAg
mRNA-targeted siRNA showing a marked dose-dependent
inhibitory effect on HDV positive strand RNAs but not on the
negative strand viral genome. The nuclear/cytoplasmic fractionated
RNA assay demonstrated that the cytosolic HDV RNAs
were more susceptible to siRNA than nuclear HDV RNAs. In
contrast to HDAg mRNA-targeted siRNAs that demonstrated
gene silencing at day 1, genome-targeted siRNAs did not
demonstrate gene silencing until day 4, hinting at differences
in kinetics between the two siRNA targeting strategies. HDV-targeted
siRNA-LNP effects were durable, with greater than 1-log
reductions of viral RNAs maintained at even 21 days after a
single treatment. Similarly, a 1-log reduction in HDAg protein
was achieved out to day 12 so far and more extended time
points are to be investigated. In conclusion, a direct HDV-targeted
siRNA-LNP approach can effectively suppress positive
and negative strand HDV RNAs and HDAg protein in vitro, and
provides a promising novel strategy to treat HDV infection. The
efficacy of direct HDV targeting relative to indirect effects from
HBV gene silencing are currently under investigation.
Disclosures:
Xin Ye - Employment: Tekmira Pharmaceuticals Corporation
Nicholas M. Snead - Employment: Tekmira Pharmaceuticals
Trisha R. Barnard - Employment: Tekmira Pharmaceuticals Corporation
Emily P. Thi - Employment: Tekmira Pharmaceuticals
Amy C. Lee - Employment: Tekmira
The following authors have nothing to disclose: Ian MacLachlan
2063
Serum hepatitis B core-related antigen as a treatment
predictor during pegylated interferon therapy in
patients with HBeAg-positive chronic hepatitis B
Natthaya Chuaypen 1 , Nawarat Posuwan 2 , Sunchai Payungporn 1 ,
Yasuhito Tanaka 3 , Yong Poovorawan 4 , Pisit Tangkijvanich 1 ; 1 Biochemistry,
Chulalongkorn University, Bangkok, Thailand; 2 Chulalongkorn
University, Bangkok, Thailand; 3 Virology and Liver unit,
Nagoya City University Graduate School of Medical Sciences,
Nagoya, Japan; 4 Paediatrics, Chulalongkorn University, Bangkok,
Thailand
Background: The role of quantitative serum hepatitis B core-related
antigen (HBcrAg) in patients with chronic hepatitis B
(CHB) receiving pegylated interferon (PEG-IFN) has never been
investigated. The aims of this study were to assess the correlation
of HBcrAg with intrahepatic covalently closed circular
DNA (cccDNA), and compare its usefulness with quantitative
serum HBsAg in patients with HBeAg-positive CHB during
PEG-IFN therapy. Method: We retrospectively analyzed data
of Thai patients with HBeAg-positive CHB treated with PEG-
IFN for 48 weeks. Virological response (VR) was defined as
HBeAg clearance and HBV DNA < 2,000 IU/mL at 24 weeks
post treatment. Paired liver biopsies at weeks 0 and 48 were
analyzed for cccDNA by real-time PCR. HBsAg and HBcrAg
levels were assessed at weeks 0, 4, 12, 24, 48, and 72 by
automated chemiluminescent immunoassays. HBV genotype
was performed by direct sequencing. Results: A total of 46
patients (31 male, mean age 33.2 years) were enrolled.
The distribution of HBV genotypes B and C was 10.9% and
89.1%, respectively. VR was achieved in 15 (32.6%) patients.
Responders had higher cccDNA decline compared with non-responders
(1.7±0.8 vs 0.4±1.1 log 10
copies/cell, P=0.001),
although baseline cccDNA were comparable between groups.
Baseline HBsAg and HBcrAg were significantly correlated
with cccDNA (Pearson correlation, r=0.450, P=0.013 and
r=0.631, P20,000 IU/mL as the cut-off level, the
negative predictive value (NPV) of achieving VR at weeks 12
and 24 were 72.7% and 100%, respectively. For HBcrAg, the
best cut-off level was log 10
8.0 IU/mL, which provided NPV at
week 12 and 24 of 89.5% and 100%, respectively. Conclusion:
These results demonstrated that the convenient quantitative
HBcrAg represented a good serum marker of intrahepatic
cccDNA in patients with HBeAg-positive CHB, which was comparable
with that of quantitative HBsAg. Monitoring HBcrAg
levels during PEG-IFN therapy may be useful to identify patients
with a very low probability of treatment response.
Disclosures:
Yasuhito Tanaka - Grant/Research Support: Chugai Pharmaceutical CO., LTD.,
MSD, Bristol-Myers Squibb; Speaking and Teaching: janssen pharma, Bristol-Myers
Squibb
The following authors have nothing to disclose: Natthaya Chuaypen, Nawarat
Posuwan, Sunchai Payungporn, Yong Poovorawan, Pisit Tangkijvanich
2064
Encapsidation and secretion of HBV RNA can be inhibited
by Core Inhibitors but not by Nucleoside Analogs
Angela Lam, Suping Ren, Christine Espiritu, Mollie Kelly, Vincent
Lau, Robert Vogel, George D. Hartman, Lalo Flores, Klaus Klumpp;
Novira Therapeutics Inc., Doyleston, PA
Background: Infectious HBV particles contain a partially double
stranded DNA genome that is replicated in infected hepatocytes
through reverse transcription of HBV pre-genomic RNA
(pgRNA). HBV capsid, which encloses pgRNA and viral polymerase,
is assembled from HBV core protein building blocks
and is the site of HBV DNA synthesis. HBV core inhibitors
are therefore able to potently inhibit HBV replication. In the
current study, the effect of the HBV core inhibitor NVR-3891 on
HBV RNA encapsidation and secretion was examined along
with other core inhibitors and nucleoside analogs Lamivudine
(LMV) or Tenofovir (TFV). Methods: Intracellular encapsidated
HBV pgRNA was examined by Northern blot. HBV DNA and
RNA levels were determined using Quantigene assays in HBV
infected HepaRG cells and human serum samples. The nature
and function of HBV RNA containing particles was evaluated
using detergent and nuclease treatments, and analytical gradient
centrifugation. Results: NVR-3891 induced HBV core
protein mis-assembly in vitro and inhibited HBV replication
with a mean EC 50
of 1.9 mM in persistently infected differentiated
HepaRG cells. Northern blot analysis of HBV infected
cells showed that pgRNA encapsidation was inhibited by NVR-
3891, while treatment of infected cells with nucleoside analogs
led to an increase in encapsidated pgRNA. Analysis of
the supernatants of infected cells and human sera from HBV
infected patients showed high levels of HBV RNA containing