studies

224A AASLD ABSTRACTS HEPATOLOGY, October, 2015
tor was examined by polymerase chain reaction (PCR) and
sequencing method, and the destruction of cccDNA was examined
by KCl precipitation, plasmid-safe ATP-dependent DNase
digestion, rolling circle amplification and quantitative PCR combined
method. Results: All of gRNAs could significantly reduce
the HBsAg or HBeAg production in the culture supernatant,
which was dependent on the region in which gRNA against.
All of dual-gRNAs could efficiently suppress the HBsAg and
HBeAg production for HBV of genotypes A-D, and the efficacy
of dual-gRNAs in suppressing HBsAg and HBeAg production
was significantly increased when compared to the single gRNA
used alone. Furthermore, with PCR direct sequencing we confirmed
that these dual-gRNAs could specifically destroy HBV
expressing template by removing the fragment between the
cleavage sites of the two used gRNAs. The gRNA-miR-HBVgRNA
ternary cassette exerted a synergistic effect in efficiently
inhibiting HBV replication and destroying HBV-expressing template
in vitro and in vivo. We determined that the optimal stem
extended length of pri-miRNA was 38 base pairs in our ternary
cassette. Most importantly, together with RNAi approach
the gRNA-miR-HBV-gRNA ternary cassette showed the potent
activity on the destruction of HBV cccDNA. Conclusions: These
results suggested that dual-gRNAs guided CRISPR/Cas9 system
could efficiently destroy HBV expressing templates (genotypes
A-D). Together with RNAi approach, the gRNA-miR-HBV-gRNA
ternary cassette showed the potent activity on the destruction
of HBV cccDNA. Since HBV cccDNA was an obstacle for the
elimination of chronic HBV infection, thus the CRISPR/Cas9 system
may be a potential tool for the clearance of HBV cccDNA.
Disclosures:
The following authors have nothing to disclose: Jie Wang, Fengmin Lu
both GSEA and IPA. Strikingly, there was a comparable transcriptional
response to TLR7-CM treatment in HBV-infected and
uninfected PHH (p0.80), including a comparable
induction of IFN-stimulated gene (ISG) signature. Notably,
APOBEC3B (A3B) mRNA levels were only weakly induced
by TLR7-CM (mean = 2.6-fold), and A3A expression was not
significantly stimulated. Consistent with the lack of viral interference
in the hepatocyte response to TLR7-CM, HBV infection
did not result in prominent global transcriptional response in
Mock-CM treated PHH. Knock-down of IFNAR1 mRNA (~90%
reduction) abrogated the antiviral activity of TLR7-CM, suggesting
that one or more of the ISGs strongly induced by treatment
(e.g. MX1, IFI16, IFIT1 and IFITM1) may be HBV restriction
factors. Conclusion: These data demonstrate that type I IFN is a
key antiviral cytokine induced by GS-9620 in PBMCs, and that
HBV does not interfere with IFN-signaling in human hepatocytes.
In addition, transcriptional profiling identified candidate
HBV restriction factors, but indicated that APOBEC3A likely
does not mediate the antiviral activity of TLR7-CM in HBV-infected
hepatocytes.
Disclosures:
Li Li - Employment: Gilead Sciences
Congrong Niu - Employment: Gilead Science
Stephane Daffis - Employment: Gilead Sciences
Hilario Ramos - Employment: Gilead Sciences
Eduardo Salas - Employment: Gilead Sciences; Stock Shareholder: Gilead Sciences
Christian Voitenleitner - Employment: Gilead Sciences
William E. Delaney - Employment: Gilead Sciences; Patent Held/Filed: Gilead
Sciences; Stock Shareholder: Gilead Sciences
Simon P. Fletcher - Employment: Gilead Sciences; Stock Shareholder: Gilead
Sciences
35
HBV Does Not Modulate the Transcriptional Response
to TLR7-Induced Cytokines in Highly Infected Primary
Human Hepatocytes
Li Li, Congrong Niu, Stephane Daffis, Hilario Ramos, Eduardo
Salas, Christian Voitenleitner, William E. Delaney, Simon P.
Fletcher; Gilead Sciences, Foster City, CA
Background & Aims: GS-9620, an oral agonist of toll-like
receptor 7 (TLR7), is in phase 2 trials for the treatment of
chronic hepatitis B (CHB). We previously demonstrated that
prolonged exposure to antiviral cytokines directly induced by
TLR7 activation, such as interferon-α (IFN-α), potently inhibited
HBV in primary human hepatocytes (PHH) in vitro. Here we
characterized the transcriptional response of PHH to TLR7-induced
cytokines in order to identify potential HBV restriction
factors and to determine whether HBV infection modulates
signaling of these cytokines in hepatocytes. Methods: PHH
from two different donors were infected with HBV or mock
infected, and 13 days later were treated with media from
healthy human PBMCs stimulated with either GS-9620 (TLR7
conditioned media; TLR7-CM) or DMSO (control; Mock-CM).
High level infection (≥75% PHH infected) was confirmed by
HBV core staining. Whole transcriptome analysis of PHH
± HBV at 8, 24 and 48 hours post-treatment with TLR7-CM
and Mock-CM was performed by RNA-Seq. Transcriptional
responses were characterized by gene set enrichment analysis
(GSEA) and Ingenuity Pathway Analysis (IPA). Knock-down of
IFN-α/β receptor 1 (IFNAR1) mRNA was performed by siRNA,
and included a negative (scramble) control. Results: TLR7-CM
induced a comparable transcriptional signature in PHH from
two independent donors. In both donors, TLR7-CM induced the
greatest transcriptional response at 8 hours post-treatment, with
IFN signaling pathways being the top induced pathways by
36
GalNAc-siRNA conjugate ALN-HBV targets a highly
conserved, pan-genotypic X-orf viral site and mediates
profound and durable HBsAg silencing in vitro and in
vivo
Laura Sepp-Lorenzino, Andrew G. Sprague, Tara Mayo, Tuyen
M. Nguyen, Svetlana Shulga Morskaya, Huilei Xu, Stuart Milstein,
Greg Hinkle, Pia Kasperkovitz, Richard G. Duncan, Natalie Keirstead,
Brenda Carito, Lauren Moran, Prasoon Chaturvedi, Krishna
C. Aluri, Husain Attarwala, Renta M. Hutabarat, Ju Liu, Chris Tran,
Qianfan Wang, Benjamin S. Brigham, Akin Akinc, Klaus B. Charisse,
Vasant Jadhav, Satya Kuchimanchi, Martin A. Maier, Muthiah
Manoharan, Rachel Meyers, Tadeusz Wyrzykiewicz, Haroon
Hashmi, Julie Donovan, Tim Mooney, Daniel Freedman, Tanya
P. Sengupta, Karin Galil, Eoin Coakley, Patrick Haslett; Alnylam
Pharmaceuticals, Cambridge, MA
Background: Despite the prevalence of chronic HBV infection
(CHB), there are no highly effective therapies allowing sustained
off-treatment viral control and/or cure of HBV infection.
An RNAi therapeutic targeting the HBV genome has the potential
to achieve a “functional cure” by effectively decreasing
expression of all viral gene products, including tolerogenic viral
antigens, e.g., HBsAg. Methods and Results: ALN-HBV is a
hepatocyte-targeted GalNAc-siRNA conjugate with Enhanced
Stabilization Chemistry (ESC) for subcutaneous (SC) administration.
It targets a site in the HBV X protein open reading
frame (ORF), thereby targeting all four HBV RNA transcripts
for degradation by RNA interference. Bioinformatic analyses
indicate a >95% perfect sequence homology with a database
of 3,950 full length HBV viral genomes incorporating genotypes
A through J, and a low potential for off-target activity
against host genes. Pan-genotypic activity was confirmed in