studies

HEPATOLOGY, VOLUME 62, NUMBER 1 (SUPPL) AASLD ABSTRACTS 695A
as shown by increased ISG induction. Interestingly, SeP was
found to regulate IFN signal transduction independent of the
JAK-STAT pathway. In vivo, the ISGs Mx-1 and Oas2 were
significantly increased in SepKO mice compared with WT mice
following PolyI:C injection. To explore the clinical significance,
hepatic gene expression was analyzed in 66 CHC patients;
SeP was upregulated in the early stage of liver fibrosis and was
significantly correlated with a poor outcome of IFN therapy.
Conclusion: In CHC, SeP impaired innate immunity. The present
findings would be useful for the development of novel therapeutic
strategies for patients with chronic liver disease with various
etiologies including NASH and CHC.
Disclosures:
Stanley M. Lemon - Advisory Committees or Review Panels: Merck, Santaris,
Abbott, Gilead; Consulting: Achillion, Idenix; Grant/Research Support: Merck,
Tibotec, Scynexis; Speaking and Teaching: Hoffman LaRoche
Shuichi Kaneko - Grant/Research Support: MDS, Co., Inc, Chugai Pharma., Co.,
Inc, Toray Co., Inc, Daiichi Sankyo., Co., Inc, Dainippon Sumitomo, Co., Inc,
Ajinomoto Co., Inc, Bristol Myers Squibb., Inc, Pfizer., Co., Inc, Astellas., Inc,
Takeda., Co., Inc, Otsuka„ÄÄPharmaceutical, Co., Inc, Eizai Co., Inc, Bayer
Japan, Eli lilly Japan
The following authors have nothing to disclose: Kazuhisa Murai, Masao Honda,
Tetsuro Shimakami, Takayoshi Shirasaki, Hirofumi Misu, Toshinari Takamura,
Seishi Murakami
991
Long noncoding RNAs regulate HCV replication and
ISGs in a Jak-STAT independent manner
Xiao Liu, Jian Hong, Dachuan Cai, Sae Hwan Lee, Dahlene Fusco,
Esperance A. Schaefer, Cynthia Brisac, Anna Lidofsky, Wenyu Lin,
Raymond T. Chung; Gastrointestinal Unit, Massachusetts General
Hospital, Harvard Medical School, Boston, MA
Background/ Aims: Long non-coding RNAs (lncRNAs) involve
in many important cellular process regulations. However, their
roles in the innate immune response including type-1 interferon
(IFN) response to HCV infection are not well understood.
We previously identified SART1, a host protein involved in
RNA splicing and pre-mRNA processing, as a regulator of
IFN antiviral effects (Lin et al, J Hepatology 2015, 62:1024).
We sought to explore lncRNA regulators of HCV replication,
the JAK-STAT pathway, and interferon-stimulated genes (ISGs).
Methods: We performed SART1 siRNA knockdown and RNA-
Seq in Huh7.5.1 cells with or without IFN treatment. Bioinformatic
Homo sapiens Genome Browser Gateway analysis was
performed to predict the location of lncRNAs and adjacent
coding genes. Selected lncRNAs, Jak-STAT pathway genes and
ISGs and their proteins, together with HCV replication, were
monitored by qRT-PCR and Western blot in the JFH1 HCV infectious
model. Results: We obtained 6699 lncRNAs regulated
by SART1 and IFN in RNA-Seq. Our bioinformatic analysis
of RNA-Seq data identified a cluster of 60 lncRNAs involved
in IFN’s antiviral regulation. qRT-PCR confirmed that expression
of 14 of these lncRNAs was consistent with the RNA-Seq
data. We then identified 4 anti-sense lncRNA related to ISGs
which negatively regulated the adjacent coding gene expression
based on previous reports. Of these, we selected lncRNA
RP11-670E13.5 (lncRNA-TRIM25) for further characterization
of its regulation of HCV. We found that knockdown of lncRNA
RP11-670E13.5 significantly increased HCV replication compared
to Neg siRNA. We also found that siRNA lncRNA RP11-
670E13.5 significantly reduced TRIM25, MX1, ISG15 mRNA
compared to Neg siRNA in JFH1-infected Huh7.5.1 cells.
Interestingly, STAT1, JAK1, OAS3, and RIG-I mRNA expression
were not affected by the knock-down of lncRNA RP11-
670E13.5. Moreover, we found that siRNA to STAT1 had no
effect on lncRNA RP11-670E13.5 expression. These data suggest
that lncRNA RP11-670E13.5 regulation of HCV is independent
of the Jak-STAT signaling pathway. Conclusion: We
found that lncRNA RP11-670E13.5 has important regulatory
functions of antiviral innate immunity in the JFH1 HCV model.
Our data suggested that lncRNA RP11-670E13.5 regulates
HCV replication independently of the JAK-STAT pathway. We
speculate that RP11-670E13.5 exerts its regulatory function on
the enhancer and promoter activation of nearby protein-coding
(target) genes such as TRIM25, MX1, and ISG15. Further characterization
of the mechanism by which selected lncRNAs that
associate with these genes regulate HCV replication and ISGs
are warranted.
Disclosures:
Dahlene Fusco - Grant/Research Support: Gilead
Raymond T. Chung - Grant/Research Support: Gilead, Mass Biologics, Abbvie,
Merck, BMS
The following authors have nothing to disclose: Xiao Liu, Jian Hong, Dachuan
Cai, Sae Hwan Lee, Esperance A. Schaefer, Cynthia Brisac, Anna Lidofsky,
Wenyu Lin
992
Evaluation of protective and therapeutic strategies
against hepatitis C virus in a syngenic transplantation
mouse model
Matti Sällberg 1 , Sepideh Levander 1 , Fredrik Holmstom 1 , Gustaf
Ahlén 1 , Lars Frelin 1 , Gang Long 2 , Daniel Rupp 2 , Ralf Bartenschlager
2 ; 1 Laboratory Medicine, Karolinska Institutet, Stockholm,
Sweden; 2 Molecular Virology and Infectious Diseases, University
of Heidelberg, Heidelberg, Germany
Key features of the hepatitis C virus (HCV) are its high genetic
plasticity and its ability to establish chronic infection in hepatocytes.
Development of prophylactic vaccines is hampered by
the lack of immune competent small animal models supporting
robust HCV replication. Herein, we devised an immune competent
mouse model that is based on the syngenic transplantation
of H-2 b -restricted Hep56 cells containing a self-replicating
subgenomic HCV replicon RNA of genotype (gt) 2a. To allow
non-invasive in vivo monitoring of tumor growth, cells were
also stably transfected with a luciferase (Luc) reporter gene.
As controls, Hep56 cells stably expressing an enzymatically
active HCV NS3/4A (gt2a) protease complex were used.
Upon subcutaneous injection, all cell lines generated palpable
tumors with sizes peaking around day 8-16 post inoculation.
Tumor growth correlated with an accumulation of inflammatory
cells and central necrotic areas, concomitant with a decrease
of HCV RNA and Luc DNA copy numbers. DNA vaccination
resulted in a robust NS3/4A(gt2a)-specific CD4+ and CD8+
T cell response protecting wild type, but not HCV NS3/4A(gt1a)-transgenic
(Tg) mice against HCV replicon cell tumors suggesting
cross-genotypic tolerance in NS3/4A(gt1a)-Tg mice.
Hep56 cells stably expressing gt2a NS3/4A primed and
boosted a strong CTL-dominated response, whereas the HCV
replicon cells did not, despite comparable levels of antigen
expression. Thus, similar to human infection, hepatocytes with
replicating HCV RNA appear to be poorly immunogenic arguing
that the replicon-cell based syngenic transplantation model
shares features of infected hepatocytes in patients. Unlike other
models, syngenic transplantation of HCV replicon cells can
be rapidly applied to genetically modified immune competent
mice of the widely used C57BL/6J background for the study
of HCV-specific cellular immune responses. This model will be
highly useful in vaccine development for hepatitis C
Disclosures:
Matti Sällberg - Consulting: Chrontech Pharma AB, Abbvie
The following authors have nothing to disclose: Sepideh Levander, Fredrik Holmstom,
Gustaf Ahlén, Lars Frelin, Gang Long, Daniel Rupp, Ralf Bartenschlager