403
Antiviral Activity of a New siRNA Targeting a
Hyper-Conserved Region in Hepatitis B X Gene
Transcripts: Preliminary Data.
Sheila Maestro1,2, Maria Francesca Cortese3,4, Carolina
González3, David Tabernero3,5, Mar Riveiro Barciela5,6,
Maria Buti5,7, Gloria González-Aseguinolaza1,2 and Francisco
Rodríguez-Frías3,5, (1)Gene Therapy and Regulation of Gene
Expression Program, Center for Applied Medical Research
(CIMA), (2)Instituto De Investigación Sanitaria De Navarra
(IdiSNA), (3)Biochemistry and Microbiology/Liver Pathology
Unit, Vall D’hebron University Hospital, (4)Liver Unit, Vall
D’hebron Research Institute, (5)Ciberehd, Instituto De Salud
Carlos III, (6)Department of Internal Medicine/Liver Unit, Vall
D’hebron University Hospital, (7)Hospital Universitari Vall
d’Hebron, Barcelona, Spain
Background: Functional cure of chronic hepatitis B (CHB)
by standard nucleos(t)ide analog-based treatment is rarely
achieved, with the risk of hepatocellular carcinoma (HCC)
remaining present. The hepatitis B X gene (HBX) plays a
key role in this process. Using next-generation sequencing
(NGS) a previous study by our group (González et al. World J
Gastroenterol 2018;24:2095) identified two hyper-conserved
regions in 5’ end of HBX [nucleotide (nt) 1255-1286; nt
1519-1603] regardless of the patients’ clinical stage or HBV
genotype. The aim of this study was to determine the antiviral
activity of a small interference RNA (siRNA) battery targeting
these regions. Methods: siRNAs were identified using the
iScore Designer. In order to evaluate their antiviral activity,
HepG2-NTCP cells were infected with purified HBV, and
48 hours post-infection they were transfected with siRNAs
(alone or in combination) using lipofectamine. A known siRNA
targeting X transcripts (siRNA4) and a non-specific scrambled
siRNA were used as controls. The expression of hepatitis
B X (HBx), core (HBc) and surface (HBs) antigen mRNAs
was determined by real time PCR at different time points.
Results: We identified 3 potential siRNAs (1-3) targeting
the described hyper-conserved regions. Mock-infected cells
were productively infected and expressed HBs, HBc and
HBx transcripts. siRNAs efficiently reduced gene expression
from 48h to 96h post-transfection (Figure). Of note, siRNA1
and siRNA4 were more potent inhibitors of viral expression
(percentage of HBx expression relative to scrambled siRNA:
18.6 and 29.5% for siRNA1 and siRNA4, respectively vs. 32.1
and 44% for siRNA2 and siRNA3 at 96h, Figure panel C). The
combination of siRNA1+4 further increased the inhibition of
gene expression with a percentage of HBV antigen expression
relative to scrambled siRNA of 27.5, 5.6 and 22% for HBc,
HBs and HBx, respectively at 96h (Figure). Conclusion: By
targeting a hyper-conserved region of HBX, siRNA1 efficiently
reduces HBV gene expression in vitro. The use of this siRNA
alone or in combination with other known siRNAs could help
to control HBV infection and prevent disease progression and
HCC development. Further in vitro and in vivo studies are
required to confirm these results. Funding: Instituto de Salud
Carlos III (grant PI15/00856) co-financed by the European
Regional Development Fund (ERDF).作者: StephenW 时间: 2018-10-16 08:33