825
GENE SILENCING BY GAPMERS: A NEW THERAPEUTIC TOOL
IN HBV INFECTION.
Maria Francesca Cortese1,2, Selene Garcia-Garcia1,2, Rosario
Casillas1,2, Rosa Lopez-Martinez1, Beatriz Pacin Ruiz2, Sara
Sopena1,2, David Tabernero1,3, Roser Maria Ferrer-Costa4, Mar
Riveiro Barciela5,6, Maria Asuncion Buti Ferret7,8 and Francisco
Rodriguez-Frías1,2,7, (1)Biochemistry and Microbiology/Liver
Pathology Unit, Vall D’hebron University Hospital, (2)Liver
Unit, Vall D’hebron Research Institute, (3)Liver Pathology
Unit Departments of Biochemistry and Microbiology, Vall
d’ Hebron University Hospital and Universitat Autonoma
De Barcelona, Barcelona, Spain, University Hospital Vall
D’hebron, (4)Department of Biochemistry, Vall D’hebron
University Hospital, Vall D’hebron University Hospital, (5)Liver
Unit, Internal Medicine Department. Hospital Universitari Vall
d’Hebron, Barcelona, Spain, University Hospital Vall D’hebron,
(6)Ciberehd, Instituto Carlos III, Madrid, (7)Centro De
Investigación Biomédica En Red, Enfermedades Hepáticas y
Digestvas (CIBERehd), Instituto De Salud Carlos III, Madrid,
Spain, (8)Liver Unit, Internal Medicine Department. Hospital
Universitari Vall d’Hebron, Barcelona, Spain
Background: Hepatitis B Virus (HBV) infection cannot
be eradicated due to the persistence of cccDNA, which
continuously supports the intracellular expression of viral
proteins Of note, a little but consistent percentage of
successfully treated patients with chronic hepatitis B develops
hepatocarcinoma, reinforcing the need of new therapeutic
strategy HBV X gene (HBX), due to its co-terminal localization,
could be an optimal target for a gene therapy strategy Here
we present a new system of viral gene silencing based on
antisense locked nucleic acid (LNA) Gapmer (GP) that
targets HBX hyperconserved regions Methods: HepG2-
NTCP cells were treated with DMSO 2 5% at least 14 days
and later infected with HBV at 500 multiplicities of genome
equivalents by spinoculation at 37ºC for 1h and incubating
overnight Infected cells were treated with GP (GP1 and/or
GP4 at 50nM, Qiagen) and/or siRNA (50nM) using TransIT-X2
(Mirus) at 48h or 5d post infection (pi) A scrambled control
gapmer was used in each experiment Cells and supernatants
were collected after 72h of treatment Pregenomic RNA
(pgRNA) was extracted from cells through RNAeasy Plus
mini kit (Qiagen) and quantified by RT-qPCR using TaqMan
probe (Roche, Lightcycler® 480) HBsAg and HBeAg were
quantified in supernatants by chemilumiscent enzyme assay
(Roche, COBAS® 8000) Results: Mock-untreated infected
cells productively expressed HBV reaching a peak of 4log
of pgRNA (copies/ng), 16IU/mL of HBeAg and 2U/mL of
HBsAg. Early treatment (at 48hpi) with GP efficiently inhibited
HBV pgRNA related to the scrambled, reaching an average
of 75 2±4 5, 54 1±19 2 and 54 3±15 5% for GP1, GP4 and
GP1+GP4, respectively Similar percentages of inhibition
related to the scrambled were obtained for protein expression
(HBeAg = 79 3±8 3, 60 2±13 8, 70 4±11 6%; HBsAg =
71 1±10 2, 60 8±15 3; 64 8±8 7% for GP1, GP4, GP1+4
respectively) The inhibition was also maintained when
treatment was carried out at 5dpi, reaching a percentage
of 81% for GP1, 78% for GP4 and 69% for siRNA Of note,
co-treatment with GP4 and siRNA partially increased the
inhibition up to 93 3% Conclusion: Gapmers targeting
hyper-conserved regions in HBX gene efficiently inhibit HBV
expression in vitro at either early or late treatment The cotreatment
of gapmers with siRNA targeting the same regions
could be a valuable strategy to inhibit HBV expression at both
nuclear and cytoplasmic level Funding: Instituto de Salud
Carlos III (grant PI18/01436), co-financed by the European
Regional Development Fund (ERDF)
Disclosures:
Mar Riveiro Barciela – AbbVie and Gilead: Speaking and Teaching
Maria Asuncion Buti Ferret – Abbvie, Gilead,Janssen: Advisory Committee
or Review Panel; Abbvie, Gilead,Janssen: Grant/Research Support; Abbvie,
Gilead,Janssen: Speaking and Teaching
The following people have nothing to disclose: Maria Francesca Cortese,
Selene Garcia-Garcia, Rosario Casillas, Rosa Lopez-Martinez, Beatriz Pacin
Ruiz, Sara Sopena, David Tabernero, Roser Maria Ferrer-Costa, Francisco
Rodriguez-Frías 作者: StephenW 时间: 2020-10-30 13:48