antiviral potencies of 21 shRNA vectors

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  Journal of Hepatology
Volume 52, Issue 6, June 2010, Pages 817-826
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doi:10.1016/j.jhep.2009.10.038 | How to Cite or Link Using DOI
Copyright © 2010 European Association for the Study of the Liver Published by
Elsevier Ireland Ltd.
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Research Article
Quantitative assessment of the antiviral potencies of 21 shRNA vectors
targeting conserved, including structured, hepatitis B virus sites


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Dianxing Sun1, 2, Christine Rösler2, Karin Kidd-Ljunggren3 and Michael
Nassal2, ,

1 Bethune International Peace Hospital, Dept. of Liver Disease, 398 West
Zhongshan Road, 050082 Shijiazhuang, PR China
2 University Hospital Freiburg, Internal Medicine II/Molecular Biology,
D-79106 Freiburg, Germany
3 Lund University, Dept. of Clinical Sciences, SE-22185 Lund, Sweden

Received 7 September 2009;  revised 16 October 2009;  accepted 19 October
2009.  Available online 1 April 2010.


Background & Aims
RNA interference (RNAi) may offer new treatment options for chronic hepatitis
B. Replicating via an RNA intermediate, hepatitis B virus (HBV) is known to be
principally vulnerable to RNAi. However, beyond delivery, the relevant issues
of potential off-target effects, target site conservation in circulating HBV
strains, and efficacy of RNAi itself have not systematically been addressed,
nor can the different existing data be quantitatively compared. The aim of
this study was to provide such information.
Methods
To focus on the intracellular RNAi process itself and minimise other variables
affecting overall RNAi efficacy, we used a robust co-transfection system to
quantitatively assess the relative potencies of 21 small-hairpin (sh) RNA
vectors, targeting conserved sites throughout the HBV genome, against viral
RNAs, proteins, nucleocapsids, and secreted virions under standardised
conditions.
Results
The approach enabled a distinct efficacy ranking, with the six most potent
shRNAs achieving 95% reductions in virion formation, sequence-specifically and
without detectable interferon induction, yet by differentially affecting
different steps. Efficacy correlated poorly with predictions and was not
principally abolished by target structure. Sequence comparisons suggest that
truly conserved, RNAi-targetable sequences comprise less than 500 nucleotides
of the circulating HBV genomes.

Conclusions
The HBV genome can harbour only a finite number of optimal target sites, but
current predictions are poorly suited to constrain the number of possible
candidates. However, the small size of the highly conserved sequence space
suggests experimental identification as a viable option.


Abbreviations: RNAi, RNA interference; siRNA, small interfering RNA; shRNA,
small-hairpin RNA; RISC, RNA-induced silencing complex; HBV, hepatitis B
virus; IFN, interferon; sgRNA, subgenomic RNA; HBeAg, hepatitis B virus e
antigen; pgRNA, pregenomic RNA; PCR, polymerase chain reaction; qPCR,
quantitative PCR; RT-PCR, real-time PCR; NAGE, native agarose gel
electrophoresis; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GFP, green
fluorescent protein; ASLS, antisense-loop-antisense; SLAS, sense-loop-antisense



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