Quantitative assessment of the antiviral potencies of 21 shRNA vectors targeting conserved, including structured, hepatitis B virus sites.
OBJECTIVE
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.
Sun
Dianxing
D
Bethune International Peace Hospital, Departmrnt of Liver Disease, 398 West Zhongshan Road, 050082 Shijiazhuang, PR China.
Rösler
Christine
C
Kidd-Ljunggren
Karin
K
Nassal
Michael
M
D001483:Base Sequence; D002460:Cell Line; D017124:Conserved Sequence; D004797:Enzyme-Linked Immunosorbent Assay; D015967:Gene Expression Regulation, Viral / Q000235:genetics; D015316:Genetic Therapy / Q000379:methods*; D016679:Genome, Viral; D006514:Hepatitis B Surface Antigens / Q000235:genetics / Q000378:metabolism; D006513:Hepatitis B e Antigens / Q000235:genetics / Q000378:metabolism; D006515:Hepatitis B virus / Q000235:genetics* / Q000254:growth & development; D019694:Hepatitis B, Chronic / Q000628:therapy* / Q000821:virology*; D006801:Humans; D016898:Interferon-alpha / Q000235:genetics; D009690:Nucleic Acid Conformation; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D034622:RNA Interference; D016372:RNA, Antisense / Q000235:genetics; D012367:RNA, Viral / Q000737:chemistry / Q000235:genetics*; D014162:Transfection / Q000379:methods; D014779:Virus Replication / Q000235:genetics
D006514:Hepatitis B Surface Antigens; D006513:Hepatitis B e Antigens; D016898:Interferon-alpha; D016372:RNA, Antisense; D012367:RNA, Viral