The short hairpin RNA driven by polymerase II suppresses both wild-type and lamivudine-resistant hepatitis B virus strains.
BACKGROUND
Chronic infection with hepatitis B virus (HBV) is widespread because of the limited availability of therapeutic treatments. Although previous reports have suggested that RNA interference has promise as a treatment for HBV infection, further studies of long-term and off-target drug effects on HBV, especially on drug-resistant strains of HBV, are needed. Therefore, seven vectors that express short hairpin RNAs (shRNAs), driven by the polymerase II promoter, pSilencer4.1/HBV, were constructed to target open reading frames (ORFs) of the HBV C and S genes from wild-type and drug-resistant strains. Treatment efficiency was also assessed.


METHODS
The pSilencer4.1/HBV vectors were investigated in HepG2.2.15 cells and transgenic mice that consistently produce wild-type HBV. Additionally, vectors that produce a lamivudine-resistant strain of HBV were developed and cotransfected, along with pSilencer/HBV, into both HepG2 cells and mice. The effects of polymerase-II-driven pSilencer4.1/HBV were compared with those of polymerase-III-driven pSilencer3.1/HBV at both the gene and protein level.


RESULTS
pSilencer4.1/HBV inhibited the expression of viral protein, DNA and HBV subtype ayw mRNA in both HepG2.2.15 cells and transgenic mice. Toxicity, as well as off-target effects, did not occur after a short- to medium-term examination. Moreover, an HBV strain resistant to lamivudine, subtype adr, was suppressed by shRNA in both HepG2 cells and mice. In contrast to polymerase III, vectors that used polymerase II could drive efficient silencing without off-target effects.


CONCLUSIONS
Silencing by shRNA dramatically inhibited HBV expression and replication regardless of strain type. ShRNA could therefore be a promising treatment for HBV infection.
Ren
Guang-Li
GL
Department of Pediatrics, General Hospital of GuangZhou Military Command of PLA, GuangZhou, China. guangliren@hotmail.com
Fang
Ying
Y
Ma
Heng-Hao
HH
Lei
Ying-Feng
YF
Wang
Dan
D
Xu
Man-Chun
MC
Wang
Ping-Zhong
PZ
Huang
Chang-Xing
CX
Nie
Oing-He
OH
Sun
Yong-Tao
YT
Bai
Xue-Fan
XF
D000818:Animals; D000998:Antiviral Agents / Q000494:pharmacology / Q000627:therapeutic use; D045744:Cell Line, Tumor; D004279:DNA, Viral; D024882:Drug Resistance, Viral; D015967:Gene Expression Regulation, Viral; D005822:Genetic Vectors; D006514:Hepatitis B Surface Antigens / Q000302:isolation & purification / Q000378:metabolism*; D006515:Hepatitis B virus / Q000187:drug effects / Q000235:genetics / Q000302:isolation & purification / Q000502:physiology*; D019694:Hepatitis B, Chronic / Q000378:metabolism / Q000628:therapy*; D006801:Humans; D019259:Lamivudine / Q000494:pharmacology* / Q000627:therapeutic use; D008099:Liver / Q000166:cytology / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D008822:Mice, Transgenic; D034622:RNA Interference; D012319:RNA Polymerase II / Q000378:metabolism; D012333:RNA, Messenger; D034741:RNA, Small Interfering / Q000627:therapeutic use*
D000998:Antiviral Agents; D004279:DNA, Viral; D006514:Hepatitis B Surface Antigens; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D019259:Lamivudine; D012319:RNA Polymerase II