Therapeutic inhibition of hepatitis B virus surface antigen expression by RNA interference

Cheng, Tsung Lin; Chang, Wen‐Wei; Su, Ih Jen; Lai, M. M. C.; Huang, Wenya; Lei, Huan Yao; Chang, Wen-Tsan

doi:10.1016/j.bbrc.2005.08.173

Cited by 21 publications

(14 citation statements)

References 58 publications

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“…Similar results were also reported in the literature using large dsRNA and long shRNA. 33,35 While we and these authors achieved high gene silencing compared to single shRNA, our strategy is somewhat different from theirs. The long shRNA comprises 62 bp with multiple G:U wobble pairings to facilitate cleavage into multiple siRNAs by DICER.…”

Section: Discussionmentioning

confidence: 86%

“…The rest three siRNAs also inhibited HBsAg to different extent. 21,33 The difference in inhibition among various siRNA may be attributed to the change of affinity between cellular siRNA binding protein and the secondary and/or tertiary structures of target mRNA participating in the induction of RNAi activities. 34 Since we achieved high levels of HBsAg gene silencing when we used a pool of chemically synthesized siRNAs, we postulated that multiple shRNAs cloned into a single U6 promoter would produce a pool of siRNAs after cleavage of dsRNA by Dicer, leading to significant increase in the intracellular siRNA levels.…”

Section: Discussionmentioning

confidence: 99%

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siRNA pool targeting different sites of human hepatitis B surface antigen efficiently inhibits HBV infection

Yong

Mahato

2008

Journal of Drug Targeting

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The main objective was to determine whether a pool of siRNAs targeting different regions of hepatitis B virus surface antigen (HBsAg) efficiently inhibits HBV infection. siRNAs targeting different regions of HBsAg were transfected into HBV-producing HepG2.2.15 cells and at 72h post transfection, the culture medium was collected for ELISA to determine HBsAg, while total RNA was isolated from the cells for real-time PCR. Three siRNA sequences which efficiently inhibited HBV infection were converted into small hairpin RNAs (shRNAs) and then cloned into a single plasmid psiSTRIKE™ driven by a single U6 promoter. These shRNA expressing plasmids were tested for HBsAg gene silencing in HepG2.2.15 cells. A pool of siRNAs targeting HBsAg efficiently inhibited HBV replication and antigen expression when transfected into HepG2.2.15 cells, compared to the use of single siRNA. Similarly, the plasmid encoding three different shRNAs driven by a single U6 promoter was more effective in silencing HBsAg at DNA, mRNA and protein levels compared to the plasmid encoding single shRNA. No apoptotic change was observed in the cells when the plasmid was transfected at a dose of 0.5-2μg/1×10 6 cells after complex formation with Lipofectamine LTX™. Furthermore, transfection with siRNA or shRNA did not increase IFN-γ release, suggesting no induction of interferon response. In conclusion, a pool of chemically synthesized siRNAs as well as the shRNA expression plasmid encoding multiple shRNAs targeting different regions of HBsAg showed high gene silencing in HepG2.2.15 cells.

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Section: Discussionmentioning

confidence: 86%

Section: Discussionmentioning

confidence: 99%

siRNA pool targeting different sites of human hepatitis B surface antigen efficiently inhibits HBV infection

Yong

Mahato

2008

Journal of Drug Targeting

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“…For this, the post-transcriptional gene silencing (PTGS) by doublestranded RNA (dsRNA) or RNA interference (RNAi) has emerged as a new tool for studying gene function in an increasing number of organisms (Fire 1999). Following its application in Caenorhabditis elegance (Fire et al 1998), the RNAi approach by introduction of dsRNA has been used in various mammalian species such as human (Brusselmans et al 2005, Cheng et al 2005, Hyslop et al 2005, mouse (Svoboda et al 2000, Wianny & ZernickaGoetz 2000, Grabarek et al 2002, Stein et al 2003, Alizadeh et al 2005, Gui & Joyce 2005, Plusa et al 2005) and porcine (Cabot & Prather 2003). Most recently, the work by our group (Nganvongpanit et al 2006) and others (Paradis et al 2005) have used the RNAi approach to suppress transcripts in bovine embryos and oocytes respectively.…”

Section: Introductionmentioning

confidence: 99%

Selective degradation of maternal and embryonic transcripts in in vitro produced bovine oocytes and embryos using sequence specific double-stranded RNA

Nganvongpanit

Müller²,

Rings³

et al. 2006

Reproduction

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RNA interference (RNAi) has been used for selective degradation of an mRNA transcript or inhibiting its translation to a functional protein in various species. Here, we applied the RNAi approach to suppress the expression of the maternal transcript C-mos and embryonic transcripts Oct-4 in bovine oocytes and embryos respectively, using microinjection of sequence-specific double-stranded RNA (dsRNA). For this, 435 bp C-mos and 341 bp Oct-4 dsRNA were synthesized and microinjected into the cytoplasm of immature oocytes and zygotes respectively. In experiment 1, immature oocytes were categorized into three groups: those injected with C-mos dsRNA, RNase-free water and uninjected controls. In experiment 2, in vitro produced zygotes were categorized into three groups: those injected with Oct-4 dsRNA, RNase-free water and uninjected controls. The developmental phenotypes, the level of mRNA and protein expression were investigated after treatment in both experiments. Microinjection of C-mos dsRNA has resulted in 70% reduction of C-mos transcript after maturation compared to the water-injected and uninjected controls (P!0.01). Microinjection of zygotes with Oct-4 dsRNA has resulted in 72% reduction in transcript abundance at the blastocyst stage compared to the uninjected control zygotes (P!0.01). Moreover, a significant reduction in the number of inner cell mass (ICM) cells was observed in Oct-4 dsRNA-injected embryos compared to the other groups. From oocytes injected with C-mos dsRNA, 60% showed the extrusion of the first polar body compared to 50% in water-injected and 44% in uninjected controls. Moreover, only oocytes injected with C-mos dsRNA showed spontaneous activation. In conclusion, our results demonstrated that sequence-specific dsRNA can be used to knockdown maternal or embryonic transcripts in bovine embryogenesis.

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“…8,14,18,40,[45][46][47][48][49][50][51][52] The in vitro studies comprise transiently shRNA-transfected cells, which were either co-transfected with plasmids encoding a full-length HBV genome, or already contained the viral DNA. The in vivo approaches were similar, in that the HBV DNA was either transiently introduced by hydrodynamic tail vein injection of plasmid DNA, or stably integrated into the genome of transgenic mice.…”

Section: Hbv As An Shrna Targetmentioning

confidence: 99%

“…This is because they do not promote HBV eradication from the infected host, thus resulting in relapse and recurrence of viremia after cessation of treatment. 4 An additional complication arises from the virus' ability to form escape mutants with prolonged treatment which are resistant to existing drugs, 14 a phenomenon that is an even greater problem with HCV infection.…”

Section: Introductionmentioning

confidence: 99%

Therapeutic short hairpin RNA expression in the liver: viral targets and vectors

Grimm

Kay

2006

Gene Ther

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Over 500 million people worldwide are infected with one or more different and unrelated types of human hepatitis virus. Such individuals are at a high risk of developing acute or chronic hepatic disease, and ultimately dying from sequelae. Although a vaccine is available for hepatitis A and B virus, treatment options for chronically infected patients are limited, and particularly ineffective in case of hepatitis C virus (HCV) infection. A promising new avenue currently being explored is to harness the power of RNA interference for development of an antiviral therapy. The timing to pursue this particular approach is excellent, with the first in vivo animal models for HCV infection becoming available, and the technology for liver-specific expression of short hairpin RNAs advancing at a rapid pace. Here, we critically review these important current developments, and discuss the next steps to bring this novel approach into the clinics.

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Therapeutic inhibition of hepatitis B virus surface antigen expression by RNA interference

Cited by 21 publications

References 58 publications

siRNA pool targeting different sites of human hepatitis B surface antigen efficiently inhibits HBV infection

siRNA pool targeting different sites of human hepatitis B surface antigen efficiently inhibits HBV infection

Selective degradation of maternal and embryonic transcripts in in vitro produced bovine oocytes and embryos using sequence specific double-stranded RNA

Therapeutic short hairpin RNA expression in the liver: viral targets and vectors

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