Inhibition of HBV replication by siRNA in a stable HBV-producing cell line

Konishi, Masayoshi; Wu, Catherine H.; Wu, George Y.

doi:10.1053/jhep.2003.50416

Cited by 71 publications

(14 citation statements)

References 39 publications

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“…R ibonucleic acid interference is a naturally occurring mechanism for silencing homologous genes. 1,2 Synthetic and plasmid-based siRNA expression systems are both effective at suppressing HBV infection, in vitro and in vivo, [3][4][5][6][7][8][9] providing the basis for a new therapeutic strategy against HBV. However, the absence of a suitable delivery system presents a major obstacle against their clinical use.…”

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confidence: 99%

Targeted inhibition of HBV gene expression by single-chain antibody mediated small interfering RNA delivery

et al. 2007

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RNA interference is highly effective at inhibiting HBV gene expression and replication. However, before small interfering RNA (siRNA) can be used in the clinic, it is essential to develop a system to target their delivery. Antibody-mediated delivery is a novel approach for targeting siRNA to appropriate cells. In this report, we asked whether this siRNA delivery strategy would be effective against HBV. Of 5 candidates, a specific siRNA that effectively inhibited HBV gene expression and replication was determined. Two fusion proteins, s-tP and sC-tP, were constructed to contain a single chain of the human variable fragment, scFv, against hepatitis B surface antigen (HBsAg), a truncated protamine (tP), and in the case of sC-tP, a constant region of the chain (C). S-tP and sC-tP were developed to provide targeted delivery of the siRNA, siRNA expressing cassettes (SEC), and siRNA-producing plasmids. Fluorescein isothiocyanate-siRNA, fluorescein isothiocyanate-SEC, and plasmid DNA were specifically delivered into HBsAg-positive cells using the sC-tP fusion protein, and effectively inhibited HBV gene expression and replication. HBV gene expression was also inhibited by siRNA or siRNA-producing plasmids in HBV transgenic mice. Conclusion: Our results describe a potential method for the targeted delivery of siRNA or siRNAproducing plasmids against HBV, using anti-HBsAg fusion proteins. (HEPATOLOGY 2007;46: 84-94.) R ibonucleic acid interference is a naturally occurring mechanism for silencing homologous genes. 1,2 Synthetic and plasmid-based siRNA expression systems are both effective at suppressing HBV infection, in vitro and in vivo, [3][4][5][6][7][8][9] providing the basis for a new therapeutic strategy against HBV. However, the absence of a suitable delivery system presents a major obstacle against their clinical use. [10][11][12] Targeted delivery of small interfering RNA (siRNA) to relevant cell populations should increase the therapeutic index and minimize potential side effects and cellular toxicity.Antibody-mediated delivery is an effective method of targeting siRNA to particular cells. This involves fusing the nucleic acid-binding domain to a Fab fragment or scFv that recognizes a membrane receptor, resulting in a fusion protein that possesses cell recognition and nucleic acid-binding abilities. The fusion protein can then bind nucleic acids and deliver them into target cells through receptor-mediated endocytosis. This antibody-mediated uptake has been successfully used to deliver plasmid DNA and antisense oligonucleotides. [13][14][15][16][17] Song et al. 18 demonstrated that siRNA targeted delivered to human immunodeficiency virus-infected cells and HER2 positive cells by this strategy executed gene silencing. However, whether this promising strategy for siRNA delivery is applicable to other diseases remains unknown. 19 In this report, we provide evidence that antibody-mediated siRNA delivery is effectively targeted to HBV-infected cells.In a previous study, we have obtained 5 human antihepatitis B sur...

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confidence: 99%

Targeted inhibition of HBV gene expression by single-chain antibody mediated small interfering RNA delivery

et al. 2007

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“…Several therapeutic strategies against HBV, such as interferon-α (3), nucleotide analogues (19-21), antisense DNA (22), ribozyme (23) and RNA interference (24,25), have been reported in clinical or laboratory investigations. However, to date no ideal treatment for HBV eradication has been reported.…”

Section: Discussionmentioning

confidence: 99%

Ultrastructural demonstration of hepatitis B virus production in a mouse model produced by hydrodynamic transfection

Konishi

Tanaka²,

Kaito³

et al. 2007

Int J Mol Med

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Abstract. A mouse model of hepatitis B virus (HBV)infection produced by hydrodynamic injection of HBV DNA has been recently established. However, the ultrastructural demonstration of HBV particles in this mouse model has not as yet been reported. In our study, plasmid DNA containing wild-type HBV DNA was rapidly injected into 8-week-old female SCID mice via the tail vein. Serum levels of HBsAg were measured by ELISA kit. Intrahepatic HBV protein expression was detected by immunohistochemistry of HBcAg. Ultrastructural study of the serum samples was performed by transmission electron microscopy and immunogold electron microscopy. Serum HBsAg and intrahepatic HBcAg were detected in HBV DNA-injected mice for at least 14 days. Spherical and filamentous particles 22 nm in diameter and double-shelled Dane-like particles 42 nm in diameter were detected in the sera of mice. The ultrastructural features of these particles were identical to HBV particles observed in serum from chronic hepatitis B patients. These particles were confirmed to be HBV particles by immunogold electron microscopy. We conclude that our present HBV mouse model using hydrodynamic transfection of HBV DNA is appropriate for production of HBV virions including Dane particles. This mouse model may be useful for screening in vivo the efficacy of antiviral drugs.

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“…Several groups have reported using siRNAs to inhibit HBV replication in cell cultures and animal models [12][13][14][15][16][17][18][19] . Although all of these reports showed that HBV replication was inhibited to various degrees by siRNAs, several factors must be taken into account before siRNAs are used to treat HBV infection.…”

Section: Discussionmentioning

confidence: 99%

“…The physiological functions of RNAi are not very clear as yet, but it has been used successively as a potent method of gene knockdown in study of gene function and in experimental treatment of some diseases [4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19] . In mammalian cells, double-stranded RNAs inducing RNAi must be short in length (<30 bp) so that they would not activate nonspecific interferon reactions.…”

Section: Introductionmentioning

confidence: 99%

Effect of vector-expressed siRNA on HBV replication in hepatoblastoma cells

Liu

Guo²,

Xue³

et al. 2004

WJG

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AIM:To study the effect of siRNA expressed from DNA vector on HBV replication. METHODS:Human U6 promoter was amplified from genomic DNA and cloned into plasmid pUC18 to construct a mammalian siRNA expression vector pUC18U6. Then oligonucleotides coding for a short hairpin RNA against HBV were cloned into pUC18U6 to form pUC18U6HBVsir which was introduced into 2.2.15 cells by using liposome-mediated transfection. 2.2.15 cells transfected by pUC18U6 and pUC18U6GFPsir which expressed siRNA against green fluorescent protein and mock-transfected 2.2.15 cells were used as controls. Concentration of HBsAg in the supernatant of the transfected cells was measured by using solid-phase radioimmunoassay. RESULTS:A mammalian siRNA expression vector pUC18U6 was constructed successfully. Compared with controls, pUC18U6HBVsir which expressed siRNA against HBV decreased concentration of HBsAg significantly by 44%(P<0.05).CONCLUSION: HBV replication in 2.2.15 cells is inhibited by siRNA expressed from the DNA vector.

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Inhibition of HBV replication by siRNA in a stable HBV-producing cell line

Cited by 71 publications

References 39 publications

Targeted inhibition of HBV gene expression by single-chain antibody mediated small interfering RNA delivery

Targeted inhibition of HBV gene expression by single-chain antibody mediated small interfering RNA delivery

Ultrastructural demonstration of hepatitis B virus production in a mouse model produced by hydrodynamic transfection

Effect of vector-expressed siRNA on HBV replication in hepatoblastoma cells

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