Long-term inhibition of hepatitis B virus in transgenic mice by double-stranded adeno-associated virus 8-delivered short hairpin RNA

RNA interference (RNAi) was reported to block hepatitis B virus (HBV) gene expression and replication in vitro and in vivo. However, it remains a technical challenge for RNAi-based therapy to achieve long-term and complete inhibition effects in chronic HBV infection, which presumably requires more extensive and uniform transduction of the whole infected hepatocytes. To increase the in vivo transfection efficiency in liver, we used a double-stranded adeno-associated virus 8-pseudotyped vector (dsAAV2/8) to deliver shRNA. HBV transgenic mice were used as an animal model to evaluate the inhibition effects of the RNAi-based gene therapy. A single administration of dsAAV2/8 vector, carrying HBV-specific shRNA, effectively suppressed the steady level of HBV protein, mRNA and replicative DNA in liver of HBV transgenic mice, leading to up to 2-3 log(10) decrease in HBV load in the circulation. Significant HBV suppression sustained for at least 120 days after vector administration. The therapeutic effect of shRNA was target sequence dependent and did not involve activation of interferon. These results underscore the potential for developing RNAi-based therapy by dsAAV2/8 vector to treat HBV chronic infection, and possibly other persistent liver infections as well.     

靶向C区基因的M1GS RNA核酶胞内抗乙型肝炎病毒的实验研究

目的探讨靶向作用于乙型肝炎病毒C区基因的M1GS RNA核酶胞内抗乙型肝炎病毒的作用。方法设计并应用PCR技术合成M1RNA核酶，应用pEGFP-C1载体构建M1GSRNA核酶的真核表达质粒，应用脂质体Lipofectamine TM 2000转染HepG2．2．15细胞，以ELISA法检测细胞培养液中病毒抗原，以反转录-聚合酶链反应法（RT-PCR）检测细胞内病毒mRNA，以荧光定量PCR法检测分泌入培养液的HBVDNA含量，用MTT法检测核酶对细胞增殖活性的影响。结果质粒载体表达的M1GS RNA核酶能明显抑制细胞培养液中HBeAg的表达及病毒mRNA的表达，抑制率分别为31．58％，32．5％。但M1GS RNA核酶对培养液中的HBV DNA含量无明显影响，亦不影响HepG2．2．15细胞的增殖活性。结论M1GS RNA核酶能特异性抑制HepG2．2．15细胞内HBV C区基因表达，是一种很有潜力的抗HBV基因治疗方法。     

Treatment of children persistently infected with hepatitis B virus: seroconversion or suppression

We have reviewed the current strategies regarding the treatment of persistent hepatitis B virus (HBV) in children and compared these with adult strategies. The options for achieving suppression of viral DNA replication versus hepatitis B e antigen to antibody seroconversion have been evaluated. The results of studies in different geographical locations have been confounded by HBV genotypes, as it is now clear that some genotypes respond better to treatment than others. Consideration needs to be given as to whether optimal treatment strategies developed for adults are directly applicable to children. In children, early seroconversion to allow improved long-term outcomes should be considered rather than embarking on the long-term complexities of managing patients on a combination of antiviral drugs to achieve viral suppression.     

核激素受体等对乙型肝炎病毒转录与复制的影响

目的建立乙型肝炎病毒(HBV)在非肝源细胞复制体系,观察核激素受体等对HBV基因转录和复制的调控作用。方法用复制型HBV重组质粒pHBV4.1与核激素受体HNF4、和RXRa/PPARa以及HNF3的表达质粒,分别共转染非肝源细胞株NIH3T3;用Northern吸印杂交检测HBV 3.5 kb、2.4/2.1 kb、0.7 kb mRNA的转录情况,Southern吸印杂交检测HBV DNA复制中间体水平。结果复制型HBV重组质粒pHBV4.1在肝癌细胞中,能检测到3.5 kb HBV RNA转录产物和HBV DNA复制中间体;用pHBV4.1转染NIH3T3细胞后,在没有核激素受体表达质粒共转染时未能检出3.5 kb HBV RNA等转录产物,也无HBV DNA复制中间体;当用核激素受体HNF4和RXRa/PPARa共转染时,能够激活3.5 kb HBV RNA转录和HBV DNA复制,而HNF3未能激活HBV复制,但在核激素受体HNF4或RXRα/PPARα介导的病毒复制体系中,同时共转染HNF3时,均可见3.5 kb HBV mRNA的转录和HBV DNA复制水平下降。结论利用核激素受体等成功地建立HBV在非肝源细胞中的转录与复制,且显示HNF4和RXRa/PPARa可支持HBV在非肝源细胞中转录与复制;HNF3抑制HBV肝特异性基因转录与复制。     

Entecavir for the long-term treatment of chronic hepatitis B

Hepatitis B virus is a major cause of chronic liver disease worldwide and is associated with an increased risk of hepatocellular carcinoma, progressive hepatic fibrosis and end-stage liver disease. Suppression of HBV replication is recognized as the primary on-treatment goal of antiviral therapy, as reduction of serum HBV DNA to low or undetectable levels is highly likely to have a positive impact on long-term clinical outcomes in HBV-associated chronic liver disease. Entecavir is an oral nucleoside analogue that effectively inhibits HBV polymerase, resulting in rapid viral suppression. Long-term data on patients receiving entecavir for chronic hepatitis B have demonstrated high potency, a low incidence of antiviral drug resistance and good tolerability, making entecavir an ideal first-line agent for the treatment of chronic hepatitis B.     

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.     

反义RNA抑制乙肝病毒复制的临床分子生物学研究

目的 研究表达反义RNA的逆转录病毒载体体外转基因抗乙肝病毒（HBV）的作用。为临床治疗乙型肝炎探索一条新途径。方法 合成互补于HBVX区（1400-1430）的X片段,互补于HBVP区（2375-2405）的P片段,分别构建表达正、反义X或P的重组逆转录病毒载体质粒PLXSN Xpos/Ppos,PLXSN X,PLXSN P,转染PA317细胞,用假病毒颗粒感染2.2.15细胞后第8天,提取细胞内DNA和RNA,用荧光PCR（FQ-PCR）定量方法检测2.2.15细胞的DNA和RNA。结果 反义RNA对HBVS、C、P三个开放读码区的DNA及RNA抑制作用大。结论 细胞内表达HBV反义RNA。结果 反义RNA对HBVS、C、P三个开放读码区的DNA及RNA抑制作用大。结论 细胞内表达HBV反义RNA是有明显抗乙肝病毒复制和表达的作用。为将来临床应用多基因区抗乙肝病毒治疗及抗变异病治疗打下基础。     

Use of RNA interference to modulate liver adenoma development in a murine model transgenic for hepatitis B virus

Chronic hepatitis B virus (HBV) infection is closely related to the development of severe liver complications, including hepatocellular carcinoma. In previous studies, we reported that in vivo long-term HBV suppression in transgenic mice can be achieved without apparent toxicity by short hairpin RNA sequentially delivered using adeno-associated viral (AAV) vectors of different serotypes. Our goal herein was to address the clinical utility of this delivery system and, in particular, to determine whether RNA interference (RNAi) and its ability to induce long-term HBV suppression will modulate the development of HBV-associated liver pathology. As a model system, we used a unique HBV transgenic mouse model, containing a 1.3 times over length of the HBV genome, on the ICR mouse background. These transgenic mice produce high serum HBV titers comparable with human chronic HBV patients, and, importantly, manifest characteristic HBV-associated pathology, including progressive hepatocellular injury and the development of hepatocellular adenoma. Using this system, we injected animals with AAV vectors expressing either HBV-specific or a control luciferase-specific short hairpin RNA and followed animals for a total of 18 months. We report herein that AAV-mediated RNAi therapy profoundly inhibits HBV replication and gene expression, with a significant reduction in hepatic regeneration, liver enzymes and, importantly, the appearance of liver adenomas. Indeed, the therapeutic effect of RNAi correlated with the reduction in HBV titers. Our data demonstrate that appropriately designed RNAi therapy has the potential to prevent formation of HBV-associated hepatocellular adenoma.     

拉米夫定单独和与α干扰素序贯处理的人肝胚瘤细胞株HepG2.2.15HBV差异复制的研究

目的 研究LAM单独和与IFN-序贯处理对人肝胚瘤细胞株HepG2.2.15细胞的HBV复制的影响,探讨LAM与IFN-α在体外抑制HBV复制的差异.方法 以未处理的HepG2.2.15细胞作为对照组;以1 000 IU/ml浓度IFN-α连续处理HepG2.2.15细胞10 d为单独IFN-α处理组;以0.2、1、5、20、100 μmol/L的LAM处理HepG2.2.15细胞为单独LAM处理组;序贯处理组则以浓度为0.04、0.2、5、25、125、200μmoL/L的LAM连续处理HepG2.2.15细胞7 d,再补充1 000 IU/ml IFN-α与LAM联合处理3 d,然后停止LAM处理,再单独以1 000 IU/ml IFN-α连续处理细胞10 d;分别用ELISA法、点杂交和Southern杂交分析不同处理时期、不同处理组HepG2.2.15细胞分泌的HBV抗原、细胞外HBV DNA、细胞内HBV复制中间体DNA以判断HepG2.2.15细胞内HBV复制情况.结果 LAM连续处理至10 d时,单独LAM处理组HepG2.2.15细胞分泌的HBsAg分别是1.77±0.22、1.65±0.25、1.95±0.19、1.34±0.11、1.07±0.05,分泌的HBeAg是1.41±0.13、1.37±0.09、1.63±0.07、1.26±0.12、1.05 ±0.09,对照组分泌的HBsAg和HBeAg分别是3.34±0.15和3.33±0.05,单独LAM处理组与对照组相比分泌的HBsAg和HBeAg下降,差异有统计学意义(HBsAg的t值为10.21、10.04、9.94、18.62、24.86,HBeAg的t值为23.87、32.97、34.22、27.57和38.35,P均＜0.05).点杂交、Southern杂交分析显示LAM连续处理10 d后,单独LAM处理组的细胞外HBV DNA和细胞内HBV复制中间体DNA不能被检测到.停止LAM处理并代之以1 000 IU/ml IFN-α序贯处理10 d,序贯处理组均有细胞内HBV复制中间体DNA的出现,且细胞外HBV抗原和HBV DNA恢复表达;即HBV颗粒在HepG2.2.15细胞内又重新恢复复制状态并分泌至细胞外.结论 LAM与IFN-α在体外细胞模型中具有不同的抗病毒效应,导致HepG2.2.15细胞内HBV复制的差异性.

Abstract：

Objective To investigate the characteristics of HBV replication in HepG2.2. 15 cells treated with LAM alone or sequentially treated with LAM and IFN-α, and to further explore the different suppressive effect on HBV replication by LAM and IFN-α in vitro. Methods Untreated HepG2. 2. 15 cells were used as control group, HepG2. 2. 15 cells treated with 1 000 IU/ml of IFN-α alone for 10 d were served as IFN-α group. The HepG2.2. 15 cells treated with 0.2,1,5,20,100 μmol/L of LAM were used as LAM groups. HepG2. 2. 15 cells treated with 0. 04,0. 2,5,25,125,200 μmol/L of LAM for 7 d, then combined with 1 000 IU/ml of IFN-α for another 3 d. Afterwards, the cells were treated with 1 000 IU/ml of IFN-α only for another 10 d. These cells were served as LAM/IFN-α sequential treatment group. The ELISA was used for analyzing the secreted HBV antigens, while the Dot blot, Southern blot were applied for analyzing the extracellular HBV DNA and intracellular HBV replicative intermediate DNA in HepG2. 2. 15 cells of different treatment groups. Results The secreted HBsAg in the LAM group were 1. 77 ± 0. 22, 1.65 ±0.25, 1.95 ±0. 19, 1.34 ±0. 11, 1.07 ±0.05, respectively, and the secretion of HBeAg were 1.41 ±0. 13, 1.37 ± 0. 09, 1.63 ± 0. 07, 1.26 ± 0. 12, 1.05 ± 0. 09. The secreted HBsAg and HBeAg in control group were 3. 34 ± 0. 15 and 3.33 ± 0. 05. Statistical analysis showed that HBsAg and HBeAg secretion in the LAM group were significantly reduced by treatment with LAM. The t values of HBsAg were 10. 21,10.04, 9.94, 18.62, 24.86, and the t values of HBeAg were 23.87, 32.97, 34.22, 27.57, 38.35,respectively, all P values were ＜ 0.05. Dot blot, Northern blot hybridization analysis indicated that the extracellular HBV DNA and intracellular HBV replicative intermediate DNA could not be detected in the LAM group after cells treated by LAM for 10 days. When LAM was replaced with treatment of 1 000 IU/ml of IFN-α alone, it could not suppress the HBV replication effectively. Moreover, the intracellular HBV replicative intermediate DNA still existed in almost all groups, accompanied with the recovered expression of HBV antigens as well as extracellular HBV DNA, which suggested that the HBV particles restored replication again and secreted extracellular in HepG2. 2. 15 cells, although the sequential treatment lasted for 10 days.Conclusion The effect of viral suppression by LAM and IFN-α in vitro were different, which attributed to the different HBV replicative characteristcs in HepG2. 2. 15 cells.     

Zinc finger protein designed to target 2-long terminal repeat junctions interferes with human immunodeficiency virus integration

Abstract Integration of the human immunodeficiency virus type 1 (HIV-1) genome into the host chromosome is a vital step in the HIV life cycle. The highly conserved cytosine-adenine (CA) dinucleotide sequence immediately upstream of the cleavage site is crucial for integrase (IN) activity. As this viral enzyme has an important role early in the HIV-1 replication cycle, interference with the IN substrate has become an attractive strategy for therapeutic intervention. We demonstrated that a designed zinc finger protein (ZFP) fused to green fluorescent protein (GFP) targets the 2-long terminal repeat (2-LTR) circle junctions of HIV-1 DNA with nanomolar affinity. We report now that 2LTRZFP-GFP stably transduced into 293T cells interfered with the expression of vesicular stomatitis virus glycoprotein (VSV-G)-pseudotyped lentiviral red fluorescent protein (RFP), as shown by the suppression of RFP expression. We also used a third-generation lentiviral vector and pCEP4 expression vector to deliver the 2LTRZFP-GFP transgene into human T-lymphocytic cells, and a stable cell line for long-term expression studies was selected for HIV-1 challenge. HIV-1 integration and replication were inhibited as measured by Alu-gag real-time PCR and p24 antigen assay. In addition, the molecular activity of 2LTRZFP-GFP was evaluated in peripheral blood mononuclear cells. The results were confirmed by Alu-gag real-time PCR for integration interference. We suggest that the expression of 2LTRZFP-GFP limited viral integration on intracellular immunization, and that it has potential for use in HIV gene therapy in the future.     

Inhibition of hepatitis viral replication by siRNA

Small interfering RNA (siRNA)-mediated sequence-specific gene silencing is a powerful tool to inhibit endogenous and exogenous gene expression, and it holds great potential to prevent and eradicate viral infection, for which existing therapy is inadequate, such as HIV, hepatitis B virus (HBV) and hepatitis C virus (HCV). A number of studies have documented the effectiveness of siRNA against HBV or HCV at various regions of the viral genome in infected human hepatoma cell lines. Selected siRNA may reduce the production of viral replicons, as well as structural or non-structural proteins by > 90%. Only a few in vivo studies that demonstrated the efficacy of siRNA in the suppression of HBV replication in mice are available. Thus, reliable models of HBV and HCV infection in small animals or non-human primates are needed to evaluate the delivery and efficacy of siRNA as a therapeutic modality for viral hepatitis.     

Inhibition of hepatitis viral replication by siRNA

Small interfering RNA (siRNA)-mediated sequence-specific gene silencing is a powerful tool to inhibit endogenous and exogenous gene expression, and it holds great potential to prevent and eradicate viral infection, for which existing therapy is inadequate, such as HIV, hepatitis B virus (HBV) and hepatitis C virus (HCV). A number of studies have documented the effectiveness of siRNA against HBV or HCV at various regions of the viral genome in infected human hepatoma cell lines. Selected siRNA may reduce the production of viral replicons, as well as structural or non-structural proteins by > 90%. Only a few in vivo studies that demonstrated the efficacy of siRNA in the suppression of HBV replication in mice are available. Thus, reliable models of HBV and HCV infection in small animals or non-human primates are needed to evaluate the delivery and efficacy of siRNA as a therapeutic modality for viral hepatitis.     

Nucleoside/nucleotide analogues in the treatment of chronic hepatitis B

The current available agents for the treatment of chronic hepatitis B (CHB) include immunomodulatory agents, such as interferon-α and pegylated interferon-α, and oral nucleoside/nucleotide analogues (NAs), including lamivudine, adefovir, telbivudine, entecavir and tenofovir. The NAs work mainly by inhibiting hepatitis B virus (HBV) DNA polymerase activity and thus suppress HBV replication. Oral NAs have become the mainstay of CHB treatment, mainly due to their profound viral suppressive effects and also due in part to the ease of single daily dosing and lack of significant side effects. One major drawback of NA therapy is the development of drug resistance mutations with long-term treatment. Lamivudine, the first oral NA approved for CHB patients, is associated with high rates of drug resistance, with resultant virological relapse and biochemical flare. Fortunately, newer and more potent NAs, such as entecavir and tenofovir, have very low resistance rates, with potent and durable viral suppression. This review is aimed at the current developments in NAs for CHB treatment, detailing the mechanisms of antiviral activity of the different agents, the efficacy of viral suppression, the achievement of treatment endpoints, the development of drug resistance and the optimal strategies for using these drugs.