RNA干扰在抗乙肝治疗中的应用及其研究进展

据世界卫生组织(WHO)报道,全世界约有20亿人曾感染过乙型肝炎病毒(HBV),其中3.5亿人为慢性HBV感染者.我国现有1.3亿乙肝病毒携带者和3 000多万乙肝患者,其中约有20%～40%的患者经过多年慢性炎症的反复发作可发展为肝硬化和肝癌.然而,至今人们仍没有找到一种能够彻底治愈慢性乙肝的特效药物.自从RNA干扰(RNA interference,RNAi)技术建立以来,人们致力于将其应用于抗病毒药物的研究与开发.研究结果表明,RNAi可有效地抑制乙肝病毒的复制,但靶向目的基因的不同RNA干扰片段所沉默的效率不同.关于将RNAi抗病毒药物应用于人体治疗的安全性和有效性还有待进一步研究,RNAi发生"脱靶"的现象是临床应用的难点之一.     

针对SARS冠状病毒重要蛋白的siRNA设计(英文)

NA干涉 (RNAinterference ,RNAi)是一种特异性地导致转录后基因沉默的现象 ,在哺乳动物细胞中小分子干扰RNA双链体 (smallinterferingRNAduplexes ,siRNAduplexes)可以有效地诱导RNAi现象 ,为一些疾病的治疗开辟了新的途径 .针对SARS冠状病毒 (SARScoronavirus ,SARS CoV)中编码 5个主要蛋白质的基因 ,用生物信息学的方法设计了3 48条候选siRNA靶标 .在理论上 ,相应的siRNA双链体能特异地抑制SARS CoV靶基因的表达 ,同时不会影响人体细胞基因的正常表达 ,这为进一步siRNA类药物的实验研究提供了理论基础     

针对SARS冠状病毒重要蛋白的siRNA设计(英)

RNA干涉(RNA interference, RNAi)是一种特异性地导致转录后基因沉默的现象,在哺乳动物细胞中小分子干扰RNA双链体(small interfering RNA duplexes, siRNA duplexes)可以有效地诱导RNAi现象,为一些疾病的治疗开辟了新的途径.针对SARS冠状病毒(SARS coronavirus, SARS-CoV)中编码5个主要蛋白质的基因,用生物信息学的方法设计了348条候选siRNA靶标.在理论上,相应的siRNA双链体能特异地抑制SARS-CoV靶基因的表达,同时不会影响人体细胞基因的正常表达,这为进一步siRNA类药物的实验研究提供了理论基础.     

小环载体介导的siRNA 稳定抑制乙肝病毒的复制和表达

【目的】尝试构建表达小干扰RNA(small interfering RNA,siRNA)的小环载体,并初步鉴定其对乙肝病毒(hepatitis B virus,HBV)复制及其基因表达的抑制作用。【方法】设计并合成靶向HBV S区的siRNA,将其克隆到小环载体pMC.BESPX-MCS2上,测序正确后将重组体pMC-H1-siHBS-U6转化入感受态E.coliZYCY10P3S2T,然后在培养基中加入L-阿拉伯糖,诱导其降解细菌骨架,获取只含有目的基因表达盒的小环RNA干扰载体pmc-H1-siHBS-U6。将小环RNA干扰载体与HBV真核表达质粒pHBV1.3共转染Huh-7细胞,分别在转染后1-7天,ELISA法检测Huh-7细胞上清中的HBsAg、HBeAg,并且通过Real-time RT-PCR法分析干扰RNA对HBV DNA及mRNA的抑制效果。【结果】成功构建了靶向HBV S基因的siRNA小环表达载体pmc-H1-siHBS-U6。该载体能显著抑制Huh-7细胞HBsAg和HBeAg分泌,并且其抑制效果能够维持2-3周时间。Real-time PCR证实HBV的DNA与mRNA水平分别降低了71%和80%,而对照siRNA及空载体则无此作用。【结论】成功构建了靶向HBV的小环RNA干扰载体,并且其能稳定、高效、特异地抑制HBV基因的表达与复制,该研究不仅对探索HBV的基因治疗提供了重要线索,而且为RNA干扰的应用提供了新的运载体系。     

Stable inhibition of hepatitis B virus expression and replication by expressed siRNA

RNA interference might be an efficient antiviral therapy for some obstinate illness. Here, we studied the effects of hepatitis B virus (HBV)-specific 21-nt small interfering RNAs (siRNA) on HBV gene expression and replication in 2.2.15 cells. Seven vectors expressing specific hairpin siRNA driven by the RNA polymerase II-promoter were constructed and transfected into 2.2.15 cells. In the cell strain that can stably express functional siRNA, the HBV surface antigen (HBsAg) and the HBV e antigen (HBeAg) secretion into culture media was inhibited by 86% and 91%, respectively, as shown by an enzyme-linked immunosorbent assay. Immunofluorescence and Western blot indicated similar results. HBV DNA was markedly restrained by 3.28-fold, as assessed by the fluorescent quantitation PCR. Moreover, the HBV mRNA was significantly reduced by 80% based on semiquantitative RT-PCR. In conclusion, the specific siRNA can knock down the HBV gene expression and replication in vitro, and the silence effects have no relationship with interferon response.     

Identification of the suppressive factors for human immunodeficiency virus type-1 replication using the siRNA mini-library directed against host cellular genes

We performed the screening to find the novel host factors affecting human immunodeficiency virus type-1 (HIV-1) replication using the siRNA mini-library consisted with 257 siRNAs directed against cellular genes. J111 cells, a human acute monocytic leukemia cell line, were transfected with individual siRNA, followed by either infected or transfected with the HIV-1 molecular clone with luciferase reporter gene in 96-well plate format. The results showed that six siRNAs significantly enhanced the HIV-1 replication in J111 cells, indicating that the target cellular genes of those siRNAs may negatively regulate HIV-1 replication in normal cell culture condition. We also discuss the possible mechanisms by which those cellular proteins regulate viral replication.     

A novel system for rapid screening of effective siRNA target sites by one step transfection with a single vector

siRNA technique has been widely used to study the gene functions and to develop disease therapeutics. One of the challenges of RNAi application is to obtain the most effective target specific siRNA sequences. Currently the process to select and validate optimal siRNA sites for a given gene, which is usually based on screening by using Western blot, Northern blot or Q-PCR, remains empirical and time consuming. Although few fluorescence-based siRNA sequence selection systems have proven useful, the rapid and efficient screening of siRNA target sites is still challenging. In the paper, we developed a quick and efficient method to screen siRNA target sites with a novel single vector system, which contains the following cassettes: (1) an eGFP reporter gene expression cassette followed by a multiple cloning site and SV40 pA for insertion of a target sequence; (2) siRNA expression cassette containing a dual PoI III promoter driving in opposite directions; and (3) an internal loading control, mCherry reporter gene. Based on this one-step transfection with single vector system, we could rapidly screen effective, target specific siRNA fragments in an unbiased manner by judging the fluorescence intensity ratio of eGFP to mCherry. The generation of this novel vector system will promote the application of siRNA in basic research and disease therapy.     

Suppression of hepatitis C virus replicon by RNA interference directed against the NS3 and NS5B regions of the viral genome

RNA interference (RNAi) is a phenomenon in which small interfering RNA (siRNA), an RNA duplex 21 to 23 nucleotides (nt) long, or short hairpin RNA (shRNA) resembling siRNA, mediates degradation of the target RNA molecule in a sequence-specific manner. RNAi is now expected to be a useful therapeutic strategy for hepatitis C virus (HCV) infection. In the present study we compared the efficacy of a number of shRNAs directed against different target regions of the HCV genome, such as 5'-untranslated region (5'UTR) (nt 286 to 304), Core (nt 371 to 389), NS3-1 (nt 2052 to 2060), NS3-2 (nt 2104 to 2122), and NS5B (nt 7326 to 7344), all of which except for NS5B are conserved among most, if not all, HCV subtype 1b (HCV-1b) isolates in Japan. We utilized two methods to express shRNAs, one utilizing an expression plasmid (pAVU6+27) and the other utilizing a recombinant lentivirus harboring the pAVU6+27-derived expression cassette. Although 5'UTR has been considered to be the most suitable region for therapeutic siRNA and/or shRNA because of its extremely high degree of sequence conservation, we observed only a faint suppression of an HCV subgenomic replicon by shRNA against 5'UTR. In both plasmid-and lentivirus-mediated expression systems, shRNAs against NS3-1 and NS5B suppressed most efficiently the replication of the HCV replicon without suppressing host cellular gene expression. Synthetic siRNA against NS3-1 also inhibited replication of the HCV replicon in a dose-dependent manner. Taken together, the present results imply the possibility that the recombinant lentivirus expressing shRNA against NS3-1 would be a useful tool to inhibit HCV-1b infection.     

pSUPER介导的RNAi抗乙肝病毒S基因的研究

目的 研究RNAi抗乙肝病毒的作用.方法 设计并合成一段针对HBV S基因的干扰序列及一段无关的序列,克隆进pSUPER载体中,分别构建pSUPEK-HBS、pSUPEK-nonsense载体,然后将pSUPER、pSUPEK-HBS、pSUPER-nonsense转染进HepG2.2.15细胞中,用ELISA方法对细胞上清中HBsAg、HBeAg进行检测.尾静脉注射HBV转基因小鼠,取血清检测.结果 pSUPER-HBS所表达的siRNA成功的抑制了HepG 2.2.15上清及HBV转基因鼠血清中的HBsAg、HBeAg,抑制率分别达到70%、51%以及60%、42%.结论 针对HBV S区的siRNA能明显抑制HBV的复制.     

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

RNA interference (RNAi) mediated inhibition of virus-specific genes has emerged as a potential therapeutic strategy against virus induced diseases. Human hepatitis B virus (HBV) surface antigen (HBsAg) has proven to be a significant risk factor in HBV induced liver diseases, and an increasing number of mutations in HBsAg are known to enhance the difficulty in therapeutic interventions. The key challenge for achieving effective gene silencing in particular for the purpose of the therapeutics is primarily based on the effectiveness and specificity of the RNAi targeting sequence. To explore the therapeutic potential of RNAi on HBV induced diseases in particular resulted from aberrant or persistent expression of HBsAg, we have especially screened and identified the most potent and specific RNAi targeting sequence that directly mediated inhibition of the HBsAg expression. Using an effective DNA vector-based shRNA expression system, we have screened 10 RNAi targeting sequences (HBsAg-1 to 10) that were chosen from HBsAg coding region, in particular the major S region, and have identified four targeting sequences that could mediate sequence specific inhibition of the HBsAg expression. Among these four shRNAs, an extremely potent and highly sequence specific HBsAg-3 shRNA was found to inhibit HBsAg expression in mouse HBV model. The inhibition was not only preventive in cotransfection experiments, but also had therapeutic effect as assessed by post-treatment protocols. Moreover, this HBsAg-3 shRNA also exhibited a great potency of inhibition in transgenic mice that constitutively expressed HBsAg. These results indicate that HBsAg-3 shRNA can be considered as a powerful therapeutic agent on HBsAg induced diseases.     

乙型肝炎病毒变异株功能基因组研究及其临床意义

综述了近年对乙型肝炎病毒（HBV）变异株的复制、免疫学特性、致病性、耐药性等功能基因组研究及其临床意义的研究进展,阐述基因组水平研究HBV变异株功能的重要性及有关结果,展望今后HBV变异株生物学特性研究的方向。     

Expression and characterization of hepatitis C virus core protein fused to hepatitis B virus core antigen

Recombinant plasmids were constructed by fusing the gene fragments encoding the full-length (1-191aa) and the truncated (1-40aa and 1-69aa) HCV core proteins (HCc) respectively to the core gene of HBV at the position of amino acid 144 and expressed in E. coli. The products were analyzed by ELISA, Western blotting as well as the immunization of the mice. The results showed that those fusion proteins (B144C191, B144C69, B144C40) possessed the dual antigenicity and immunogenicity of both hepatitis B virus core antigen (HBcAg) and hepatitis C virus core protein (HCc). Analysis by electron microscopy and CsCl density gradient ultra-centrifugation revealed that similar to the HBcAg itself, all fusion proteins were able to form particles. Comparison of the antigenicity and immunogenicity of those fusion proteins showed that the length of HCc gene fused to HBeAg had no much effect on the antigenicity and immunogenicity of HBcAg, however, B144C69 and B144C40 induced higher titres antibodies against HCc than B14d