基因沉默的维持——长效RNA干扰

自揭示了哺乳动物细胞中的RNA干扰（RNAi）现象以来,对其进行了大量的研究与资金投入,期望将其发展成一种切实可行的治疗手段。毫无疑问,RNAi是最具发展潜力的基因治疗策略。由于许多采用RNAi治疗的疾病需要长期用药,因此,有关RNAi类药物长期应用的安全性问题成为关注重点。     

果蝇抗病毒免疫

果蝇是遗传和发育研究的重要模式生物,对果蝇抗病毒免疫研究有助于了解宿主是如何与病毒互相作用以及宿主几条免疫路径在抗病毒中所起的作用.本文从两方面综述了果蝇抗病毒免疫,即果蝇抗病毒先天免疫路径和核糖核酸干扰(RNAi)抗病毒免疫,这些分析能帮助我们更好地了解果蝇抗病毒免疫的机制和特点.     

006 基于RNAi的新型药物浮出水面

RNA干扰的治疗前景RNA干扰（RNAi）是一种高度保守的内源性基因沉默机制，发现于1998年，为双链RNA介导的序列专一性降解mRNA。科学家们很快就认识到了RNAi的治疗潜力。     

RNAi药物的临床研究进展

RNA干扰（RNAi）是指在细胞内由双链RNA（double-stranded RNA,dsRNA）介导的降解同源序列的mRNA,从而抑制相应基因表达的现象。它是转录后基因沉默（PTGS）的一种。RNA干扰不仅是基础研究的热点．也是临床应用研究的热点．小干扰RNA（siRNA）药物虽然在国际上已有部分进入Ⅰ-Ⅲ期临床研究,但是siR—NA的脱靶效应、siRNA的导入等实际问题一直是RNA干扰临床治疗的最大障碍。核酸药物比传统的化学分子甚至比蛋白质更容易被降解,而RNA干扰药物的有效传递依旧是RNAi药物临床应用的拦路虎,该技术一旦被攻克．将会被广泛应用于基因性疾病、传染性疾病及恶性肿瘤的临床治疗。本文就RNA干扰的作用机制、临床应用、存在问题及广阔前景作一简要综述。     

RNA干扰与恶性肿瘤治疗

RNA干扰（RNA interference,RNAi）是指在进化中高度保守的、由双链RNA（double-stranded RNA,dsRNA）诱发的、同源mRNA高效特异性降解的现象。它已在许多不同种属的生物体中被发现,并在生物体细胞之间进行传递,具有抵抗病毒入侵和维持基因组稳定的作用。由于使用RNAi技术可以特异性剔除或关闭特定基因表达．     

RNA干扰技术

RNA干扰首次发现于美丽线虫,siRNAs(小干扰RNA)的产生可诱导特异内源性mRNA的降解,现在被认为是真核细胞在翻译后水平抑制蛋白产生的主要途径.典型的内源性siRNA是由19-23个碱基构成的双链寡核苷酸RNA,由RNase蛋白复合物聚集降解靶mRNA所产生.RNA干扰最近常被用作逆转录基因工具来沉默多倍体有机体中的基因表达.表达siRNAs的方法日新月异,已经由最初的在体内或者体外利用病毒载体转染合成的siRNA至细胞,发展为在不同型细胞和有机体中建立不同功能的特异蛋白.RNA干扰的方法较之前的方法(反义DNA或抗体封闭技术)在抑制基因表达方面有着明显的优点.RNAi序列特异性的抑制效应是有选择性的、长期的,系统地调节靶向基因.不论是直接转染siRNA或者由RNA载体表达产生,RNAi都可抑制哺乳动物中的特异基因,这无疑加速了基因功能的研究速度,而且极有潜力成为高效的基因特异性治疗方法.药理学家一直梦寐以求可有方法能够选择性的拮抗或剔除个体特异蛋白的功能,RNAi十分有望使其梦想成真.     

Alnylam公司与葛兰素史克公司合作应用VaxiRNA技术提高疫苗生产能力

日前Alnylam制药公司就利用其开发的RNA干扰（RNAi）技术VaxiRNA生产疫苗产品,与葛兰素史克（GSK）公司达成合作伙伴协议。VaxiRNA技术通过采用小分子干扰RNA（siRNA）来增加疫苗生产过程中病毒滴度,从而提高疫苗生产能力。Alnylam公司CEO John Maraganore博士称,     

RNA干扰抗HBV的研究进展

RNA干扰是由长的双链RNA（double—stranded RNA．dsRNAl引发的降解与之同源的信使RNA（messager RNA，mRNA）的过程，dsRNA进入真核细胞．在胞浆内经限制性核酸内切酶Dicer作用，被切割成21—23bp的大小的小分子干扰RNA片段（small interfering RNAs,siRNhs），siRNAs与核酶复合物结合从而形成所谓的RNA诱导沉默复合物（RNA—induced silencing complex。RISC）。激活的RISC可以精确降解与siRNAs序列同源的mRNA．完全抑制了该基因在细胞内的翻译和表达。RNAi可以用来降低或清除内源性异常基因如癌基因表达和抵抗外源性入侵遗传学分子如转座子、转基因、病毒等．被认为是一种古老的保护体免受病毒入侵的机制．因此对传染性疾病的治疗可以说是RNAi最具前景的应用。     

RNAi的分子机制及其应用的研究进展

RNA干扰（RNA interference,RNAi）是最近几年发现和发展起来的一门新兴的在转录水平上的基因阻断技术。它与反义RNA技术有很大不同，是一种双链RNA（Doublestranded RNA,dsRNA）分子在mRNA水平上关闭相应序列基因的表达或使其沉默的过程，也就是序列特异性的转录后基因沉默。     

RNA interference: its use as antiviral therapy

RNA interference (RNAi) is a sequence-specific gene-silencing mechanism that has been proposed to function as a defence mechanism of eukaryotic cells against viruses and transposons. RNAi was first observed in plants in the form of a mysterious immune response to viral pathogens. But RNAi is more than just a response to exogenous genetic material. Small RNAs termed microRNA (miRNA) regulate cellular gene expression programs to control diverse steps in cell development and physiology. The discovery that exogenously delivered short interfering RNA (siRNA) can trigger RNAi in mammalian cells has made it into a powerful technique for generating genetic knock-outs. It also raises the possibility to use RNAi technology as a therapeutic tool against pathogenic viruses. Indeed, inhibition of virus replication has been reported for several human pathogens including human immunodeficiency virus, the hepatitis B and C viruses and influenza virus. We reviewed the field of antiviral RNAi research in 2003 (Haasnoot et al. 2003), but many new studies have recently been published. In this review, we present a complete listing of all antiviral strategies published up to and including December 2004. The latest developments in the RNAi field and their antiviral application are described.     

Effective inhibition of hepatitis E virus replication in A549 cells and piglets by RNA interference (RNAi) targeting RNA-dependent RNA polymerase

RNA interference (RNAi) is a natural mechanism for suppressing or silencing expression of aberrant or foreign genes. It is a powerful antiviral strategy that has been widely employed to protect hosts from viral infection. Hepatitis E (HE) is an acute fulminant hepatitis in adults that has particularly high mortality in pregnant women. At this point in time, there is no vaccine or antiviral treatment that is effective against the infectious agent, HEV. The nonstructural polyprotein region possesses an RNA-dependent RNA polymerase (RdRp) that is responsible for the replication of the viral RNA genome. RdRp is therefore regarded as one of the most attractive candidates for RNA interference (RNAi). In the present study, the high efficiency and specificity of siRNA were evaluated by Real-Time quantitative PCR and Western blot assays. Protective effects against HEV infection were achieved in A549 cells and in piglets. In piglets treated with a shRNA-RdRp-1 expression plasmid prior to HEV inoculation, HEV antigens were significantly reduced in the liver, spleen, and kidneys, and the activities of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and total bilirubin (TBIL) were clearly decreased. These results suggested that RNAi is a potentially effective antiviral strategy against HEV replication and infection.     

Combination of soluble coxsackievirus-adenovirus receptor and anti-coxsackievirus siRNAs exerts synergistic antiviral activity against coxsackievirus B3

Coxsackievirus B3 (CVB-3) is a major causative agent of chronic heart muscle infections. The present study describes a cell culture system with an ongoing virus infection to evaluate two novel inhibitory strategies, either individually or combined: (1) RNA interference (RNAi) to degrade cytoplasmatic CVB-3 RNA and (2) a vector-delivered soluble variant of the coxsackievirus-adenovirus receptor fused to a human immunoglobulin (sCAR-Fc), which inhibits cellular uptake of CVB-3. Both approaches were capable of inhibiting CVB-3 in persistently infected human myocardial fibroblasts. The antiviral effect of a single treatment lasted for up to one week and could be extended by repeated applications. Each of the single treatments initially reduced the virus titer by approximately 1-log, whereas the combination of both approaches resulted in 4-log inhibition and retained substantial antiviral activity at later time points, when the effect of sCAR-Fc or siRNAs alone had already disappeared. Further analysis revealed that sCAR-Fc protects cells from virus-induced lysis but does not diminish the virus load. Reduction of the virus titer was only achieved with additional destruction of viral RNA by RNAi. Taken together, combination of RNAi and a protein-based antiviral strategy was found to result in a strong synergistic inhibition of an ongoing virus infection.     

Potentiality of small interfering RNAs (siRNA) as recent therapeutic targets for gene-silencing

In recent years, RNA interference (RNAi) is one of the most important discoveries. RNAi is an evolutionarily conserved mechanism for silencing gene expression by targeted degradation of mRNA. Short double-stranded RNAs, known as small interfering RNAs (siRNA), are incorporated into an RNA-induced silencing complex that directs degradation of RNA containing a homologous sequence. siRNA has been shown to work in mammalian cells, and can inhibit viral infection and control tumor cell growth in vitro. Recently, it has been shown that intravenous injection of siRNA or of plasmids expressing sequences processed to siRNA can protect mice from autoimmune and viral hepatitis. In this review, we have discussed about the discovery of RNAi and siRNA, mechanism of siRNA mediated gene silencing, mediated gene silencing in mammalian cells, vectored delivery of siRNA, pharmaceutical potentiality of siRNA from mice to human. We have also discussed about promise and hurdles of siRNA or RNAi that could provide an exciting new therapeutic modality for treating infection, cancer, neurodegenerative disease, antiviral diseases (like viral hepatitis and HIV-1), huntington's disease, hematological disease, pain research and therapy, sarcoma research and therapy and many other illness in details. It will be a tool for stem cell biology research and now, it is a therapeutic target for gene-silencing.     

Molecular strategies to design an escape-proof antiviral therapy

Two antiviral approaches against the human immunodeficiency virus type 1 (HIV-1) were presented at the Antivirals Congress in Amsterdam. The common theme among these two separate therapeutic research lines is the wish to develop a durable therapy that prevents viral escape. We will present a brief overview of these two research lines and focus on our efforts to design an escape-proof anti-HIV therapy. The first topic concerns the class of HIV-1 fusion inhibitors, including the prototype T20 peptide and the improved versions T1249 and T2635, which were all developed by Trimeris–Roche. The selection of T20-resistant HIV-1 strains is a fairly easy evolutionary process that requires a single amino acid substitution in the peptide binding site of the viral envelope glycoprotein (Env) target. The selection of T1249-resistant HIV-1 strains was shown to require a more dramatic amino acid substitution in the viral Env protein, in particular the introduction of charged amino acid residues that cause resistance by charge-repulsion of the antiviral peptide. The third generation peptide T2635 remains active against all these HIV-1 escape variants because the charged residues within this peptide are “masked” by an introduced intra-helical salt bridge. This charge masking concept could facilitate the future design of escape-proof antiviral peptides. The second topic concerns the mechanism of RNA interference (RNAi) that we are currently employing to develop an antiviral gene therapy. One can make human T cells resistant to HIV-1 infection by a stable RNAi-inducing gene transfer, but the virus escapes under therapeutic pressure of a single inhibitor. Several options for a combinatorial RNAi attack to prevent viral escape will be discussed. The simultaneous use of multiple RNAi inhibitors turns out to be the most effective and durable strategy.     

Can siRNA technology provide the tools for gene therapy of the future?

A new era in genetics has started 15 years ago, when co-suppression in petunia has been discovered. Later, co-suppression was identified as RNA interference (RNAi) in many plant and lower eukaryote animals. Although an ancient antiviral host defense mechanism in plants, the physiologic role of RNAi in mammals is still not completely understood. RNAi is directed by short interfering RNAs (siRNAs), one subtype of short double stranded RNAs. In this review we summarize the history and mechanisms of RNAi. We also aim to highlight the correlation between structure and efficacy of siRNAs. Delivery is the most important obstacle for siRNA based gene therapy. Viral and nonviral deliveries are discussed. In vivo delivery is the next obstacle to clinical trials with siRNAs. Although hydrodynamic treatment is effective in animals, it cannot be used in human therapy. One possibility is organ selective catheterization. The known side effects of synthesized siRNAs are also discussed. Although there are many problems to face in this new field of gene therapy, successful in vitro and in vivo experiments raise hope for treating human disease with siRNA.     

Engineered virus-encoded pre-microRNA (pre-miRNA) induces sequence-specific antiviral response in addition to nonspecific immunity in a fish cell line: Convergence of RNAi-related pathways and IFN-related pathways in antiviral response

Transfection with synthesized virus-specific small interfering RNAs (siRNAs) efficiently inhibits viral replication in viral-infected fish cell lines, implying the involvement of RNA interference (RNAi)-related pathways in the antiviral response of fish cells. Here, we demonstrate that plasmid expressing virus-encoded pre-microRNAs (pre-miRNAs) can also inhibit viral replication through these pathways. By incorporating sequences encoding miRNAs specific to major capsid protein (MCP) gene of red sea bream iridovirus (RSIV) and a miRNA specific to hirame rhabdovirus (HIRRV) genome into a murine miR-155 pre-miRNA backbone, we were able to intracellularly express viral pre-miRNAs (miR-MCPs and miR-HIRRV) in a fish cell line. The miR-MCPs and miR-HIRRV, delivered as pre-miRNA precursors in transfected cells, inhibited viral replication when these cells were infected with the target virus. Although this may suggest sequence-specific interference, inhibitory effect on viral replication was also observed in cells transfected with a plasmid expressing pre-miRNA targeting β-galactosidase gene (miR-LacZ) that served as a specificity control. Expression of pre-miRNAs was found to activate interferon (IFN)-related pathways, correlating with upregulation of the antiviral IFN-induced Mx protein. The antiviral effects of viral-miRNAs observed here were partly the result of the antiviral miRNA-related pathways and partly the result of the antiviral IFN-related pathways. We propose that engineered virus-encoded pre-miRNA can engage not only RNAi-related pathways but also IFN-related pathways to induce potent antiviral responses in fish cells.