RNA干扰抑制乙型肝炎病毒复制及基因表达研究进展

RNA干扰(RNAinterference,RNAi)是指由21～23个核苷酸组成的双链RNA(dsRNA)所引发的生物细胞内同源基因转录后沉默的现象,是生物体在进化过程中普遍存在的一种基因调控机制。目前对由乙型肝炎病毒(HBV)引起的病毒性肝炎尚无令人满意的治疗效果,而RNA干扰技术的出现为各类慢性HBV感染的治疗开辟了新的途径。本文对RNA干扰抑制HBV复制及基因表达的研究现状、存在问题及应用前景进行了综述。     

RNAi的抗病毒机制及应用

RNA干扰(RNA interference,RNAi)能够在转录水平、翻译水平和转录后水平上抑制病毒的复制,这为临床治疗病毒性疾病奠定了理论基础。近年来,RNAi技术发展迅速,使得RNAi技术应用于病毒性疾病的治疗成为可能。本文从RNAi抗病毒的作用机制及RNAi在抗动物病毒性疾病方面的应用等方面进行综述,并对RNAi在抗病毒方面的应用前景进行展望。     

RNA干扰及其抗病毒应用的可行性探讨

RNA干扰是存在于动植物细胞中的,由双链RNA介导的,序列特异性的mRNA降解过程。在哺乳动物细胞里,RNAi可以由21￣25个核苷酸长度的双链小干扰RNA(siRNA)触发。目前,以RNAi为策略来抑制病毒复制的方法已获得了巨大成功。但RNAi具有高度序列特异性,而多种病毒,如HIV-1、HBV A等却具有迅速变异的倾向。本文就病毒进化出的多种逃避RNA干扰的机制以及如何降低病毒对RNAi的耐受力等问题进行了多方面的探讨。     

RNA干扰与抗病毒感染

RNA干扰(RNAi)是由小干扰RNA(siRNA)引发的生物细胞内同源基因的转录后基因沉默(PTGS)现象,是一种古老的生物抵抗外在感染的防御机制。RNAi因其在维持基因组稳定、调控基因表达和保护基因组免受外源核酸侵入等方面发挥的重要作用,已被广泛用于探索基因功能、基因治疗和新药的研发。外源导入siRNA引发的RNAi可以特异性抑制病毒的复制与感染,为抗病毒感染治疗开辟了一条新的途径。     

人类免疫缺陷病毒1型逃避RNA干扰的研究

用RNA干扰(RNAi)技术来阻断特定基因的表达,为治疗诸如人类免疫缺陷病毒1型(HIV—1)等慢性病毒感染提供了一种新的手段。虽然,通过小干扰RNA(siRNA)以降解序列特异性的mRNA可选择性地抑制一些在HIV—1复制过程中起关键作用的病毒蛋白,但RNAi技术仍然存在着许多问题亟待解决。本文就HIV—1特异性的RNAi技术作用的潜力和持久性作一研究。     

RNA干扰用于基因治疗的研究进展

RNA干扰(RNAi)是20世纪末才被人们认识和重视的一种通过双链RNA抵御病毒入侵或抑制转座子活动的生物防御机制。随着RNA干扰分子机制研究的深入及其应用研究的发展,人们发现RNA干扰技术在基因功能研究及人类疾病的基因治疗上具有广阔的应用前景。本文在简述RNAi分子机制的基础上,综述了RNAi在抗病毒治疗及抗肿瘤治疗方面的研究和应用概况。     

RNA干扰与植物抗病毒

RNA干扰是多种生物体内由双链RNA介导的同源mRNA降解现象,是植物体内天然的抗病毒机制。然而病毒在长期进化过程中也获得了通过编码沉默抑制蛋白来对抗植物体RNAi系统的能力。本文对RNA干扰过程、病毒编码的沉默抑制蛋白及利用干扰技术进行抗病毒基因工程研究进行简要综述。     

脊椎动物病毒与微RNA

抗病毒作用是RNA干扰(RNAi)在植物及低等动物中的一个重要功能。一方面,宿主细胞编码并表达短干扰RNA(siRNA),对入侵细胞的病毒产生抑制作用;另一方面,病毒编码并表达特定的RNA或蛋白质,以对抗宿主细胞的RNAi。在部分脊椎动物病毒中已经发现多种由病毒编码的微RNA(miRNA),它们对病毒及细胞基因的表达有重要的调节作用。同时,某些细胞miRNA也可影响脊椎动物病毒的复制。然而,RNAi在脊椎动物细胞中是否具有广谱抗病毒活性、脊椎动物病毒又是否普遍编码miRNA及普遍具备拮抗RNAi的机制?目前尚无定论,有待于进一步的研究加以阐明。     

RNA 干扰技术抑制病毒复制的研究进展

RNA干扰是指dsRNA抑制细胞内同源基因表达的现象,RNA干扰现象自发现以来在短短的几年里,已成功地用于不同种属的生物研究。dsRNA不仅参与内源基因表达调控,而且能够抑制宿主内病原微生物基因的表达,参与构筑生物体的防御机制。近年来,运用RNAi技术在哺乳动物中的研究不断深入,尤其是抑制病毒复制的研究成果令人欣喜,这为人类动物抗病毒治疗提供了新的思路。     

RNA干扰的抗病毒作用及其影响因素

自从在哺乳动物细胞中发现存在RNA干扰现象以来,RNA干扰技术也应用于抗病毒研究.通过多种形式诱发的RNA干扰对细胞中病毒的复制或病毒基因的表达都产生了有效的抑制,为病毒病的预防和治疗提供了新的途径.然而,一些因素制约着RNA干扰的潜在应用,如在RNA干扰压力下病毒逃逸株的出现,有的病毒可以编码RNA干扰抑制因子以及小干扰RNA的脱靶效应等.     

Inhibition of virus replication by RNA interference

RNA interference (RNAi) is a sequence-specific gene-silencing mechanism in eukaryotes, which is believed to function as a defence against viruses and transposons. Since its discovery, RNAi has been developed into a widely used technique for generating genetic knock-outs and for studying gene function by reverse genetics. Additionally, inhibition of virus replication by means of induced RNAi has now been reported for numerous viruses, including several important human pathogens such as human immunodeficiency virus type 1, hepatitis C virus, hepatitis B virus, dengue virus, poliovirus and influenza virus A. In this review, we will summarize the current data on RNAi-mediated inhibition of virus replication and discuss the possibilities for the development of RNAi-based antiviral therapeutics.     

Transiently expressed short hairpin RNA targeting 126 kDa protein of tobacco mosaic virus interferes with virus infection

RNA-interference (RNAi) silences gene expression by'guiding mRNA degradation in asequence-specific fashion.Small interfering RNA (siRNA),an intermediate of the RNAi pathway,has beenshown to be very effective in inhibiting virus infection in mammalian cells and cultured plant cells.Here,wereport that Agrobacterium tumefaciens-mediated transient expression of short hairpin RNA (shRNA) couldinhibit tobacco mosaic virus (TMV) RNA accumulation by targeting the gene encoding the replication-asso-ciated 126 kDa protein in intact plant tissue.Our results indicate that transiently expressed shRNA efficientlyinterfered with TMV infection.The interference observed is sequence-specific,and time-and site-dependent.Transiently expressed shRNA corresponding to the TMV 126 kDa protein gene did not inhibit cucumbermosaic virus (CMV),an unrelated tobamovirus.In order to interfere with TMV accumulation in tobaccoleaves,it is essential for the shRNA constructs to be infiltrated into the same leaves as TMV inoculation.Ourresults support the view that RNAi opens the door for novel therapeutic procedures against virus diseases.We propose that a combination of the RNAi technique and Agrobacterium-mediated transient expressioncould be employed as a potent antiviral treatment in plants.     

Silencing SARS-CoV Spike protein expression in cultured cells by RNA interference

The severe acute respiratory syndrome (SARS) has been one of the most epidemic diseases threatening human health all over the world. Based on clinical studies, SARS-CoV (the SARS-associated coronavirus), a novel coronavirus, is reported as the pathogen responsible for the disease. To date, no effective and specific therapeutic method can be used to treat patients suffering from SARS-CoV infection. RNA interference (RNAi) is a process by which the introduced small interfering RNA (siRNA) could cause the degradation of mRNA with identical sequence specificity. The RNAi methodology has been used as a tool to silence genes in cultured cells and in animals. Recently, this technique was employed in anti-virus infections in human immunodeficiency virus and hepatitis C/B virus. In this study, RNAi technology has been applied to explore the possibility for prevention of SARS-CoV infection. We constructed specific siRNAs targeting the S gene in SARS-CoV. We demonstrated that the siRNAs could effectively and specifically inhibit gene expression of Spike protein in SARS-CoV-infected cells. Our study provided evidence that RNAi could be a tool for inhibition of SARS-CoV.     

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.     

RNA干扰技术在几项研究领域的应用

作为一项新的反向遗传学技术 ,RNA干扰技术正在越来越多地应用于包括鉴定基因功能、疾病治疗、植物病毒抗性研究在内的多项研究领域。在鉴定基因功能研究中 ,由于该技术的操作简便性 ,使得在基因组水平进行大范围的基因功能鉴定成为可能 ;而针对致病相关基因、致病病毒基因组进行RNA干扰 ,可以有效抑制病情恶化 ,有可能成为未来疾病治疗的重要手段 ;同时 ,将RNA干扰技术应用于植物抗病毒研究 ,为工程化植物抗病毒遗传育种提供了一个高效、特异的抗性获得手段。对RNA干扰技术在上述三个研究领域的应用作简要综述 ,并对应用过程中需注意的问题进行了探讨 。     

RNA干扰抗病毒感染

RNA干扰是由双链RNA诱导的、关闭同源序列基因表达的机制。它是一种自然存在于植物、线虫、果蝇等真核细胞生物中的抵抗病毒感染方式。随着在哺乳动物细胞培养中成功地诱导RNA干扰,利用RNA干扰预防、治疗病毒感染已成为新的研究热点,并取得了有希望的成果。在未来,有望成为抗病毒感染的有效方法。     

Inhibition of hepatitis B virus replication with linear DNA sequences expressing antiviral micro-RNA shuttles

RNA interference (RNAi) may be harnessed to inhibit viral gene expression and this approach is being developed to counter chronic infection with hepatitis B virus (HBV). Compared to synthetic RNAi activators, DNA expression cassettes that generate silencing sequences have advantages of sustained efficacy and ease of propagation in plasmid DNA (pDNA). However, the large size of pDNAs and inclusion of sequences conferring antibiotic resistance and immunostimulation limit delivery efficiency and safety. To develop use of alternative DNA templates that may be applied for therapeutic gene silencing, we assessed the usefulness of PCR-generated linear expression cassettes that produce anti-HBV micro-RNA (miR) shuttles. We found that silencing of HBV markers of replication was efficient (>75%) in cell culture and in vivo. miR shuttles were processed to form anti-HBV guide strands and there was no evidence of induction of the interferon response. Modification of terminal sequences to include flanking human adenoviral type-5 inverted terminal repeats was easily achieved and did not compromise silencing efficacy. These linear DNA sequences should have utility in the development of gene silencing applications where modifications of terminal elements with elimination of potentially harmful and non-essential sequences are required.