Strategy for a generic resistance to geminiviruses infecting tomato and papaya through in silico siRNA search

Use of siRNA is a powerful methodology to particularly knockdown the targeted genes in a sequence specific manner. The potential of siRNA can be harnessed for silencing specific geminiviral genes in papaya and tomato plant hosts, thus making them resistant to the respective viruses. The challenge is in designing exogenous siRNA which can trigger silencing of viral genes irrespective of the genetic variability in different viral isolates and at the same time the selected siRNA does not target any plant gene (off target silencing). In this study, we have designed siRNA from the most conserved regions of viral coat protein (AV1) and replicase (AC1) genes retrieved from different isolates of geminiviruses infecting papaya (PLCV), and tomato (TLCV & TLCV, Northern India), so as to give a broad spectrum resistance and efficient silencing as it is highly homology-dependent strategy. Software siRNA finder (Ambion) was used on the selected conserved sequences in order to select only those putative siRNA oligonucleotides which fulfill all the basic criteria required as per the algorithm. Finally, a cross search using BLAST was performed to confirm that the designed siRNAs do not have any homology to plant genome sequences. The putative siRNA sequences thus designed to target essential genes of geminiviruses and introduced into the plants may facilitate developing papaya and tomato crops with generic resistance to geminiviruses.     

Simultaneous gene transduction and silencing using stimuli-responsive viral/nonviral chimeric nanoparticles

Despite viral vectors' predominant use in clinical trials, due to higher gene delivery efficiency than nonviral counterparts, intrinsic immunogenicity and limited tunability for multi-modal effects are major concerns for their usage in gene therapy. An adeno-associated viral (AAV) particle was shielded with acid-degradable, siRNA-encapsulating polyketal (PK) shell, resulting in core-shell viral/nonviral chimeric nanoparticles (ChNPs). The AAV core of a ChNP is protected from immune responses by the PK shell which also facilitates the intracellular trafficking of the AAV core and efficiently releases the encapsulated siRNA into the cytoplasm. ChNPs led to significantly enhanced gene transduction, compared to unmodified free AAVs, and simultaneous silencing of a target gene, while avoiding inactivation by recognition from the immune system. Furthermore, conjugation of sialic acid (SA) on the surface of ChNPs enabled receptor-mediated targeted gene delivery to CD22-expressing cells. The ChNPs developed in this study combine the advantages of both viral and nonviral vectors and are a promising platform for targeted co-delivery of DNA and siRNA in inducing synergistic therapeutic effects by simultaneous expression and silencing of multiple genes.     

siRNA及其导入体内外方法的研究进展

RNAi是一种有效的基因封闭工具，siRNA诱导RNAi效应的发挥。siRNA将哺乳动物细胞内特异基因的mRNA 特异性降解而使基因失活，引起转录后基因沉默。siRNA有效作用的发挥关键是依赖它本身的性能及能否较高效率地转染入靶细胞后与靶基因结合。在体外常通过脂质体、聚乙烯亚胺及电穿孔等介导siRNA的转染。siRNA导入体内的过程则要复杂的多，到目前为止仍缺少有效的方法，常用脂质体和聚合物等来介导转染，依赖靶组织和靶细胞的类型来选用转导途径。     

siRNA efficiency: structure or sequence-that is the question

The triumphant success of RNA interference (RNAi) in life sciences is based on its high potency to silence genes in a sequence-specific manner. Nevertheless, the first task for successful RNAi approaches is the identification of highly active small interfering RNAs (siRNAs). Early on, it has been found that the potency of siRNAs can vary drastically. Great progress was made when thermodynamic properties that influence siRNA activity were discovered. Design algorithms based on these parameters enhance the chance to generate potent siRNAs. Still, many siRNAs designed accordingly fail to silence their targeted gene, whereas others are highly efficient despite the fact that they do not fulfil the recommended criteria. Therefore, the accessibility of the siRNA-binding site on the target RNA has been investigated as an additional parameter which is important for RNAi-mediated silencing. These and other factors which are crucial for successful RNAi approaches will be discussed in the present review.     

Inhibition of hepatitis C virus replication and expression by small interfering RNA targeting host cellular genes

Summary. Small interfering RNA (siRNA) is a powerful tool for functional genomics and gene therapy. Viral replication and gene expression are strongly inhibited by siRNA treatment of infected mammalian cells. However, the high sequence specificity of siRNAs, combined with prolonged treatment, promote the emergence of siRNA-resistant virus variants, especially among viruses that encode a polymerase lacking proofreading capabilities, indicating that the antiviral properties of specific siRNAs are not as effective as expected. To investigate the silencing effect of siRNAs against selected host cellular proteins that promote replication of hepatitis C virus (HCV), several siRNAs against human VAMP-associated protein (hVAP-A), La antigen and polypyrimidine-tract-binding protein (PTB) were evaluated. The data show that several siRNAs markedly decreased the expression levels of corresponding cellular genes that inhibited HCV replication in Huh-7 cells. These treatments were also shown to have no impact upon cell viability. These findings provide an alternative approach for blocking HCV replication. Hence, combination therapies with siRNAs against both the virus and host genes that support virus replication are likely to be a potent approach in the treatment of chronic hepatitis C. First and second authors contributed equally to this work.     

Efficient RNAi-based gene family knockdown via set cover optimization

OBJECTIVE: We address the problem of selecting an efficient set of initiator molecules (siRNAs) for RNA interference (RNAi)-based gene family knockdown experiments. Our goal is to select a minimal set of siRNAs that (a) cover a targeted gene family or a specified subset of it, (b) do not cover any untargeted genes, and (c) are individually highly effective at inducing knockdown. METHODS AND MATERIAL: We give two formalizations of the gene family knockdown problem. First, we show that the problem, minimizing the number of siRNAs required to knock down a family of genes, is NP-Hard via a reduction to the set cover problem. Second, we generalize the basic problem to incorporate additional biological constraints and optimality criteria. To solve the resulting set-cover variants, we modify the classical branch-and-bound algorithm to include some of these biological criteria. We find that in many typical cases these constraints reduce the search space enough that we are able to compute exact minimal siRNA covers within reasonable time. For larger cases, we propose a probabilistic greedy algorithm for finding minimal siRNA covers efficiently. We apply our methods to two different gene families, the FREP genes from Biomphalaria glabrata and the olfactory genes from Caenorhabditis elegans. RESULTS AND CONCLUSION: Our computational results on real biological data show that the probabilistic greedy algorithm produces siRNA covers as good as the branch-and-bound algorithm in most cases. Both algorithms return minimal siRNA covers with high predicted probability that the selected siRNAs will be effective at inducing knockdown. We also examine the role of "off-target" interactions: the constraint of avoiding covering untargeted genes can, in some cases, substantially increase the complexity of the resulting solution. Overall, we find that in many common cases our approach significantly reduces the number of siRNAs required in gene family knockdown experiments, as compared to knocking down genes independently.     

Surface functionalized hollow manganese oxide nanoparticles for cancer targeted siRNA delivery and magnetic resonance imaging

Multifunctional hollow manganese oxide nanoparticles (HMON) were produced by a bio-inspired surface functionalization approach, using 3,4-dihydroxy-L-phenylalanine (DOPA) as an adhesive moiety, for cancer targeted delivery of therapeutic siRNA and simultaneous diagnosis via magnetic resonance imaging (MRI). Cationic polyethylenimine-DOPA conjugates were stably immobilized onto the surface of HMON due to the strong binding affinity of DOPA to metal oxides, as examined by Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy. These nanoparticles were subsequently functionalized with a therapeutic monoclonal antibody, Herceptin, to selectively target cancer cells. Confocal microscopy and MR imaging studies revealed that the surface functionalized HMON enabled the targeted detection of cancer cells in T(1)-weighted MRI as well as the efficient intracellular delivery of siRNA for cell-specific gene silencing. These nanomaterials are expected to be widely exploited as multifunctional delivery vehicles for cancer therapy and imaging applications.     

人HSV-1 UL40基因重组质粒的构建及其在siRNA筛选中的应用

目的： 快速筛选出高效沉默人HSV-1 UL40基因的siRNA，为进一步应用RNA干扰的相关研究奠定基础。方法: 应用RT-PCR扩增人HSV-1 F株UL40基因，将其连接到pEGFP-N1构建pEGFP-N1-UL40融合蛋白表达质粒。将化学合成的针对UL40基因的siRNA与重组质粒共转染Vero细胞，通过观察绿色荧光蛋白报告基因的表达筛选高效沉默人HSV-1 UL40基因的siRNA。结果: 倒置荧光显微镜观察发现设计的4对siRNA中siRNA3和siRNA4能明显降低绿色荧光蛋白的表达，流式细胞仪检测证明siRNA3和siRNA4对目的基因沉默效率分别达到76.99％和84.00％。结论: 成功构建了人HSV-1 UL40基因融合表达载体pEGFP-N1-UL40，并利用该载体筛选出2对高效沉默人HSV-1 UL40基因的siRNA。     

Evaluation of single and dual siRNAs targeting rabies virus glycoprotein and nucleoprotein genes for inhibition of virus multiplication in vitro

Small interfering RNAs (siRNAs) targeting rabies virus (RV) glycoprotein (G) and nucleoprotein (N) genes were evaluated as antiviral agents against rabies virus in vitro in BHK-21 cells. To select effective siRNAs targeting RV-G, a plasmid-based transient co-transfection approach was used. In this, siRNAs were expressed as short hairpin RNAs (shRNAs), and their ability to inhibit RV-G gene expression was evaluated in cells transfected with a plasmid expressing RV-G. The nine different siRNAs designed to target RV-G exhibited varying degrees of knockdown of RV-G gene expression. One siRNA (si-G7) with considerable effect in knockdown of RV-G expression also demonstrated significant inhibition of RV multiplication in BHK-21 cells after in vitro challenge with the RV Pasteur virus-11 (PV-11) strain. A decrease in the number of fluorescent foci in siRNA-treated cells and a reduction (86.8 %) in the release of RV into infected cell culture supernatant indicated the anti-rabies potential of siRNA. Similarly, treatment with one siRNA targeting RV-N resulted in a decrease in the number of fluorescent foci and a reduction (85.9 %) in the release of RV. As a dual gene silencing approach where siRNAs targeting RV-G and RV-N genes were expressed from single construct, the anti-rabies-virus effect was observed as an 87.4 % reduction in the release of RV. These results demonstrate that siRNAs targeting RV-G and N, both in single and dual form, have potential as antiviral agent against rabies.     

Inhibition of hepatitis C virus genotype 3a by siRNAs targeting envelope genes

Hepatitis C virus (HCV) genotype 3a is considered a significant risk factor for the development of liver diseases and hepatocellular carcinoma for most of the cases in Pakistan. Because of the limited efficiency of the current therapy, RNA interference (RNAi), which results in sequence-specific degradation of HCV RNA, has potential as a powerful alternative molecular therapeutic approach. The envelope genes (E1 and E2) of HCV come in immediate contact with cells during infection and therefore might be a relevant target for new drug development. In the present study, the expression of E1 and E2 genes of HCV genotype 3a was dramatically reduced at both the mRNA and protein level using gene-specific small interfering RNAs (siRNA) when compared to mock-transfected and cells treated with control siRNAs. The potential of siRNAs to inhibit HCV-3a replication in serum-infected Huh-7 cells was also demonstrated by combined treatment of siRNAs against the E1 and E2 genes, which resulted in a significant decrease in HCV viral copy number. This clearly demonstrates that the RNAi-mediated silencing of HCV E1 and E2 is among the first of its type for the development of an effective siRNA-based therapeutic option against HCV-3a.     

Down-regulation of viral replication by adenoviral-mediated expression of siRNA against cellular cofactors for hepatitis C virus

Small interfering RNA (siRNA) is currently being evaluated not only as a powerful tool for functional genomics, but also as a potentially promising therapeutic agent for cancer and infectious diseases. Inhibitory effect of siRNA on viral replication has been demonstrated in multiple pathogenic viruses. However, because of the high sequence specificity of siRNA-mediated RNA degradation, antiviral efficacy of siRNA directed to viral genome will be largely limited by emergence of escape variants resistant to siRNA due to high mutation rates of virus, especially RNA viruses such as poliovirus and hepatitis C virus (HCV). To investigate the therapeutic feasibility of siRNAs specific for the putative cellular cofactors for HCV, we constructed adenovirus vectors expressing siRNAs against La, polypyrimidine tract-binding protein (PTB), subunit gamma of human eukaryotic initiation factors 2B (eIF2Bgamma), and human VAMP-associated protein of 33 kDa (hVAP-33). Adenoviral-mediated expression of siRNAs markedly diminished expression of the endogenous genes, and silencing of La, PTB, and hVAP-33 by siRNAs substantially blocked HCV replication in Huh-7 cells. Thus, our studies demonstrate the feasibility and potential of adenoviral-delivered siRNAs specific for cellular cofactors in combating HCV infection, which can be used either alone or in combination with siRNA against viral genome to prevent the escape of mutant variants and provide additive or synergistic anti-HCV effects.     

siRNA对单纯疱疹病毒2型ICP4基因抑制作用的研究

目的 探讨RNA干扰对单纯疱疹病毒2型(HSV2)ICP4基因表达和HSV2 DNA复制的抑制作用.方法 化学合成靶向作用于HSV2 ICP4的四对siRNA和阴性对照siRNA,分别命名为siRNA-1,siRNA-2,siRNA-3,siRNA-4及siRNA-N,转染Vero细胞,过夜后用HSV2 HG52标准毒株感染上述Veto细胞,1、2、3、4d和5d收集Vero细胞和上清液,荧光定量RT-PCR检测ICP4 mRNA的表达水平,荧光定量PCR检测HSV2 DNA拷贝数,Western-Blot检测ICP4蛋白表达水平.结果 与阴性对照相比,四对siRNA对HSV2 ICP4 mRNA和蛋白均有不同程度的抑制作用,以siRNA-2抑制作用最强,并且均可显著降低HSV2 DNA拷贝数.结论 靶向作用于HSV2 ICP4的siRNA可显著抑制ICP4mRNA及蛋白表达和HSV2 DNA拷贝数,提示siRNA可通过抑制HSV2 ICP4基因表达而抑制HSV2的复制.     

In vivo comparative study of RNAi methodologies by in ovo electroporation in the chick embryo.

The combination of emergent RNA interference (RNAi) technology with in ovo electroporation in the chick embryo has the potential to provide a powerful and rapid means for functional analyses of novel genes in vivo. In this study, we show that electroporation of short 21-bp RNA duplexes (siRNAs) is a quick and simple method for silencing exogenous and endogenous gene expression in vivo. Quantitative comparisons with two other RNAi protocols that use long double-stranded RNA duplexes and endonuclease-digested duplexes (esiRNAs) demonstrate that siRNAs are significantly more effective at reducing gene expression. Furthermore, we also find that much higher amounts of siRNA are required for silencing of endogenous gene expression relative to plasmid-borne reporter constructs. In short, these results demonstrate that siRNAs are the most effective type of double-stranded RNA duplex for silencing gene expression and suggest that there might be important differences between silencing endogenous and exogenous genes. Finally, we review the parameters for each of these RNA-based methods of RNAi and the controls required to analyze RNAi data in the context of the developing vertebrate embryo.     

单纯疱疹病毒1型 US12 基因siRNA筛选及其对病毒增殖的影响

目的: 筛选高效沉默单纯疱疹病毒1型(HSV-1) US12 基因的siRNA,研究siRNA沉默 US12 基因后对HSV-1增殖的影响。方法: 构建pEGFP-N1-US12融合表达质粒,设计并化学合成3对靶向 US12 基因的siRNA,与pEGFP-N1-US12融合表达质粒共转染Vero细胞,流式细胞术筛选高效抑制pEGFP-N1-US12融合蛋白的siRNA,实时荧光定量PCR检测siRNA对细胞内US12 mRNA表达水平的抑制效果,空斑减数实验评价siRNA对HSV-1增殖的抑制效果。结果: 共转染实验筛选出高效抑制pEGFP-N1-US12融合蛋白的siRNAS121、siRNAS122及siRNAS123。3对siRNA均能显著降低感染细胞US12 mRNA的表达水平,但空斑减数实验均未发现3对siRNA对HSV-1的增殖有显著抑制效果。结论: 成功构建pEGFP-N1-US12融合表达质粒,筛选到高效抑制 US12 基因表达的3对siRNA,但是对病毒的体外增殖无显著影响,表明 US12 表达的立即早期蛋白ICP47的功能可能与HSV-1体外增殖无直接相关。     

Delivery systems for the direct application of siRNAs to induce RNA interference (RNAi) in vivo

RNA interference (RNAi) is a powerful method for specific gene silencing which may also lead to promising novel therapeutic strategies. It is mediated through small interfering RNAs (siRNAs) which sequence-specifically trigger the cleavage and subsequent degradation of their target mRNA. One critical factor is the ability to deliver intact siRNAs into target cells/organs in vivo. This review highlights the mechanism of RNAi and the guidelines for the design of optimal siRNAs. It gives an overview of studies based on the systemic or local application of naked siRNAs or the use of various nonviral siRNA delivery systems. One promising avenue is the the complexation of siRNAs with the polyethylenimine (PEI), which efficiently stabilizes siRNAs and, upon systemic administration, leads to the delivery of the intact siRNAs into different organs. The antitumorigenic effects of PEI/siRNA-mediated in vivo gene-targeting of tumor-relevant proteins like in mouse tumor xenograft models are described.