miRNA cassettes in viral vectors: problems and solutions

The discovery of RNA interference (RNAi), an evolutionary conserved gene silencing mechanism that is triggered by double stranded RNA, has led to tremendous efforts to use this technology for basic research and new RNA therapeutics. RNAi can be induced via transfection of synthetic small interfering RNAs (siRNAs), which results in a transient knockdown of the targeted mRNA. For stable gene silencing, short hairpin RNA (shRNA) or microRNA (miRNA) constructs have been developed. In mammals and humans, the natural RNAi pathway is triggered via endogenously expressed miRNAs. The use of modified miRNA expression cassettes to elucidate fundamental biological questions or to develop therapeutic strategies has received much attention. Viral vectors are particularly useful for the delivery of miRNA genes to specific target cells. To date, many viral vectors have been developed, each with distinct characteristics that make one vector more suitable for a certain purpose than others. This review covers the recent progress in miRNA-based gene-silencing approaches that use viral vectors, with a focus on their unique properties, respective limitations and possible solutions. Furthermore, we discuss a related topic that involves the insertion of miRNA-target sequences in viral vector systems to restrict their cellular range of gene expression. This article is part of a Special Issue entitled: MicroRNAs in viral gene regulation.     

Minimizing variables among hairpin-based RNAi vectors reveals the potency of shRNAs

RNA interference (RNAi) is a cellular process regulating gene expression and participating in innate defense in many organisms. RNAi has also been utilized as a tool to query gene function and is being developed as a therapeutic strategy for several diseases. Synthetic small interfering (siRNAs) or expressed stem–loop RNAs (short-hairpin RNAs [shRNAs] or artificial microRNAs [miRNAs]) have been delivered to cultured cells and organisms to inhibit expression of a variety of genes. A persistent question in the field, however, is which RNAi expression system is most suitable for distinct applications. To date, shRNA- and artificial miRNA-based strategies have been compared with conflicting results. In prior comparisons, sequences required for efficient RNAi processing and loading of the intended antisense strand into the RNAi-induced silencing complex (RISC) were not considered. We therefore revisited the shRNA–miRNA comparison question. Initially, we developed an improved artificial miRNA vector and confirmed the optimal shRNA configuration by altering structural features of these RNAi substrates. Subsequently, we engineered and compared shRNA- and miRNA-based RNAi expression vectors that would be processed to yield similar siRNAs that exhibit comparable strand biasing. Our results demonstrate that when comparison variables are minimized, the shRNAs tested were more potent than the artificial miRNAs in mediating gene silencing independent of target sequence and experimental setting (in vitro and in vivo). In addition, we show that shRNAs are expressed at considerably higher levels relative to artificial miRNAs, thus providing mechanistic insight to explain their increased potency.     

Conditional gene silencing in mammalian cells mediated by a stress-inducible promoter

MicroRNAs (miRNAs) are endogenous non-coding small RNAs, which negatively regulate gene expression in a sequence-specific manner through the RNA interference (RNAi) pathway. Here we describe a new miRNA-based conditional RNAi expression system that relies on cellular stress-response mechanisms in mammalian cells. In our constructs, expression of miRNA mimics is tightly controlled by a heat shock-inducible promoter. This system is highly effective in silencing permanently or conditionally expressed luciferase. The stress inducible vectors also effectively deplete co-expressed pro-apoptotic protein CHOP with heat shock. Furthermore, we demonstrate cloning of a protein-coding sequence between the stress-inducible promoter and the miRNA expression cassette allows simultaneous silencing of a target gene and activation of synthesis of a protein of choice in response to stress stimulation. This new conditional gene silencing approach could be an invaluable tool for various areas of basic and applied research and for therapeutic intervention.     

RNA干扰在疾病治疗方面的应用研究

RNA干扰是由双链RNA引起的序列特异的基因沉默现象。由于RNA干扰能在细胞组织及动物模型中沉默疾病相关基因,因此,RNA干扰也是各种疾病治疗的有效手段。在哺乳动物细胞内诱导RNA干扰可以通过导入小干扰RNA（siRNA）,或是以质粒、病毒为载体表达短的发夹RNA（shRNA）而实现。本文介绍了RNA干扰在疾病治疗方面的应用,并就其面临的挑战进行讨论。     

RNA interference in mammalian cells using siRNAs synthesized with T7 RNA polymerase

Methods that allow the specific silencing of a desired gene are invaluable tools for research. One of these is based on RNA interference (RNAi), a process by which double-stranded RNA (dsRNA) specifically suppresses the expression of a target mRNA. Recently, it has been reported that RNAi also works in mammalian cells if small interfering RNAs (siRNAs) are used to avoid activation of the interferon system by long dsRNA. Thus, RNAi could become a major tool for reverse genetics in mammalian systems. However, the high cost and the limited availability of the short synthetic RNAs and the lack of certainty that a designed siRNA will work present major drawbacks of the siRNA technology. Here we present an alternative method to obtain cheap and large amounts of siRNAs using T7 RNA polymerase. With multiple transfection procedures, including calcium phosphate co-precipitation, we demonstrate silencing of both exogenous and endogenous genes.     

Gene silencing using micro-RNA designed hairpins

During RNA interference (RNAi), long dsRNA is processed to approximately 21 nt duplexes, short interfering RNAs (siRNAs), which silence genes through a mRNA degradation pathway. Small temporal RNAs (stRNAs) and micro-RNAs (miRNAs) are approximately 21 nt RNAs that are processed from endogenously encoded hairpin-structured precursors, and function to silence genes via translational repression. Here we report that synthetic hairpin RNAs that mimic siRNAs and miRNA precursor molecules can target a gene for silencing, and the mechanism of silencing appears to be through mRNA degradation and not translational repression. The sequence and structural configuration of these RNAs are important, and even slight modification in structure can affect the silencing activity of the hairpins. Furthermore, these RNAs are active when expressed by DNA vectors containing polymerase III promoters, opening the possibility for new approaches in stable RNAi-based loss of function studies.     

Gene Silencing Efficiency and INF-β Induction Effects of Splicing miRNA 155-Based Artificial miRNA with Pre-miRNA Stem-Loop Structures

Artificial microRNA (miRNA) expression vectors have been developed and used for RNA interference. The secondary structure of artificial miRNA is important for RNA interference efficacy. We designed two groups of six artificial splicing miRNA 155-based miRNAs (SM155-based miRNAs) with the same target in the coding region or 3' UTR of a target gene and studied their RNA silencing efficiency and interferon β (IFN-β) induction effects. SM155-based miRNA with a mismatch at the +1 position and a bulge at the +11, +12 positions in a miRNA precursor stem-loop structure showed the highest gene silencing efficiency and lowest IFN-β induction effect (increased IFN-β mRNA level by 10% in both target cases), regardless of the specificity of the target sequence, suggesting that pSM155-based miRNA with this design could be a valuable miRNA expression vector.     

A plasmid-based system for expressing small interfering RNA libraries in mammalian cells

Background  

RNA interference (RNAi) is an evolutionarily conserved process that functions to inhibit gene expression. The use of RNAi in mammals as a tool to study gene function has rapidly developed in the last couple of years since the discovery that the function-inhibiting units of RNAi are short 21–25 nt double-stranded RNAs (siRNAs) derived from their longer template. The use of siRNAs allows for gene-specific knock-down without induction of the non-specific interferon response in mammalian cells. Multiple systems have been developed to introduce siRNAs into mammals. One of the most appealing of these techniques is the use of vectors containing polymerase III promoters to drive expression of hairpin siRNAs. However, there are multiple limitations to using hairpin siRNA vectors including the observation that some are unstable in bacteria and are difficult to sequence.     

Small interfering RNAs against the TAR RNA binding protein, TRBP, a Dicer cofactor, inhibit human immunodeficiency virus type 1 long terminal repeat expression and viral production

RNA interference (RNAi) is now widely used for gene silencing in mammalian cells. The mechanism uses the RNA-induced silencing complex, in which Dicer, Ago2, and the human immunodeficiency virus type 1 (HIV-1) TAR RNA binding protein (TRBP) are the main components. TRBP is a protein that increases HIV-1 expression and replication by inhibition of the interferon-induced protein kinase PKR and by increasing translation of viral mRNA. After HIV infection, TRBP could restrict the viral RNA through its activity in RNAi or could contribute more to the enhancement of viral replication. To determine which function will be predominant in the virological context, we analyzed whether the inhibition of its expression could enhance or decrease HIV replication. We have generated small interfering RNAs (siRNAs) against TRBP and found that they decrease HIV-1 long terminal repeat (LTR) basal expression 2-fold, and the LTR Tat transactivated level up to 10-fold. In the context of HIV replication, siRNAs against TRBP decrease the expression of viral genes and inhibit viral production up to fivefold. The moderate increase in PKR expression and activation indicates that it contributes partially to viral gene inhibition. The moderate decrease in micro-RNA (miRNA) biogenesis by TRBP siRNAs suggests that in the context of HIV replication, TRBP functions other than RNAi are predominant. In addition, siRNAs against Dicer decrease viral production twofold and impede miRNA biogenesis. These results suggest that, in the context of HIV replication, TRBP contributes mainly to the enhancement of virus production and that Dicer does not mediate HIV restriction by RNAi.     

TRBP, a regulator of cellular PKR and HIV-1 virus expression, interacts with Dicer and functions in RNA silencing

Dicer is a key enzyme involved in RNA interference (RNAi) and microRNA (miRNA) pathways. It is required for biogenesis of miRNAs and small interfering RNAs (siRNAs), and also has a role in the effector steps of RNA silencing. Apart from Argonautes, no proteins are known to associate with Dicer in mammalian cells. In this work, we describe the identification of TRBP (human immunodeficiency virus (HIV-1) transactivating response (TAR) RNA-binding protein) as a protein partner of human Dicer. We show that TRBP is required for optimal RNA silencing mediated by siRNAs and endogenous miRNAs, and that it facilitates cleavage of pre-miRNA in vitro. TRBP had previously been assigned several functions, including inhibition of the interferon-induced double-stranded RNA-regulated protein kinase PKR and modulation of HIV-1 gene expression by association with TAR. The TRBP-Dicer interaction shown raises interesting questions about the potential interplay between RNAi and interferon-PKR pathways.     

A quick and efficient approach for gene silencing by using triple putative microRNA-based short hairpin RNAs

The RNA interference (RNAi) technique has been widely used in gene function studies. It is typical to screen for effective siRNAs by knocking down targeted genes since a single gene can be suppressed by several siRNAs to varying degrees. The miRNA-based short hairpin RNA (shRNA) is a natural inducer of RNAi and has been used in siRNA expression strategies. We investigated the potential application of multiple putative microRNA-based shRNAs for gene silencing and studied the inhibition efficiency of exogenous GFP and firefly luciferase (luc) by triple human mir155-based shRNA expression vectors. A total of three candidate siRNA sequences targeted against GFP or luc were selected based on an online prediction program. Single and triple miRNA-155-based shRNAs targeted against GFP or luc were transfected into HEK293 cells mediated by the pcDNA₃ vector with an RNA polymerase II-type CMV (cytomegalovirus) promoter. Comparisons with negative control shRNAs revealed that GFP levels were markedly reduced by the triple miRNA-155-based GFP shRNA by fluorescent microscopy. Consistent results from the dual luciferase assay and real-time quantitative RT-PCR revealed that the triple miRNA-155-based GFP shRNA significantly suppressed GFP expression (P < 0.01), without significant differences from the most effective single miRNA-155-based GFP shRNA (P > 0.05). Results from the dual luciferase assay and real-time quantitative RT-PCR revealed that the triple miRNA-155-based luc shRNA significantly suppressed luc expression as the most effective single miRNA-155-based luc shRNA (P < 0.05). These studies demonstrated the gene silencing efficiency mediated by the triple putative miRNA-155-based shRNAs. This suggested that multiple miRNA-based shRNAs are quick and valuable strategies for gene silencing.     

Design of siRNAs producing unstructured guide-RNAs results in improved RNA interference efficiency

In RNA interference (RNAi), guide RNAs direct RNA-induced silencing complexes (RISC) to their mRNA targets, thus enabling the cleavage that leads to gene silencing. We describe a strong inverse correlation between the degree of guide-RNA secondary structure formation and gene silencing by small interfering (si)RNA. Unstructured guide strands mediate the strongest silencing whereas structures with base-paired ends are inactive. Thus, the availability of terminal nucleotides within guide structures determines the strength of silencing. A to G and C to U base exchanges, which involve wobble base-pairing with the target but preserve complementarity, turned inactive into active guide structures, thereby expanding the space of functional siRNAs. Previously observed base degenerations among mature micro (mi)RNAs together with the data presented here suggest a crucial role of the guide-RNA structures in miRNA action. The analysis of the effect of the secondary structures of guide-RNA sequences on RNAi efficiency provides a basis for better understanding RNA silencing pathways and improving the design of siRNAs.