MicroRNAs are a fundamental class of small RNAs involved in post-transcriptional gene regulation; however, the mechanism by which microRNAs regulate their gene targets in animals remains poorly understood. Practically, a mechanistic understanding of microRNA binding and regulation is crucial for the rational design of microRNA-based vectors for RNA interference. In this report, we focus on the largest known class of microRNA targets, the canonical seed targets, and explore the factors involved in modulating target downregulation in vivo at the protein level. Using an in vivo sensor assay in the ascidian Ciona intestinalis, we quantify miR-124-mediated downregulation of 38 canonical seed targets cloned from the Ciona genome as well as 10 control non-targets. Supporting previous findings, we observed that the seed type and number of seed sites are correlated with downregulation. However, up to a 50% variation in downregulation levels was observed for targets within the same seed class, indicating a role of non-seed factors in modulating downregulation. Although we did not observe a significant correlation of previously reported non-seed determinants with downregulation levels at saturation in our assay, our data suggest that two previously identified factors, secondary structure and 3'end complementarity, may play a role in the initial kinetics of microRNA-target binding. Importantly, using different concentrations of miR-124 we show that dose-dependent target downregulation profiles follow Michaelis-Menten kinetics. In summary, our findings emphasize the importance of non-seed factors as well as the importance of cellular concentrations of microRNAs relative to their targets when studying the mechanisms of endogenous microRNA regulation.
The tenth RNAi conference was held at St. Hilda's College Oxford on the 24-26 March 2015. The conference offered researchers from all over the world the chance to present, discuss and discover work pertaining to the field of RNAi. RNAi has become an essential technique in genomic research for functional validation as well as an exciting avenue to explore in therapeutic medicine. Emerging techniques such as CRISPR as well as improvements in efficiency of existing techniques and expansions in libraries have cemented the importance of RNAi at the cutting edge of research. Featured presentations and posters showcased recent research in the field ranging from RNA detection in bio fluids through to potential oligonucleotide therapies.
A key goal of our recent research efforts has been to develop novel 'triggerable nanoparticle' systems with real potential utility in vivo. These are designed to be stable from the point of administration until a target site of interest is reached, then triggered for the controlled release of therapeutic agent payload(s) at the target site by changes in local endogenous conditions or through the application of some exogenous stimulus. Here we describe investigations into the use of enzymes to trigger RNAi-mediated therapy through a process of enzyme-assisted nanoparticle triggerability. Our approach is to use PEG(2000)-peptidyl lipids with peptidyl moieties sensitive to tumour-localized elastase or matrix metalloproteinase-2 digestion, and from these prepare putative enzyme-triggered PEGylated siRNA-nanoparticles. Our results provide initial proof of concept in vitro. From these data, we propose that this concept should be applicable for functional delivery of therapeutic nucleic acids to tumour cells in vivo, although the mechanism for enzyme-assisted nanoparticle triggerability remains to be fully characterized.
RNA interference (RNAi) is an important biological process that ultimately leads to suppression of gene expression. Activators of RNAi are typically small interfering RNAs (siRNA) and microRNAs (miRNA) that offer considerable therapeutic potnetial. However, a major obstacle to take these these molecules to the clinic is the absence of safe and reliable means for their specific delivery to target cells. In this regard, a highly promising class of molecules is represented by nucleic acid aptamers. These are short, structured, single-stranded RNAs or DNAs oligonucleotides that, by binding with high specificity to target molecules, provide high affinity ligands and potential antagonists of disease-associated proteins. Further, because of the high binding specificity, aptamers represent a powerful tool for the selective delivery of therapeutic cargos, including mi/siRNAs, chemotherapeutics, toxins and nanoparticles to cancer cells or tissues, thus potentially increasing the efficacy of a given therapy as well as reducing toxicity. In this review, we will focus on recent advances in the field of aptamer-mediated mi/siRNA delivery, discussing their potential and challenges in cancer therapy.
Low biological activity and inefficient targeted delivery in vivo have hindered RNA interference (RNAi)-based therapy from realising its full clinical potential. To overcome these hurdles, progresses have been made to develop new technologies optimizing oligonucleotides chemistry on one hand and achieving its effective delivery on the other hand. In this report, we achieved, by using the electropulsation technique (EP), efficient cellular delivery of chemically-modified oligonucleotide: The locked nucleic acid (LNA)/DNA oligomer. We used single cell level confocal fluorescence microscopy to follow the spatial and temporal distribution of electrotransferred cyanine 5 (Cy5)-labeled LNA/DNA oligomer. We observed that EP allowed LNA/DNA oligomer cellular uptake providing the oligomer a rapid access to the cytoplasm of HeLa cells. Within a few minutes after electrotransfer, Cy5-LNA/DNA oligomers shuttle from cytoplasm to nucleus whereas in absence of pulses application, Cy5-LNA/DNA oligomers were not detected. We then observed a redistribution of the Cy5 fluorescence that accumulated over time into cytoplasmic organelles. To go further and to identify these compartments, we used the HeLa GFP-Rab7 cell line to visualise late endosomes, and lysosomal or mitochondrial specific markers. Our results showed that the EP technique allowed direct entry into the cytoplasm of the Cy5-LNA/DNA oligomer bypassing the endocytosic pathway. However, in absence of pulses application, Cy5-LNA/DNA oligomer were able to enter cells through the endocytosic pathway. We demonstrated that EP is an efficient technique for LNA-based oligonucleotides delivery offering strong advantages by avoiding the endolysosomal compartmentalization, giving a rapid and free access to the cytoplasm and the nucleus where they can find their targets.
The eighth annual RNAi international conference and exhibition, RNAi2013 was hosted at St Hilda's College, Oxford, UK (19-21 March 2013), and provided a platform for congregation of researchers with both academic and industrial backgrounds to share and discuss their most recent work in the fast advancing field of RNAi. RNAi has been recognised as a fundamental method for functional genomic investigations and has great potential as a therapeutic intervention for several human diseases. RNA-induced gene expression inhibition mechanisms were discussed for both the benefit of research and clinical therapeutics. The conference conveyed an impressive series of presentations given by national and international RNAi research leaders. Additionally, research posters were exhibited for the entirety of the conference. Furthermore, technology workshops were provided by Sigma-Aldrich, Eupheria Biotech GmbH and Carl Zeiss enabling conference attendees to learn about their most advanced RNAi expertise. These companies also participated in a trade exhibition along with Exiqon to promote the latest commercial RNAi products.
AMP-activated protein kinase (AMPK) is a master metabolic regulator that responds to the AMP: ATP ratio and promotes ATP production when the cell is low on energy. There are two isoforms of the catalytic alpha subunit, AMPKα1 and AMPKα2. Here, we describe the production of a small interfering RNA (siRNA) and a short hairpin RNA (shRNA) targeting both catalytic isoforms of AMPK in human, mouse, and rat. Multiple loop sequences were tested to generate the most effective shRNA. The shRNA causes significant knockdown of both isoforms of AMPKα in mouse and human cells. The shRNA effectively knocked down AMPKα1 and AMPKα2 protein levels, compared to a five basepair mismatch-control shRNA in mouse fibroblast NIH3T3 cells and significantly knocked down AMPKα1 (63%) and AMPKα2 (72%) levels compared to control in human embryonic kidney cells, HEK293s. The shRNA also causes a significant reduction in AMPK activity, measured as phosphorylation of acetyl-CoA carboxylase (ACC), a direct phosphorylation target. While the protein levels of total ACC remained the same between the AMPKα1and α2 shRNA and control shRNA-treated cells, there was a 41% reduction in phospho-ACC protein levels. The generation of this AMPKα1and α2 shRNA can be used to stably knock down protein levels and activity of both catalytic isoforms of AMPK in different species to assess function.
The PML-RARα fusion gene disrupts the retinoic acid differentiation signal in a range of leukaemia types, promoting proliferation. We designed a shRNA to target the fusion mRNA, and the shRNA expression cassette was delivered via lentiviral transduction. Delivery of this shRNA significantly down regulated the target mRNA, with effects also evident at the protein level. When combined with ATRA administration, this down regulation of the fusion gene strongly inhibited proliferation in the NB4 PML cell line.
A key goal of our research is the targeted delivery of functional biopharmaceutical agents of interest, such as small interfering RNA (siRNA), to selected cells by means of receptor-mediated nanoparticle technologies. Recently, we described how pH-triggered, PEGylated siRNA-nanoparticles (pH triggered siRNA-ABC nanoparticles) were able to mediate the passive targeting of siRNA to liver cells in vivo. In addition, PEGylated siRNA nanoparticles enabled for long-term circulation (LTC siRNA-ABC nanoparticles, LEsiRNA nanoparticles) were shown to do the same to tumour cells in vivo. Further gains in the efficiency of siRNA delivery are expected to require active targeting with nanoparticles targeted for delivery and cellular uptake by means of attached biological ligands. Here we report on the development of a new synthetic chemistry and a bioconjugation methodology that allows for the controlled formulation of PEGylated nanoparticles which surface-present integrin-targeting peptides unambiguously and so enable integrin receptor-mediated cellular uptake. Furthermore, we present delivery data that provide a clear preliminary demonstration of physical principles that we propose should underpin successful, bonefide receptor-mediated targeted delivery of therapeutic and/or imaging agents to cells.
ABC transporters export clinically-relevant drugs and their over-expression causes multidrug resistance. In order to knock-down ABC transporters, ABCC1 and ABCC2, 13 shRNAs were developed. Four shRNA candidates were tested in vivo using self-complementary adeno-associated virus serotype 8. A strong, specific knock-down of Abbc2 was observed in mice liver, but at the cost of toxicity caused by oversaturation of the RNAi machinery due to high shRNA expression. Subsequent generation of artificial miRNAs showed better efficacy profile. These results demonstrate the feasibility of knocking down Abbc2 via AAV-delivered shRNAs to the liver, and encourage the use of miRNA in further therapeutics development.
RNA interference (RNAi) is a post-transcriptional pathway in which double-stranded RNA (dsRNA) triggers the degradation of complementary mRNA in the cytoplasm of eukaryotic cells. In plants and in some animals, including Caenorhabditis elegans, initiation of RNAi in one cell can lead to sequence-specific RNA silencing in another cell, a phenomenon referred to as non-cell-autonomous RNAi. Until recently, this phenomenon had not been observed in mammalian cells. Here, we review emerging data demonstrating that non-cell-autonomous RNAi occurs in cultured mammalian cells. We discuss possible mechanisms for the transfer of RNAi between mammalian cells and highlight the implications of this phenomenon for the development of in vivo cell-based RNAi delivery.
Rhizopus oryzae is a filamentous fungus, belonging to the order Mucorales. It can ferment a wide range of carbohydrates hydrolyzed from lignocellulosic materials and even cellobiose to produce ethanol. However, R. oryzae also produces lactic acid as a major metabolite, which reduces the yield of ethanol. In this study, we show that significant reduction of lactic acid production could be achieved by short (25nt) synthetic siRNAs targeting the ldhA gene. The average yield of lactic acid production by R. oryzae during the batch fermentation process, where glucose had been used as a sole carbon source, diminished from 0.07gm/gm in wild type to 0.01gm/gm in silenced samples. In contrast, the average yield of ethanol production increased from 0.39gm/gm in wild type to 0.45gm/gm in silenced samples. These results show 85.7% (gm/gm) reduction in lactic acid production as compared with the wild type R. oryzae, while an increase of 15.4% (gm/gm) in ethanol yield.
RNAi2011: Gene Regulation by Small RNAs, the 6th international Oxford RNAi conference, was held at St Hilda's College, University of Oxford (UK), between 29 and 31 March, 2011. Sessions covered a variety of topics and provided an excellent opportunity to discuss and reflect upon recent developments in this fast-moving and promising field. A number of conference posters were also on display throughout the conference, and a number of trade stands gave conference participants the opportunity to learn about the latest product developments.
[This corrects the article on p. 386 in vol. 6, PMID: 20628499.].
The xylem feeding leafhopper Homalodisaca vitripennis (H. vitripennis) is an unusually robust and efficient vector of Xylella fastidiosa, a Gram-negative bacterium which causes several very important plant diseases. Here we investigated RNA interference (RNAi) to target actin, a key component of insect cells and whole bodies, in H. vitripennis cells. RNAi effectors were delivered via lipid based transfection and real-time RT-PCR, RNA hybridization, and microscopic analyses were employed to verify RNAi effects. When actin dsRNAs were used, a 10-fold decrease in the target H. vitripennis actin mRNA level was seen in cells. Altered phenotypic effects also were evident in transfected cells, as were small interfering RNAs, hallmarks of RNAi. The use of H. vitripennis cells and RNAi offers new opportunities to research hemipterans, the most important insect vectors of plant pathogens.
The use of chemically-synthesized short interfering RNAs (siRNAs) is the key method of choice to manipulate gene expression in mammalian cell cultures and in vivo. Several previous studies have aimed at inducing cell-specific RNA interference (RNAi) in order to use siRNA molecules as therapeutic reagents. Here, we used peptide-inhibited siRNAs that were activated after cleavage by cell-specific peptidases. We show that siRNAs with bound peptide at the antisense strand could be activated in target cells and were able to induce RNAi in a cell-specific manner. Green Fluorescent Protein (GFP) and Signal Transducer and Activator of Transcription (STAT)-3 gene expression were selectively reduced in a JEG-3 human choriocarcinoma cell line expressing the activating enzyme caspase-4, whereas the effect was absent in HEK cells which lacked the enzyme. In JEG-3 cells, reduction of STAT3 gene expression by conventional and peptide-inhibited siRNA led to a decrease in cell proliferation. This suggests that peptide-inhibited siRNAs provide improved cell specificity and offers new opportunities for their therapeutic use.
Transgenic technologies can provide important animal models for studying drug-metabolizing enzymes. Our overall aim was to generate versatile cell and animal systems that exhibited varying levels of cytochrome P450 oxidoreductase (POR) activity, more accurately modelling the human population for pharmacological and toxicology studies. Towards this goal we evaluated RNA-interference constructs designed for use in vitro and in vivo for reducing POR activity in hepatocytes. This study clearly demonstrates that both POR protein level and reductase activity can be significantly knocked down in Hepa-1 cells in vitro, while highlighting the difficulty in predicting knockdown efficiency in transgenic animals. The high levels of embryonic lethality observed, and inability to produce multi-copy transgenic animals indicates that high levels of shRNA expression may be detrimental to embryonic development.
RNA interference (RNAi) is a widely used molecular biology technique to investigate the importance of specific genes in molecular pathways. Since mammalian cells are equipped with endogenous RNAi processing machinery, it has become common practice to transfect constructs that encode for short hairpin RNAs that are then cleaved to form the active RNAi sequences that bind to target mRNAs. Given the profit potential of this research approach, companies have developed retroviral libraries of shRNA constructs targeting the majority of the human genes. Recent technologic advances have allowed the rapid improvement of the vectors carrying the shRNA constructs while the silencing sequences remain the same. Therefore, sub-cloning of shRNA sequences from more obsolete vectors to newer vectors is a straightforward way to take advantage of newer delivery technologies. We describe here a streamlined procedure to transfer shRNA sequences from the pSM2 retroviral vector to a newer pGIPZ vector that is more stable, contains a GFP cassette and allows the preparation of high titer viral particles for transduction of cells and in vivo use. We demonstrate that our protocol provides a cost-effective and fast method to successfully sub-clone shRNA from a pSM2 retroviral vector to a pGIPZ lentiviral vector making it a useful tool for the investigators that have purchased pSM2 vectors in the past and wish now to upgrade their constructs by inserting them in more versatile vectors.
The interleukin (IL)-13 pathway and its associated transcription factor, signal transducer and activator of transcription 6 (STAT6), have been clearly implicated in the pathogenesis of bronchial asthma. We have developed a system to effectively screen the STAT6 gene for targeting with small interfering (si) RNA molecules. By incorporating an in silico and in vitro screening system we were able to identify fourteen siRNA molecules suitable for pre-clinical drug development. Furthermore, we were able to demonstrate that modification of certain siRNAs, designed to improve in vivo longevity, was possible without significant loss of target knockdown efficacy and that the siRNA produced by our selection process did not induce demonstrable interferon responses. These data suggest that several STAT6-targeting siRNA suitable for pre-clinical development are available for potential use in the treatment of asthma.
