Journal of RNAi and gene silencing : an international journal of RNA and gene targeting research最新文献

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.

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.

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.

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 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.

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.

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.

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.