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

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.

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.

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.

[This corrects the article on p. 386 in vol. 6, PMID: 20628499.].

RNA interference plays a significant role in manipulating cellular and viral mechanisms to maintain latency during HIV-1 infection. HIV-1 produces several microRNAs including one from the TAR element which alter the host's response to infection. Since cyclin/cdk complexes are important for viral transcription, these studies focus on the possible cdk inhibitors that inhibit viral transcription, without affecting normal cellular mechanisms. Roscovitine and Flavopiridol are well-studied cdk inhibitors that are effective at suppressing their target cdks at a low IC50. These cdk inhibitors and possibly future generations of drugs are affected by microRNA mechanisms. From our studies, we developed a third generation derivative called CR8#13. In cells that lack Dicer there was a higher level of basal viral LTR-reporter transcription. When drugs, specifically Flavopiridol and CR8#13 were added, the transcriptional inhibition of the LTR was less potent in cells that lacked Dicer. Also, after transfection with HIV-1 clone (pNL4.3), CR8 and CR8#13 derivatives were shown to be more effective viral transcription inhibitors in cell lines that contained Dicer (T-cells) as compared to Dicer deficient lines (monocytes). We next asked whether the addition of CR8 or CR8#13 could possibly increase levels of TAR microRNA in HIV-1 LTR containing cells. We demonstrate that the 3'TAR microRNA is produced in higher amounts after drug treatment, resulting in microRNA recruitment to the LTR. MicroRNA recruitment results in chromatin alteration, changes in Pol II phosphorylation and viral transcription inhibition. In conclusion, our results indicate that viral microRNA, specifically the TAR microRNA produced from the HIV-1 LTR is responsible for maintaining latent infections by manipulating host cell mechanisms to limit transcription from the viral LTR promoter. With the microRNA machinery present, cdk inhibitors are able to significantly increase the amount of TAR microRNA, leading to downregulation of viral LTR transcription.

MicroRNAs (miRNAs) are small RNA molecules that modulate gene expression through degradation of specific mRNAs and/or repression of their translation. miRNAs are involved in both physiological and pathological processes, such as apoptosis and cancer. Their presence has been demonstrated in several organisms as well as in viruses. Virus encoded miRNAs can act as viral gene expression regulators, but they may also interfere with the expression of host genes. Viral miRNAs may control host cell proliferation by targeting cell-cycle and apoptosis regulators. Therefore, they could be involved in cancer pathogenesis. Computational prediction of miRNA/target pairs is a fundamental step in these studies. Here, we describe the use of miRiam, a novel program based on both thermodynamics features and empirical constraints, to predict viral miRNAs/human targets interactions. miRiam exploits target mRNA secondary structure accessibility and interaction rules, inferred from validated miRNA/mRNA pairs. A set of genes involved in apoptosis and cell-cycle regulation was identified as target for our studies. This choice was supported by the knowledge that DNA tumor viruses interfere with the above processes in humans. miRNAs were selected from two cancer-related viruses, Epstein-Barr Virus (EBV) and Kaposi-Sarcoma-Associated Herpes Virus (KSHV). Results show that several transcripts possess potential binding sites for these miRNAs. This work has produced a set of plausible hypotheses of involvement of v-miRNAs and human apoptosis genes in cancer development. Our results suggest that during viral infection, besides the protein-based host regulation mechanism, a post-transcriptional level interference may exist. miRiam is freely available for downloading at http://ferrolab.dmi.unict.it/miriam.