Functional inhibition of chemokine receptor CCR2 by dicer-substrate-siRNA prevents pain development.

BACKGROUND: Accumulating evidence suggests that the C-C chemokine ligand 2 (CCL2, or monocyte chemoattractant protein 1) acts as a neuromodulator in the central nervous system through its binding to the C-C chemokine receptor 2 (CCR2). Notably, it is well established that the CCL2/CCR2 axis plays a key role in neuron-glia communication as well as in spinal nociceptive transmission. Gene silencing through RNA interference has recently emerged as a promising avenue in research and drug development, including therapeutic management of chronic pain. In the present study, we used 27-mer Dicer-substrate small interfering RNA (DsiRNA) targeting CCR2 and assessed their ability to reverse the nociceptive behaviors induced by spinal CCL2 injection or following intraplantar injection of complete Freund's adjuvant. RESULTS: To this end, we first developed high-potency DsiRNAs designed to target different sequences distributed across the rat CCR2 (rCCR2) messenger RNA. For optimization, methyl groups were added to the two most potent DsiRNA candidates (Evader and M7 2'-O-methyl modified duplexes) in order to improve in vivo duplex stability and to reduce potential immunostimulatory activity. Our results demonstrated that all modified candidates formulated with the cell-penetrating peptide reagent Transductin showed strong RNAi activity following intrathecal delivery, exhibiting >50% rCCR2 knockdown in lumbar dorsal root ganglia. Accordingly, we found that these DsiRNA duplexes were able to reduce spinal microglia activation and were effective at blocking CCL2-induced mechanical hypersensitivity. Along with similar reductions of rCCR2 messenger RNA, both sequences and methylation patterns were similarly effective in inhibiting the CCL2 nociceptive action for the whole seven days testing period, compared to mismatch DsiRNA. DsiRNAs against CCR2 also reversed the hypernociceptive responses observed in the complete Freund's adjuvant-induced inflammatory chronic pain model. CONCLUSION: Altogether, these results validate CCR2 as a an appropriate molecular target for pain control and demonstrate that RNAi-based gene therapy represent an highly specific alternative to classical pharmacological approaches to treat central pathologies such as chronic pain.

Spinal NTS2 receptor activation reverses signs of neuropathic pain.

Management of painful peripheral neuropathies remains challenging, since patients with chronic pain respond poorly to the available pharmacopeia. In recent years, the G-protein-coupled receptor neurotensin (NT) type 2 (NTS2) emerged as an attractive target for treating transitory pain states. To date, however, there is no evidence for its role in the regulation of chronic peripheral neuropathies. Here, we found that NTS2 receptors were largely localized to primary afferent fibers and superficial dorsal horns. Changes in the time course of the gene expression profile of NT, NTS1, and NTS2 were observed over a 28-d period following the sciatic nerve constriction [chronic constriction injury (CCI) model]. We next determined the effects of central delivery of selective-NTS2 agonists to CCI-treated rats on both mechanical allodynia (evoked withdrawal responses) and weight-bearing deficits (discomfort and quality-of-life proxies). The NTS2 analogs JMV431, levocabastine, and ß-lactotensin were all effective in reducing ongoing tactile allodynia in CCI-treated rats. Likewise, amitriptyline, pregabalin, and morphine significantly attenuated CCI-induced mechanical hypersensitivity. NTS2 agonists were also efficient in reversing weight-bearing and postural deficits caused by nerve damage, unlike reference analgesics currently used in the clinic. Thus, NTS2 agonists may offer new treatment avenues for limiting pain associated with peripheral neuropathies and improve functional rehabilitation and well-being.

Post-transcriptional regulation of cystic fibrosis transmembrane conductance regulator expression and function by microRNAs.

MicroRNAs (miRNAs) are increasingly recognized as important posttranscriptional regulators of gene expression, and changes in their actions can contribute to disease states. Little is understood regarding miRNA functions in the airway epithelium under normal or diseased conditions. We profiled miRNA expression in well-differentiated primary cultures of human cystic fibrosis (CF) and non-CF airway epithelia, and discovered that miR-509-3p and miR-494 concentrations were increased in CF epithelia. Human non-CF airway epithelia, transfected with the mimics of miR-509-3p or miR-494, showed decreased cystic fibrosis transmembrane conductance regulator (CFTR) expression, whereas their respective anti-miRs exerted the opposite effect. Interestingly, the two miRNAs acted cooperatively in regulating CFTR expression. Upon infecting non-CF airway epithelial cells with Staphylococcus aureus, or upon stimulating them with the proinflammatory cytokines TNF-α or IL-1ß, we observed an increased expression of both miRNAs and a concurrent decrease in CFTR expression and function, suggesting that inflammatory mediators may regulate these miRNAs. Transfecting epithelia with anti-miRs for miR-509-3p and miR-494, or inhibiting NF-κB signaling before stimulating cells with TNFα or IL-1ß, suppressed these responses, suggesting that the expression of both miRNAs was responsive to NF-κB signaling. Thus, miR-509-3p and miR-494 are dynamic regulators of CFTR abundance and function in normal, non-CF airway epithelia.

A microRNA network regulates expression and biosynthesis of wild-type and DeltaF508 mutant cystic fibrosis transmembrane conductance regulator.

Production of functional proteins requires multiple steps, including gene transcription and posttranslational processing. MicroRNAs (miRNAs) can regulate individual stages of these processes. Despite the importance of the cystic fibrosis transmembrane conductance regulator (CFTR) channel for epithelial anion transport, how its expression is regulated remains uncertain. We discovered that miRNA-138 regulates CFTR expression through its interactions with the transcriptional regulatory protein SIN3A. Treating airway epithelia with an miR-138 mimic increased CFTR mRNA and also enhanced CFTR abundance and transepithelial Cl(-) permeability independent of elevated mRNA levels. An miR-138 anti-miR had the opposite effects. Importantly, miR-138 altered the expression of many genes encoding proteins that associate with CFTR and may influence its biosynthesis. The most common CFTR mutation, ΔF508, causes protein misfolding, protein degradation, and cystic fibrosis. Remarkably, manipulating the miR-138 regulatory network also improved biosynthesis of CFTR-ΔF508 and restored Cl(-) transport to cystic fibrosis airway epithelia. This miRNA-regulated network directs gene expression from the chromosome to the cell membrane, indicating that an individual miRNA can control a cellular process more broadly than recognized previously. This discovery also provides therapeutic avenues for restoring CFTR function to cells affected by the most common cystic fibrosis mutation.

Protection against TNFα-dependent liver toxicity by intraperitoneal liposome delivered DsiRNA targeting TNFα in vivo.

Tumor necrosis factor-alpha (TNFα) is a classic proinflammatory cytokine implicated in the pathogenesis of several autoimmune and inflammatory diseases including viral encephalitis. Macrophages being major producers of TNFα are thus attractive targets for in vivo RNA interference (RNAi) mediated down regulation of TNFα. The application of RNAi technology to in vivo models however presents obstacles, including rapid degradation of RNA duplexes in plasma, insufficient delivery to the target cell population and toxicity associated with intravenous administration of synthetic RNAs and carrier compounds. We exploited the phagocytic ability of macrophages for delivery of Dicer-substrate small interfering RNAs (DsiRNAs) targeting TNFα (DsiTNFα) by intraperitoneal administration of lipid-DsiRNA complexes that were efficiently taken up by peritoneal macrophages and other phagocytic cells. We report that DsiTNFα-lipid complexes delivered intraperitoneally altered the disease outcome in an acute sepsis model. Down-regulation of TNFα in peritoneal CD11b+ monocytes reduced liver damage in C57BL/6 mice and significantly delayed acute mortality in mice treated with low dose LPS plus d-galactosamine (D-GalN).

Synthesis and characterization of mannosylated pegylated polyethylenimine as a carrier for siRNA.

Regulation of gene expression using small interfering RNA (siRNA) is a promising strategy for research and treatment of numerous diseases. In this study, we develop and characterize a delivery system for siRNA composed of polyethylenimine (PEI), polyethylene glycol (PEG), and mannose (Man). Cationic PEI complexes and compacts siRNA, PEG forms a hydrophilic layer outside of the polyplex for steric stabilization, and mannose serves as a cell binding ligand for macrophages. The PEI-PEG-mannose delivery system was constructed in two different ways. In the first approach, mannose and PEG chains are directly conjugated to the PEI backbone. In the second approach, mannose is conjugated to one end of the PEG chain and the other end of the PEG chain is conjugated to the PEI backbone. The PEI-PEG-mannose delivery systems were synthesized with 3.45-13.3 PEG chains and 4.7-3.0 mannose molecules per PEI. The PEI-PEG-Man-siRNA polyplexes displayed a coarse surface in Scanning Electron Microscopy (SEM) images. Polyplex sizes were found to range from 169 to 357 nm. Gel retardation assays showed that the PEI-PEG-mannose polymers are able to efficiently complex with siRNA at low N/P ratios. Confocal microscope images showed that the PEI-PEG-Man-siRNA polyplexes could enter cells and localized in the lysosomes at 2h post-incubation. Pegylation of the PEI reduced toxicity without any adverse reduction in knockdown efficiency relative to PEI alone. Mannosylation of the PEI-PEG could be carried out without any significant reduction in knockdown efficiency relative to PEI alone. Conjugating mannose to PEI via the PEG spacer generated superior toxicity and gene knockdown activity relative to conjugating mannose and PEG directly onto the PEI backbone.

T-cell activation is enhanced by targeting IL-10 cytokine production in toll-like receptor-stimulated macrophages.

Toll-like receptor (TLR) agonists represent potentially useful cancer vaccine adjuvants in their ability to stimulate antigen-presenting cells (APCs) and subsequently amplify the cytotoxic T-cell response. The purpose of this study was to characterize APC responses to TLR activation and to determine the subsequent effect on lymphocyte activation. We exposed murine primary bone marrow-derived macrophages to increasing concentrations of agonists to TLRs 2, 3, 4, and 9. This resulted in a dose-dependent increase in production of not only tumor necrosis factor-alpha (TNF-α), a surrogate marker of the proinflammatory response, but also interleukin 10 (IL-10), a well-described inhibitory cytokine. Importantly, IL-10 secretion was not induced by low concentrations of TLR agonists that readily produced TNF-α. We subsequently stimulated lymphocytes with anti-CD3 antibody in the presence of media from macrophages activated with higher doses of TLR agonists and observed suppression of interferon gamma release. Use of both IL-10 knockout macrophages and IL-10 small-interfering RNA (siRNA) ablated this suppressive effect. Finally, IL-10 siRNA was successfully used to suppress CpG-induced IL-10 production in vivo. We conclude that TLR-mediated APC stimulation can induce a paradoxical inhibitory effect on T-cell activation mediated by IL-10.

RNase H-dependent PCR (rhPCR): improved specificity and single nucleotide polymorphism detection using blocked cleavable primers.

BACKGROUND: The polymerase chain reaction (PCR) is commonly used to detect the presence of nucleic acid sequences both in research and diagnostic settings. While high specificity is often achieved, biological requirements sometimes necessitate that primers are placed in suboptimal locations which lead to problems with the formation of primer dimers and/or misamplification of homologous sequences. RESULTS: Pyrococcus abyssi (P.a.) RNase H2 was used to enable PCR to be performed using blocked primers containing a single ribonucleotide residue which are activated via cleavage by the enzyme (rhPCR). Cleavage occurs 5'-to the RNA base following primer hybridization to the target DNA. The requirement of the primer to first hybridize with the target sequence to gain activity eliminates the formation of primer-dimers and greatly reduces misamplification of closely related sequences. Mismatches near the scissile linkage decrease the efficiency of cleavage by RNase H2, further increasing the specificity of the assay. When applied to the detection of single nucleotide polymorphisms (SNPs), rhPCR was found to be far more sensitive than standard allele-specific PCR. In general, the best discrimination occurs when the mismatch is placed at the RNA:DNA base pair. CONCLUSION: rhPCR eliminates the formation of primer dimers and markedly improves the specificity of PCR with respect to off-target amplification. These advantages of the assay should find utility in challenging qPCR applications such as genotyping, high level multiplex assays and rare allele detection.

Chemical modification patterns compatible with high potency dicer-substrate small interfering RNAs.

Dicer-substrate small interfering RNAs (DsiRNAs) are synthetic RNA duplexes that are processed by Dicer into 21-mer species and show improved potency as triggers of RNA interference, particularly when used at low dose. Chemical modification patterns that are compatible with high potency 21-mer small interfering RNAs have been reported by several groups. However, modification patterns have not been studied for Dicer-substrate duplexes. We therefore synthesized a series of chemically modified 27-mer DsiRNAs and correlated modification patterns with functional potency. Some modification patterns profoundly reduced function although other patterns maintained high potency. Effects of sequence context were observed, where the relative potency of modification patterns varied between sites. A modification pattern involving alternating 2'-O-methyl RNA bases was developed that generally retains high potency when tested in different sites in different genes, evades activation of the innate immune system, and improves stability in serum.

Central delivery of Dicer-substrate siRNA: a direct application for pain research.

RNA interference (RNAi) is gaining acceptance as a potential therapeutic strategy against peripheral disease, and several clinical trials are already underway with 21-mer small-interfering RNA (siRNA) as the active pharmaceutical agent. However, for central affliction like pain, such innovating therapies are limited but nevertheless crucial to improve pain research and management. We demonstrate here the proof-of-concept of the use of 27-mer Dicer-substrate siRNA (DsiRNA) for silencing targets related to CNS disorders such as pain states. Indeed, low dose DsiRNA (0.005 mg/kg) was highly efficient in reducing the expression of the neurotensin receptor-2 (NTS2, a G-protein-coupled receptor (GPCR) involved in ascending nociception) in rat spinal cord through intrathecal (IT) administration formulated with the cationic lipid i-Fect. Along with specific decrease in NTS2 mRNA and protein, our results show a significant alteration in the analgesic effect of a selective-NTS2 agonist, reaching 93% inhibition up to 3-4 days after administration of DsiRNA. In order to ensure that these findings were not biased by unsuspected off-target effects (OTEs), we also demonstrated that treatment with a second NTS2-specific DsiRNA also reversed NTS2-induced antinociception, and that NTS2-specific 27-mer duplexes did not alter signaling through NTS1, a closely related receptor. Altogether, DsiRNAi represents a potent tool for dissecting nociceptive pathways and could further lead to a new class of central active drugs.

Advances in molecular and cellular therapies for hearing loss.

Development of effective therapeutics for hearing loss has proven to be a slow and difficult process, evidenced by the lack of restorative medicines and technologies currently available to the otolaryngologist. In large part this is attributable to the limited regenerative potential in cochlear cells and the secondary degeneration of the cochlear architecture that commonly follows sensorineural hearing impairment. Therapeutic advances have been made using animal models, particularly in regeneration and remodeling of spiral ganglion neurons, which retract and die following hair cell loss. Natural regeneration in avian and reptilian systems provides hope that replacement of hair cells is achievable in humans. The most exciting recent advancements in this field have been made in the relatively new areas of cellular replacement and gene therapy. In this review we discuss recent developments in gene- and cell-based therapy for hearing loss, including detailed analysis of therapeutic mechanisms such as RNA interference and stem cell transplantation, as well as in utero delivery to the mammalian inner ear. We explore the advantages and limitations associated with the use of these strategies for inner ear restoration.

Advances in Molecular and Cellular Therapies for Hearing Loss.

Development of effective therapeutics for hearing loss has proven to be a slow and difficult process, evidenced by the lack of restorative medicines and technologies currently available to the otolaryngologist. In large part this is attributable to the limited regenerative potential in cochlear cells and the secondary degeneration of the cochlear architecture that commonly follows sensorineural hearing impairment. Therapeutic advances have been made using animal models, particularly in regeneration and remodeling of spiral ganglion neurons, which retract and die following hair cell loss. Natural regeneration in avian and reptilian systems provides hope that replacement of hair cells is achievable in humans. The most exciting recent advancements in this field have been made in the relatively new areas of cellular replacement and gene therapy. In this review we discuss recent developments in gene- and cell-based therapy for hearing loss, including detailed analysis of therapeutic mechanisms such as RNA interference and stem cell transplantation, as well as in utero delivery to the mammalian inner ear. We explore the advantages and limitations associated with the use of these strategies for inner ear restoration.

Characterization of modified antisense oligonucleotides in Xenopus laevis embryos.

A wide variety of modified oligonucleotides have been tested as antisense agents. Each chemical modification produces a distinct profile of potency, toxicity, and specificity. Novel cationic phosphoramidate-modified antisense oligonucleotides have been developed recently that have unique and interesting properties. We compared the relative potency and specificity of a variety of established antisense oligonucleotides, including phosphorothioates (PS), 2'-O-methyl (2'OMe) RNAs, locked nucleic acids (LNAs), and neutral methoxyethyl (MEA) phosphoramidates with new cationic N,N-dimethylethylenediamine (DMED) phosphoramidate-modified antisense oligonucleotides. A series of oligonucleotides was synthesized that targeted two sites in the Xenopus laevis survivin gene and were introduced into Xenopus embryos by microinjection. Effects on survivin gene expression were examined using quantitative real-time PCR. Of the various modified oligonucleotide designs tested, LNA/PS chimeras (which showed the highest melting temperature) and DMED/phosphodiester chimeras (which showed protection of neighboring phosphate bonds) were potent in reducing gene expression. At 40 nM, overall specificity was superior for the LNA/PS-modified compounds compared with the DMED-modified oligonucleotides. However, at 400 nM, both of these compounds led to significant degradation of survivin mRNA, even when up to three mismatches were present in the heteroduplex.

Functional polarity is introduced by Dicer processing of short substrate RNAs.

Synthetic RNA duplexes that are substrates for Dicer are potent triggers of RNA interference (RNAi). Blunt 27mer duplexes can be up to 100-fold more potent than traditional 21mer duplexes. Not all 27mer duplexes show increased potency. Evaluation of the products of in vitro dicing reactions using electrospray ionization mass spectrometry reveals that a variety of products can be produced by Dicer cleavage. Use of asymmetric duplexes having a single 2-base 3'-overhang restricts the heterogeneity that results from dicing. Inclusion of DNA residues at the ends of blunt duplexes also limits heterogeneity. Combination of asymmetric 2-base 3'-overhang with 3'-DNA residues on the blunt end result in a duplex form which directs dicing to predictably yield a single primary cleavage product. It is therefore possible to design a 27mer duplex which is processed by Dicer to yield a specific, desired 21mer species. Using this strategy, two different 27mers can be designed that result in the same 21mer after dicing, one where the 3'-overhang resides on the antisense (AS) strand and dicing proceeds to the 'right' ('R') and one where the 3'-overhang resides on the sense (S) strand and dicing proceeds to the 'left' ('L'). Interestingly, the 'R' version of the asymmetric 27mer is generally more potent in reducing target gene levels than the 'L' version 27mer. Strand targeting experiments show asymmetric strand utilization between the two different 27mer forms, with the 'R' form favoring S strand and the 'L' form favoring AS strand silencing. Thus, Dicer processing confers functional polarity within the RNAi pathway.

Synthetic dsRNA Dicer substrates enhance RNAi potency and efficacy.

RNA interference (RNAi) is the process of sequence-specific post-transcriptional gene silencing triggered by double-stranded RNAs. In attempts to identify RNAi triggers that effectively function at lower concentrations, we found that synthetic RNA duplexes 25-30 nucleotides in length can be up to 100-fold more potent than corresponding conventional 21-mer small interfering RNAs (siRNAs). Some sites that are refractory to silencing by 21-mer siRNAs can be effectively targeted by 27-mer duplexes, with silencing lasting up to 10 d. Notably, the 27-mers do not induce interferon or activate protein kinase R (PKR). The enhanced potency of the longer duplexes is attributed to the fact that they are substrates of the Dicer endonuclease, directly linking the production of siRNAs to incorporation in the RNA-induced silencing complex. These results provide an alternative strategy for eliciting RNAi-mediated target cleavage using low concentrations of synthetic RNA as substrates for cellular Dicer-mediated cleavage.

Hybridization kinetics and thermodynamics of molecular beacons.

Molecular beacons are increasingly being used in many applications involving nucleic acid detection and quantification. The stem-loop structure of molecular beacons provides a competing reaction for probe-target hybridization that serves to increase probe specificity, which is particularly useful when single-base discrimination is desired. To fully realize the potential of molecular beacons, it is necessary to optimize their structure. Here we report a systematic study of the thermodynamic and kinetic parameters that describe the molecular beacon structure-function relationship. Both probe and stem lengths are shown to have a significant impact on the binding specificity and hybridization kinetic rates of molecular beacons. Specifically, molecular beacons with longer stem lengths have an improved ability to discriminate between targets over a broader range of temperatures. However, this is accompanied by a decrease in the rate of molecular beacon-target hybridization. Molecular beacons with longer probe lengths tend to have lower dissociation constants, increased kinetic rate constants, and decreased specificity. Molecular beacons with very short stems have a lower signal-to-background ratio than molecular beacons with longer stems. These features have significant implications for the design of molecular beacons for various applications.