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PubMed2026

Target, silence, replace: a review on RNA-based drugs in modern medicine.

RNA therapies have evolved into a revolutionary approach in contemporary medicine for treating various diseases by directly targeting RNA molecules engaged in disease pathogenesis. These therapeutic agents regulate biological processes through diverse mechanisms, including modulation of RNA function and gene expression. Medical applications of RNA are greatly enhanced by its structure, adaptability, and capacity for targeted binding. Among these traits is its ability to bind to certain molecules unique to those chemicals. RNA-based treatments have emerged from advancements in the production, modification, and cellular transport of RNA molecules. Several RNA drugs have been approved whereas some are under trial for few diseases. RNA therapeutics can function at the level of RNAs, DNAs and proteins. The evolution of mRNA vaccines during the COVID-19 epidemic emphasizes the exciting potential of RNA therapies in the treatment of diseases. This article provides a comprehensive overview of the several forms of RNA therapies, including small-interfering RNA (siRNA), messenger RNA (mRNA), and antisense-oligonucleotides (ASOs), together with information on their action mechanisms and delivery strategies that improve cellular absorption and shield RNA molecules from degradation. Further, CRISPR-based editing of the genome can be employed for modification of target RNA sequences for various disorders. Development of RNA aptamers have also been identified as pivotal RNA-therapeutic candidate. Additionally, we have explained mechanistic details and examples of drugs approved for RNA therapy. Emphasizing their potential to enhance patient outcomes and fulfil unmet medical requirements, we also highlight the clinical development of RNA therapies in treating cancer and other infectious diseases.

RNA interference↗
PubMed2001

Glycyrrhizin enhances interleukin-12 production in peritoneal macrophages.

Interleukin-12 (IL-12) is a monocyte/macrophage-derived cytokine that plays a prominent role in the development of T helper type 1 (Th1) cell-mediated immune responses. Glycyrrhizin (GL), an aqueous extract of liquorice root, used as Chinese medicine, is known to have various immunomodulating activities. In this study, GL showed a dose-dependent priming effect on lipopolysaccharide (LPS)-induced IL-12 p40 and IL-12 p70 (heterodimer of p40 and p35) protein production by peritoneal macrophages (PM). The maximal effect was observed when GL was intraperitoneally administered 12 hr before the PM were harvested and stimulated in vitro with LPS. The increases in IL-12 p70 and p40 protein production were primarily due to up-regulated transcription of IL-12 p35 and p40 messenger RNAs (mRNAs), as demonstrated by RNase protection assay. The augmentation of IL-12 p40 mRNA expression induced by GL pretreatment was associated with increased NF-kappaB activation. Moreover, GL exhibited the same priming effect on IL-12 production in interferon-gamma knockout (IFN-gamma-/-) mice. The production of granulocyte-macrophage colony-stimulating factor (GM-CSF) was not induced at any time point after GL pretreatment. These findings demonstrated the ability of GL to enhance LPS-induced IL-12 production by peritoneal macrophages, and indicated that the priming effect of GL on IL-12 production was independent of both IFN-gamma and GM-CSF.

Animals↗
PubMed1996

Internal initiation of translation directed by the 5'-untranslated region of the mRNA for eIF4G, a factor involved in the picornavirus-induced switch from cap-dependent to internal initiation.

The eIF4 group initiation factors carry out recognition of the mRNA cap, unwinding of mRNA secondary structure, and binding of mRNA to the 43 S preinitiation complex. Infection by picornaviruses results in proteolytic cleavage of one of these factors, eIF4G, an event that severely restricts cap-dependent translation but permits cap-independent initiation to proceed from internal ribosome entry sequences in picornaviral RNAs. The 5'-untranslated region (5'-UTR) of eIF4G mRNA resembles such picornaviral sequences in being unusually long and containing multiple open reading frames and a polypyrimidine tract. When inserted upstream of a luciferase reporter gene, this 5'-UTR served as a translational enhancer in four different cell lines. Mutation of all four upstream ATG codons to AAG did not alter the translational enhancement. The presence of the eIF4G 5'-UTR between an RNA hairpin and the luciferase cistron stimulated expression 119-fold. Similarly, the presence of the 5'-UTR between the two cistrons of a bicistronic mRNA stimulated expression of the downstream cistron 42-fold. These results indicate that the eIF4G 5'-UTR directs internal initiation. The ability to continue synthesis of eIF4G when the cell is unable to carry out normal cap-dependent translation may represent an autoregulatory mechanism or be part of the cellular response to stresses that interrupt cap-dependent translation.

Base Sequence↗
PubMed2004

Oxidant hypersensitivity of Fanconi anemia type C-deficient cells is dependent on a redox-regulated apoptotic pathway.

Fanconi anemia is a genetic disorder characterized by bone marrow failure. Significant evidence supports enhanced apoptosis of hematopoietic stem/progenitor cells as a critical factor in the pathogenesis of bone marrow failure in Fanconi anemia. However, the molecular mechanism(s) responsible for the apoptotic phenotype are incompletely understood. Here, we tested whether alterations in the activation of a redox-dependent pathway may participate in the pro-apoptotic phenotype of primary Fancc -/- cells in response to oxidative stress. Our data indicate that Fancc -/- cells are highly sensitive to oxidant stimuli and undergo enhanced oxidant-mediated apoptosis compared with wild type controls. In addition, antioxidants preferentially enhanced the survival of Fancc -/- cells. Because oxidative stress activates the redox-dependent ASK1 pathway, we assessed whether Fancc -/- cells exhibited increased oxidant-induced ASK1 activation. Our results revealed ASK1 hyperactivation in H2O2-treated Fancc -/- cells. Furthermore, using small interfering RNAs to decrease ASK1 expression and a dominant negative ASK1 mutant to inhibit ASK1 kinase activity, we determined that H2O2-induced apoptosis was ASK1-dependent. Collectively, these data argue that the predisposition of Fancc -/- hematopoietic stem/progenitor cells to apoptosis is mediated in part through altered redox regulation and ASK1 hyperactivation.

Apoptosis↗
PubMed1992

Auxin and growth regulation of rice coleoptile segments : molecular analysis.

Auxin-stimulated elongation of apical segments of rice (Oryza sativa L. cv Arborio) coleoptiles occurring in the first 4 hours of treatment has been studied. Cell extension promoted in the first 2 hours by 10 micromolar indole-3-acetic acid (IAA) is specifically auxin-dependent, whereas after 4 hours, elongation also depends on endogenous production of ethylene. Similar to other systems, rice coleoptile cell elongation stimulated by auxin requires continuous synthesis of RNA and protein. Two-dimensional gel analysis of the in vitro translation products obtained from polyadenylated RNAs extracted from treated and untreated segments after 1 or 4 hours from the initial addition of IAA shows few transcriptional differences. At 60 minutes of treatment, the level of three mRNAs coding for proteins of 22.5, 25, and 33 kilodaltons was moderately enhanced while the disappearance of a 38 kilodalton translation product was observed. Additional repression of another mRNA coding for a 28 kilodalton product begins to show by this time, but becomes more evident after 4 hours treatment. At 4 hours, four IAA-specific mRNA enhancements coding for proteins with molecular masses ranging between 35 to 40 kilodaltons were also observed. We discuss these data in relation to the possible involvement of IAA-mediated transcriptional regulation in growth promotion of rice coleoptiles and, more widely, in control of cell elongation.

Journal Article↗
PubMed2004

Effects of silencing leukocyte-type 12/15-lipoxygenase using short interfering RNAs.

The leukocyte-type 12/15-lipoxygenase (12/15-LO) has been implicated in the pathogenesis of atherosclerosis, hypertension, and diabetes. 12/15-LO and its products are associated with LDL oxidation, cellular growth, migration, adhesion, and inflammatory gene expression in monocytes/macrophages, endothelial cells, and vascular smooth muscle cells (VSMCs). Our objective, therefore, was to develop novel expression vectors for short interfering RNAs (siRNAs) targeting 12/15-LO to evaluate its functional relevance in macrophages and VSMCs. We used a PCR-based approach to rapidly identify effective siRNA target sites on mouse 12/15-LO and initially tested their efficacy on a fusion construct of 12/15-LO cDNA and enhanced green fluorescent protein. We then cloned these U6 promoter+siRNA PCR products into plasmid vectors [short hairpin siRNAs (shRNAs)] to knockdown endogenous 12/15-LO expression in mouse macrophages and also rat and mouse VSMCs. Furthermore, the functional effects of shRNA-mediated 12/15-LO knockdown were noted by the reduced oxidant stress and chemokine [monocyte chemoattractant protein-1 (MCP-1)] expression in a differentiated mouse monocytic cell line as well as by the reduced cellular adhesion and fibronectin expression in VMSCs. Knocking down 12/15-LO expression also reduced the expression of inflammatory genes, MCP-1, vascular cell adhesion molecule-1, and interleukin-6 in VSMCs. Our results illustrate the functional relevance of 12/15-LO activation in macrophages and VSMCs and its relationship to oxidant stress and inflammation.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
PubMed1991

[Effect of double-stranded RNA and interferon type I on reproduction of cytomegalovirus in human fibroblasts].

Double-stranded RNAs (dsRNA) - larifan and ridostin, and recombinant interferons-alpha-2 and beta-1, manufactured in the USSR, inhibited the reproduction of CMV in cell culture. Antiviral effect depended on concentration of preparations, time of administration and m. o. i. DEAE-dextran enhances antiviral effect dsRNA. DsRNA and interferons added to cells infected with CMV at high m. o. i. enhance the viral reproduction. Among studied preparations highest antiviral effect was shown by natural leucocyte interferon (Egis). It was 10 times more active, than recombinant IFNs and in concentration of 1000 U/ml was comparable to activity of larifan (200 mg/ml).

Antiviral Agents↗
PubMed1997

The protein cofactor allows the sequence of an RNase P ribozyme to diversify by maintaining the catalytically active structure of the enzyme.

To study the effect proteins have on the catalysis and evolution of RNA enzymes, we simulated evolution of RNase P catalytic M1 RNA in vitro, in the presence and absence of its C5 protein cofactor. In the presence of C5, functional M1 sequence variants (not catalytically active in the absence of C5) were selected in addition to those identical to M1. C5 maintains the catalytically active structure of the variants and allows for an enhanced spectrum of M1 molecules to function in the context of a ribonucleoprotein (RNP) complex. The generation of an RNP enzyme, requiring both RNA and protein components, from a catalytically active RNA molecule has implications for how modern RNP complexes evolved from ancestral RNAs.

Bacterial Proteins↗
PubMedDate not supplied

Enhanced expression of S8, L12, L23a, L27 and L30 ribosomal protein mRNAs in human hepatocellular carcinoma.

Differential display (DD) analysis using surgically resected human hepatocellular carcinoma (HCC) and adjacent non-tumorous liver tissues was performed. We identified 5 cDNAs up-regulated in human hepatocellular carcinoma, encoding S8, L12, L23a, L27 and L30 ribosomal protein mRNAs. Northern blot analysis, using total RNAs from thirteen pairs of HCC and abjacent non-tumorous liver tissues demonstrated that these mRNA levels were up-regulated along with the histological grading of tumors. The expression of these mRNAs was also high in three human HCC cell lines (HuH-7, HepG2 and HLF), irrespective of the growth state. These results suggest that activation of these genes is an important manifestation of HCC phenotypes.

Blotting, Northern↗
PubMed1996

Oxidant-inducible adapt 15 RNA is associated with growth arrest- and DNA damage-inducible gadd153 and gadd45.

We have recently described a novel RNA, designated adapt15, which is strongly induced by a pretreatment concentration of hydrogen peroxide in hamster HA-1 fibroblasts under conditions where a protective "adaptive response" occurs. adapt15 may therefore be involved in protecting cells against the damaging effects of oxidative stress. Since two other known sequences, gadd45 and gadd153m were also found to be induced under our pretreatment conditions and are known to be growth arrest and DNA damage inducible, we decided to also assess the possible association of adapt15 with growth arrest and DNA damage. We found that, like gadd45 and gadd153, the levels of adapt15 RNA were low during proliferation, but high during density saturation- and low serum (G0)-growth arrests. Exposure of HA-1 cells to DNA-damaging agents revealed significant induction of adapt15 RNA by methylmeth- anesulfonate and cis-platinum but not X-irradiation. Near identical responses were also observed for gadd45 and gadd153 RNAs, suggesting coordinate regulation of adapt15, gadd45, and gadd153. All three RNAs were also increased relative to control following heat shock, a nongenotoxic treatment. Finally, the induction of adapt15 by hydrogen peroxide was strongly dependent upon calcium, a hallmark of gadd153 induction. The coordinate inductions of adapt15, gadd45, and gadd153 by multiple agents, and their induction by an adaptive- but not by a nonadaptive-response level of hydrogen peroxide, suggest these RNAs may act in concert to protect cells against the damaging effects of oxidative stress.

Adaptation, Biological↗
PubMed2005

RNA interference targeting of Bcr-Abl increases chronic myeloid leukemia cell killing by 17-allylamino-17-demethoxygeldanamycin.

17-Allylamino-17-demethoxygeldanamycin (17-AAG) induces degradation of Hsp90 client proteins, including Bcr-Abl, however, its clinical use as an anti-tumor agent may be limited by toxicity and modest efficacy. We reasoned that Bcr-Abl targeting by RNA interference (RNAi) might selectively increase the activity of 17-AAG against Bcr-Abl+ leukemia cells. 17-AAG in combination with targeting small interfering RNAs (siRNAs) reduced Bcr-Abl protein levels, triggered increases in markers of apoptosis and decreased cell viability more effectively than did control siRNA and 17-AAG together, or Bcr-Abl targeting siRNA alone. Combination targeting strategies such as this may therefore achieve enhanced therapeutic potency.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed2004

A Dicer-2-dependent 80s complex cleaves targeted mRNAs during RNAi in Drosophila.

We use native gel electrophoresis to characterize complexes that mediate RNA interference (RNAi) in Drosophila. Our data reveal three distinct complexes (R1, R2, and R3) that assemble on short interfering RNAs (siRNAs) in vitro. To form, all three complexes require Dicer-2 (Dcr-2), which directly contacts siRNAs in the ATP-independent R1 complex. R1 serves as a precursor to both the R2 and R3 complexes. R3 is a large (80S), ATP-enhanced complex that contains unwound siRNAs, cofractionates with known RNAi factors, and binds and cleaves targeted mRNAs in a cognate-siRNA-dependent manner. Our results establish an ordered biochemical pathway for RISC assembly and indicate that siRNAs must first interact with Dcr-2 to reach the R3 "holo-RISC" complex. Dcr-2 does not simply transfer siRNAs to a distinct effector complex, but rather assembles into RISC along with the siRNAs, indicating that its role extends beyond the initiation phase of RNAi.

Adenosine Triphosphate↗
PubMed1999

Expression patterns of matrix metalloproteinases and their inhibitors in parenchymal and non-parenchymal cells of rat liver: regulation by TNF-alpha and TGF-beta1.

BACKGROUND/AIMS: Although matrix metalloproteinases (MMPs) and their specific inhibitors (TIMPs) play an essential role in liver injury associated with tissue remodeling, the cellular origin of MMPs/TMPs within the liver remains to be clarified. METHODS: Different liver cell populations were analysed with respect to their expression by reverse transcription-polymerase chain reaction, Northern blot analysis and zymography. RESULTS: MMP and TIMP coding transcripts were detectable in all liver cell types by reverse transcription-polymerase chain reaction; however, the cellular expression levels were markedly different as assessed by Northern blot analysis. Gelatinase-B was predominantly expressed in Kupffer cells, gelatinase-A in hepatic stellate cells and rat liver myofibroblasts and stromelysins-1, -2 as well as collagenase in hepatic stellate cells. Membrane type-1 MMP (MMP-14) was found in significant amounts in all liver cells. TIMP-1 coding m-RNAs were present mainly in hepatic stellate cells and rat liver myofibroblasts, TIMP-2 additionally in Kupffer cells, while TIMP-3 expression was detectable only in hepatocytes. During in vitro activation of hepatic stellate cells, MMP expression was mostly downregulated, while TIMP expression was enhanced, thereby providing an explanation for matrix accumulation co-localised with these cells during chronic liver injury. In general, TNF-alpha stimulated both MMP and TIMP expression of hepatic stellate cells, while TGF-beta1 induced TIMP expression only. CONCLUSIONS: Collectively these data demonstrate that all resident liver cells are involved in matrix degradation to some extent and that hepatic stellate cells play an important role in matrix breakdown in addition to matrix synthesis. The cytokine-specific regulation of MMP/TIMP expression in hepatic stellate cells suggests that the initial matrix breakdown following liver injury might be enhanced by TNF-alpha, while diminished matrix degradation during chronic tissue injury might be due to the action of TGF-beta1 through TIMP induction.

Animals↗
PubMed2005

Predicting candidate genomic sequences that correspond to synthetic functional RNA motifs.

Riboswitches and RNA interference are important emerging mechanisms found in many organisms to control gene expression. To enhance our understanding of such RNA roles, finding small regulatory motifs in genomes presents a challenge on a wide scale. Many simple functional RNA motifs have been found by in vitro selection experiments, which produce synthetic target-binding aptamers as well as catalytic RNAs, including the hammerhead ribozyme. Motivated by the prediction of Piganeau and Schroeder [(2003) Chem. Biol., 10, 103-104] that synthetic RNAs may have natural counterparts, we develop and apply an efficient computational protocol for identifying aptamer-like motifs in genomes. We define motifs from the sequence and structural information of synthetic aptamers, search for sequences in genomes that will produce motif matches, and then evaluate the structural stability and statistical significance of the potential hits. Our application to aptamers for streptomycin, chloramphenicol, neomycin B and ATP identifies 37 candidate sequences (in coding and non-coding regions) that fold to the target aptamer structures in bacterial and archaeal genomes. Further energetic screening reveals that several candidates exhibit energetic properties and sequence conservation patterns that are characteristic of functional motifs. Besides providing candidates for experimental testing, our computational protocol offers an avenue for expanding natural RNA's functional repertoire.

Algorithms↗
PubMed2005

Ribozymes, DNAzymes and small interfering RNAs as therapeutics.

Selective gene silencing by nucleic acid enzymes has provided researchers with a new strategy to block gene expression and drug target validation. Ribozymes, DNAzymes and small interfering RNAs (siRNAs) are being explored as genetic inhibitors of gene expression as well as potential therapeutics against viral infections, inflammatory disorders, haematological diseases and cancers. We review the mode of action of these molecules, with special emphasis on their construction and the possibility to enhance their serum half-lives via specific chemical modifications. Their potential use in cell cultures and in animal models for disease is also highlighted.

Animals↗
PubMed1992

Differential expression of cytokines in human blood monocyte subpopulations.

Cytokine expression was analyzed in CD14++ regular monocytes and in the novel subset of CD14+/CD16+ small monocytes. Biologic activity for tumor necrosis factor (TNF), interleukin-1 (IL-1), and IL-6 in the supernatant of elutriator-enriched, cell sorter-purified small monocytes was about 10-fold lower compared with regular monocytes when stimulated with lipopolysaccharide (LPS) for 12 hours. In CD14++ regular monocytes levels were 1,157 U x 10(-3)/mL, 158 U/mL, and 1,337 U/mL for TNF, IL-1, and IL-6, respectively. By contrast, CD14+/CD16+ small monocytes exhibited 137 U x 10(-3)/mL, 14 U/mL, and 60 U/mL for TNF, IL-1, and IL-6, respectively. Additional treatment with interferon-gamma enhanced production of TNF in both subsets, but CD14+/CD16+ small monocytes still exhibited lower levels. Stimulation of the monocyte subsets by platelet-activating factor gave the same pattern of results. Hybridization with 32P-labeled oligonucleotides specific for the respective cytokine messenger RNAs (mRNAs) showed a 10-fold lower prevalence of transcripts for TNF, IL-1, and IL-6, as well. By contrast, the constitutive expression of Glyceraldehyde-3-phosphate-dehydrogenase mRNA was 1.7-fold higher in the CD14+/CD16+ small monocytes. These data indicate that the novel subset of small monocytes is selectively suppressed in the expression of the cytokines TNF, IL-1, and IL-6, suggesting that these cells may comprise a deactivated type of cell. The expression of class II transcripts in the small monocytes is, however, similar to the regular monocytes, and the cell surface expression of class II protein about threefold increased. Thus, the novel subset of small monocytes appears to be a functionally distinct type of cell.

Antigens, CD↗
PubMed1994

Glutamate-induced overexpression of NMDA receptor messenger RNAs and protein triggered by activation of AMPA/kainate receptors in rat hippocampus following forebrain ischemia.

Severe forebrain ischemia induces a large increase in expression of NMDA receptor subunits in rat brain. One week after ischemia, levels of NMDA-R1 mRNAs in the CA1 pyramidal cells of hippocampus are 7 times higher than those observed in control rats. At 7 days postischemia, an enhanced immunostaining of the NMDA-R1 subunit was observed in all hippocampal structures indicating that changes in mRNA levels are accompanied by changes in receptor protein level. Riluzole, a potent inhibitor of glutamate release and CNQX, a selective AMPA/kainate antagonist, drastically reduced the ischemia-induced expression of mRNAs for the three NMDA receptor subunits while D-AP5, a selective NMDA antagonist, had essentially no effect. Therefore ischemia-induced expression of NMDA receptor subunits is associated with glutamate release and proceeds via an AMPA/kainate pathway. These results together with those of other groups concerning ischemia effects on AMPA and GABAA receptor levels, suggest an important role of the induced expression of NMDA receptor subunits in the deleterious effects of ischemia.

6-Cyano-7-nitroquinoxaline-2,3-dione↗
PubMed1998

Differential upregulation of rat Na-K-Cl cotransporter, rBSC1, mRNA in the thick ascending limb of Henle in different pathological conditions.

BACKGROUND: Na-Cl cotransport across the apical membrane of epithelial cells in the thick ascending limb of the loop of Henle (TAL) plays a major role in salt accumulation for hypertonic medullary interstitium. The electroneutral, rat bumetanide-sensitive sodium transporter, rBSC1, is involved in this process. We studied the level of rBSC1 mRNA in dehydration and cardiac failure, since sodium transport in TAL may be enhanced in both conditions in spite of the difference in extracellular fluid accumulation. METHODS: Male Sprague-Dawley rats were deprived of water for 24 hours and myocardial infarction of about 40% of left ventricular circumference was induced in another group of rats that later developed congestive heart failure (CHF). Digoxigenin-labeled cRNA probe for rBSC1 was constructed using polymerase chain reaction (PCR), and Northern blot analysis was performed using RNAs from renal outer medulla. By inducing a point mutation at the middle of PCR product, we compared the amount of rBSC1 transcripts in the renal cortex using competitive PCR, since TAL represents a small fraction of the total cortical tissue. RESULTS: Northern analysis showed a significant increase in rBSC1 mRNA in the renal outer medulla of both dehydrated and CHF rats. In the renal cortex, however, the increase was noted only in CHF by competitive PCR. In situ hybridization using the riboprobe for northern analysis demonstrated that the transcript signal in dehydrated rats was intensified segmentally in TAL located in the inner stripe of outer medulla. Western analysis and immunohistochemistry using a specific antibody against rBSC1 confirmed the distinct segmental enhancement of apical protein expression in dehydration and diffuse enhanced expression in CHF. CONCLUSIONS: rBSC1 is differentially upregulated in different pathological conditions.

Animals↗