Transcription Factor Decoy Research Articles

Restoring vancomycin activity against resistant Enterococcus faecalis using a transcription factor decoy as a vanA operon-inhibitor.

Vancomycin-resistant enterococci (VRE) represent a public health threat due to the few available treatments. Such alarm has triggered worldwide initiatives to develop effective antimicrobial compounds and novel delivery and therapeutic strategies. vanA operon is responsible for most cases of acquired vancomycin resistance in enterococci. Development of a transcription factor decoy (TFD) system as a vanA gene transcription-inhibitor. Vancomycin MIC was determined in the presence of TFD-lipoplexes. Additionally, the effect of TFD-lipoplexes on the expression level of the vanA gene and the growth pattern of E. faecalis was evaluated. The haemolytic activity of the developed TFD-lipoplexes and their cytotoxicity were examined. TFD-lipoplexes efficiency in treating vancomycin-resistant E. faecalis (VREF) infection was tested in vivo using a systemic mice infection model. A reduction in vancomycin MIC against VRE from 256 mg/L (resistant) to 16 mg/L (intermediate susceptible), in the presence of TFD-lipoplexes, was recorded. The developed TFD-lipoplexes lacked any effect on E. faecalis growth and significantly reduced the transcription level of the vanA gene by about 3-fold. In an initial evaluation of the safety of TFD-lipoplexes, they were found not to be overtly haemolytic to human blood or cytotoxic to human skin fibroblast cells. The co-administration of TFD-lipoplexes and vancomycin efficiently eradicated VREF infection in vivo. The developed TFD-lipoplexes successfully restored vancomycin activity against VREF. They offer a safe effective unconventional therapy against this stubborn organism and present a revolution in gene therapy that can be applied to other resistance-encoding genes in various organisms.

Development of a transcription factor decoy-nanocarrier system as a successful inhibitor of Enterococcus faecalis virulence in vitro and in vivo

Enterococcus faecalis is a troublesome nosocomial pathogen that acquired resistance to most available antimicrobial agents. Antivirulence agents represent an unconventional treatment approach. Here, transcription factor decoy (TFD)-loaded cationic liposomes (TLL) were developed as an inhibitor of the Fsr quorum-sensing system and its associated virulence traits, in E. faecalis. The consensus sequence of the FsrA binding site was found conserved among 651 E. faecalis annotated genomes. The TFD was synthesized as an 82 bp DNA duplex, containing the conserved binding sequence, and loaded onto cationic liposomes. The optimum loading capacity, mean particle size, and zeta potential of the TLL were characterized. The developed TLL lacked any effect on E. faecalis growth and significantly inhibited the in vitro production of the proteolytic enzymes controlled by the Fsr system; gelatinase and serine protease, in a concentration-dependent manner. This inhibition was accompanied by a significant reduction in the transcription levels of FsrA-regulated genes (fsrB, gelE, and sprE). The developed TLL were safe as evidenced by the nonhemolytic effect on human RBCs and the negligible cytotoxicity on human skin fibroblast cells. Moreover, in the larvae infection model, TLL displayed a significant abolish in the mortality rates of Galleria mellonella larvae infected with E. faecalis. In conclusion, the developed TLL offer a new safe strategy for combating E. faecalis infection through the inhibition of quorum-sensing-mediated virulence; providing a platform for the development of similar agents to combat many other pathogens.

Roles of Glutathione and AP-1 in the Enhancement of Vitamin D-Induced Differentiation by Activators of the Nrf2 Signaling Pathway in Acute Myeloid Leukemia Cells.

Active vitamin D derivatives (VDDs)-1α,25-dihydroxyvitamin D3/D2 and their synthetic analogs-are well-known inducers of cell maturation with the potential for differentiation therapy of acute myeloid leukemia (AML). However, their dose-limiting calcemic activity is a significant obstacle to using VDDs as an anticancer treatment. We have shown that different activators of the NF-E2-related factor-2/Antioxidant Response Element (Nrf2/ARE) signaling pathway, such as the phenolic antioxidant carnosic acid (CA) or the multiple sclerosis drug monomethyl fumarate (MMF), synergistically enhance the antileukemic effects of various VDDs applied at low concentrations in vitro and in vivo. This study aimed to investigate whether glutathione, the major cellular antioxidant and the product of the Nrf2/ARE pathway, can mediate the Nrf2-dependent differentiation-enhancing activity of CA and MMF in HL60 human AML cells. We report that glutathione depletion using L-buthionine sulfoximine attenuated the enhancing effects of both Nrf2 activators concomitant with downregulating vitamin D receptor (VDR) target genes and the activator protein-1 (AP-1) family protein c-Jun levels and phosphorylation. On the other hand, adding reduced glutathione ethyl ester to dominant negative Nrf2-expressing cells restored both the suppressed differentiation responses and the downregulated expression of VDR protein, VDR target genes, as well as c-Jun and P-c-Jun levels. Finally, using the transcription factor decoy strategy, we demonstrated that AP-1 is necessary for the enhancement by CA and MMF of 1α,25-dihydroxyvitamin D3-induced VDR and RXRα protein expression, transactivation of the vitamin D response element, and cell differentiation. Collectively, our findings suggest that glutathione mediates, at least in part, the potentiating effect of Nrf2 activators on VDDs-induced differentiation of AML cells, likely through the positive regulation of AP-1.

Elicitation for activation of the actinomycete genome's cryptic secondary metabolite gene clusters.

This review summarizes the recent advances in the elicitation approaches used to activate the actinomycete genome's cryptic secondary metabolite gene clusters and shows the diversity of natural products obtained by various elicitation methods up to June 2022, such as co-cultivation of actinomycetes with actinomycetes, other non-actinomycete bacteria, fungi, cell-derived components, and/or algae. Chemical elicitation and molecular elicitation as transcription factor decoys, engineering regulatory genes, the promoter replacement strategy, global regulatory genes, and reporter-guided mutant selection were also reported. For researchers interested in this field, this review serves as a valuable resource for the latest studies and references.

Exosome-mediated delivery of RBP-J decoy oligodeoxynucleotides ameliorates hepatic fibrosis in mice.

Rationale: Macrophages play multi-dimensional roles in hepatic fibrosis. Studies have implicated Notch signaling mediated by the transcription factor RBP-J in macrophage activation and plasticity. Additionally, we have previously shown that myeloid-specific disruption of RBP-J can ameliorate hepatic fibrosis in mice. Accordingly, we next asked whether blocking Notch signaling in macrophages could serve as a therapeutic strategy to treat hepatic fibrosis. In this study, we used a combination of transcription factor decoy oligodeoxynucleotides (ODNs) and exosomes to test this possibility.Methods: Hairpin-type decoy oligodeoxynucleotides (ODNs) were designed for the transcription factor RBP-J. The effects of RBP-J decoy ODNs on Notch signaling were evaluated by western blot, quantitative RT-PCR, luciferase reporter assays, and electrophoretic mobility shift assays. ODNs were loaded into HEK293T-derived exosomes by electroporation. A hepatic fibrosis mouse model was established by the intraperitoneal injection of carbon tetrachloride or bile duct ligation. Mice with hepatic fibrosis were administered exosomes loaded with RBP-J decoy ODNs via tail vein injection. The in vivo distribution of exosomes was analyzed by fluorescence labeling and imaging. Liver histology was examined using hematoxylin and eosin, Sirius red, and Masson staining, as well as immunohistochemical staining for Col1α1 and αSMA.Results: We found that RBP-J decoy ODNs could be efficiently loaded into exosomes and inhibit the activation of Notch signaling. Furthermore, exosomes administered via the tail vein were found to be primarily taken up by hepatic macrophages in mice with liver fibrosis. Importantly, RBP-J decoy ODNs delivered by exosomes could efficiently inhibit Notch signaling in macrophages and ameliorate hepatic fibrosis in mice.Conclusions: Combined, our data showed that the infusion of exosomes loaded with RBP-J decoy ODNs represents a promising therapeutic strategy for the treatment of hepatic fibrosis.

Programmable gene regulation for metabolic engineering using decoy transcription factor binding sites.

Transcription factor decoy binding sites are short DNA sequences that can titrate a transcription factor away from its natural binding site, therefore regulating gene expression. In this study, we harness synthetic transcription factor decoy systems to regulate gene expression for metabolic pathways in Escherichia coli. We show that transcription factor decoys can effectively regulate expression of native and heterologous genes. Tunability of the decoy can be engineered via changes in copy number or modifications to the DNA decoy site sequence. Using arginine biosynthesis as a showcase, we observed a 16-fold increase in arginine production when we introduced the decoy system to steer metabolic flux towards increased arginine biosynthesis, with negligible growth differences compared to the wild type strain. The decoy-based production strain retains high genetic integrity; in contrast to a gene knock-out approach where mutations were common, we detected no mutations in the production system using the decoy-based strain. We further show that transcription factor decoys are amenable to multiplexed library screening by demonstrating enhanced tolerance to pinene with a combinatorial decoy library. Our study shows that transcription factor decoy binding sites are a powerful and compact tool for metabolic engineering.

Role of Oct4-Sox2 complex decoy oligodeoxynucleotides strategy on reverse epithelial to mesenchymal transition (EMT) induction in HT29-ShE encompassing enriched cancer stem-like cells.

Cancer stem cells are commonly tolerant toward chemotherapy and radiotherapy. Oct4 and Sox2 transcription factors are shown to be overexpressed in various cancers. At the current research, inhibition of Oct4 and Sox2 transcription factors was performed through application of decoy oligodeoxynucleotides (ODNs) strategy via repressing stemness properties in HT29-ShE cells encompassing enriched cancer stem-like cells. Designed Oct4-Sox2 complex decoy ODNs were transfected into HT29-ShE cells with Lipofectamine reagent. At the next step, ODNs efficiency transfection and subcellular localization were determined via flow cytometry and fluorescence microscopy, respectively. Further investigations such as cell proliferation and apoptosis analysis, colonosphere formation, invasion and migration, and real-time PCR assays were also carried out. Obtained results shed light on the fact that the designed complex decoys were effectively transfected into HT29-ShE cells, and they were found to be localized in subcellular compartments. Oct4-Sox2 decoy ODNs led to decreased cell viability, arresting the cell cycle in G0/G1 phases,increasing apoptosis, inhibition of migration/invasion and colonosphere formation ability of HT29-ShE cells in comparison with control and scramble groups. Furthermore, Oct4-Sox2 complex decoy could modulate the MET process via alteration of mRNA expression of downstream genes. It could be concluded that application of Oct4-Sox2 transcription factor decoy strategy in cells with stemness potential could lead to inhibiting the cell growth and triggering differentiation. Therefore, this technique could be applied along with usual remedies(chemotherapy and radiotherapy) as high potential method for treating cancer.

Co-delivery of free vancomycin and transcription factor decoy-nanostructured lipid carriers can enhance inhibition of methicillin resistant Staphylococcus aureus (MRSA).

Bacterial resistance to antibiotics is widely regarded as a major public health concern with last resort MRSA treatments like vancomycin now encountering resistant strains. TFDs (Transcription Factor Decoys) are oligonucleotide copies of the DNA-binding sites for transcription factors. They bind to and sequester the targeted transcription factor, thus inhibiting transcription of many genes. By developing TFDs with sequences aimed at inhibiting transcription factors controlling the expression of highly conserved bacterial cell wall proteins, TFDs present as a potential method for inhibiting microbial growth without encountering typical resistance mechanisms. However, the efficient protection and delivery of the TFDs inside the bacterial cells is a critical step for the success of this technology. Therefore, in our study, specific TFDs against S. aureus were complexed with two different types of nanocarriers: cationic nanostructured lipid carriers (cNLCs) and chitosan-based nanoparticles (CS-NCs). These TFD-carrier nanocomplexes were characterized for size, zeta potential and TFD complexation or loading efficiency in a variety of buffers. In vitro activity of the nanocomplexes was examined alone and in combination with vancomycin, first in methicillin susceptible strains of S. aureus with the lead candidate advancing to tests against MRSA cultures. Results found that both cNLCs and chitosan-based carriers were adept at complexing and protecting TFDs in a range of physiological and microbiological buffers up to 72 hours. From initial testing, chitosan-TFD particles demonstrated no visible improvements in effect when co-administered with vancomycin. However, co-delivery of cNLC-TFD with vancomycin reduced the MIC of vancomycin by over 50% in MSSA and resulted in significant decreases in viability compared with vancomycin alone in MRSA cultures. Furthermore, these TFD-loaded particles demonstrated very low levels of cytotoxicity and haemolysis in vitro. To our knowledge, this is the first attempt at a combined antibiotic/oligonucleotide-TFD approach to combatting MRSA and, as such, highlights a new avenue of MRSA treatment combining traditional small molecules drugs and bacterial gene inhibition.

Rational design of TFDs as novel oligonucleotide antimicrobials to treat Gram-negative infections

We have designed a new Gram-negative antimicrobial- it is an oligonucleotide Transcription Factor Decoy (TFD) that binds to and inhibits bacterial transcription factors controlling genes essential for growth and pathogenicity. The TFD is highly effective in vitro and in vivo, tested in Galleria mellonella survival models and in a mouse model of intra-abdominal infection. TFDs are formulated as nanoparticles, composed of a proprietary lipidic molecule (CM2), that binds to essential prokaryotic phospholipids, such as Cardiolipin, to deliver the oligonucleotide across the bacterial membrane. Studies with models of bacterial membranes showed that translocation was dependent on the presence of Cardiolipin but occurred in the absence of ATP or a pH gradient. Flow Cytometry studies found that the efficiency of TFD delivery to bacterial cells was high, and Live/Dead staining confirmed that cells were not being lysed by the nanoparticles. Further investigation of the mechanism of delivery used a proteomics and metabolomics study of the response of E. coli to nanoparticulate delivery. A number of highly induced transcription factors were identified consistent with stress induced by disruption of respiratory centres bound by Cardiolipin within the membrane. A TFD was designed to inhibit one of the induced transcription factors. This TFD was shown to bind tightly to its cognate transcription factor, have a potent MIC in vitro and be efficacious in murine models of infection. Hence, the work demonstrates that TFDs can be rationally designed to create new antimicrobials to efficiently target bacterial transcription factors.

Enterobacteriaceae-selective modification of the intestinal microbiota using oligonucleotide antimicrobials

The human gut microbiota is complex, dense, and hugely influential to health. Imbalances in the microbiota have been associated with numerous disease states, in many cases due to the overgrowth of Enterobacteriaceae, such as Escherichia coli. We describe an oligonucleotide antimicrobial that selectively reduces levels of Enterobacteriaceae in vitro, in a model of the human colonic microbiota, and in vivo in a murine study, whilst leaving the core microbiota intact. The antimicrobials are Transcription Factor Decoys (TFDs) that bind to and competitively inhibit an identified transcription factor necessary for growth in the intestine. This is highly conserved amongst Enterobacteriaceae and controls anaerobic respiration and response to nitrosative stress caused by the innate immune response of the host. A nanoparticulate formulation delivers the TFDs to the cytoplasm of E. coli, as visualized by confocal microscopy, and rapidly kills the bacteria under microaerobic conditions. When applied to anin vitro model of the human intestinal microbiota the TFD produced a decrease up to log10 6 c.f.u. ml−1 in coliforms within the Enterobacteriaceae family while other families remain intact. When delivered orally to the intestines of mice similar results were seen: Enterobacteriaceae were decreased or cleared from the wild-type intestinal microbiota while the remaining bacteria were unaffected. This demonstrates that TFDs can be used to make precise changes to the microbiota and has utility in testing associations between dysbiosis and disease and developing microbiota targeted therapeutics.

Oligonucleotide transcription factor decoys as tools to control bacterial transcription

Transcription Factor Decoys (TFDs) are short synthetic oligonucleotides that contain the binding site for specific bacterial transcription factors. When translocated into bacterial cytoplasm they can rapidly kill cells when targeted against essential bacterial pathways. Translocation is currently achieved by combination of the TFD with a proprietary lipidic delivery agent, CM2, to form nanoparticles. These interact with highly conserved anionic phospholipids, such as Cardiolipin, to effect delivery to both Gram-positive and Gram-negative bacteria. Simplifying translocation would be an advance that would allow serial screening of large libraries of TFDs to delineate genetic regulatory networks in numerous types of bacteria, including emerging strains. To achieve this we have combined key chemical moieties of the CM2 delivery molecule to the oligonucleotide conjugate by Click chemistry and show that these discrete conjugates are capable of translocation. Confocal laser scanning microscopy was used to monitor the uptake of the TFD-conjugates to E. coli and in parallel their effect on the targeted genetic pathways was confirmed with reporter strains and plating under selective conditions. Hence, it was confirmed that these conjugates can be used as tools to efficiently and specifically modify gene expression by inhibition of selected transcription factors.

Development of biofilm-penetrant antimicrobial delivery system to counter Burkholderia infections

Melioidosis is caused by the Gram-negative, intracellular bacterium Burkholderia pseudomallei. The epidemiology of the disease is unclear- it is limited to tropical regions, where monitoring can be sparse and further complicated by the multifaceted nature of the condition. The most common indication of acute melioidosis is pneumonia (51 %) that can progress to acute fulminant sepsis with multifocal infiltrates with high rates of mortality. Meliodosis has been estimated to cause 89 000 deaths annually with an estimated mortality rate of approximately 50 %. Due to ease of the dissemination of the pathogen, its ability to form biofilms, the high degree of antimicrobial resistance and paucity of effective treatments, controlling the infection is a major challenge. Our approach is to develop nanoparticles capable of effectively delivering antimicrobials to biofilms formed by Burkholderia species. Formulations of a proprietary lipidic delivery agent, CM2, have been tested against two species: B. cepacia UCB717 that has no capsule and B. thailandensis (that is used as a surrogate for B. pseudomallei), including strains with and without capsules both of which readily form biofilms. Confocal laser scanning microscopy was used to monitor the uptake of the nanoparticles and the MIC and MBEC of CM2 alone (and analogues), in combination with a panel of antimicrobials and a novel oligonucleotide antimicrobial, termed a Transcription Factor Decoy (TFD), were measured. The most efficacious combinations will be formulated for delivery by inhalation prior to testing in animal models.

Activation of silent biosynthetic gene clusters using transcription factor decoys.

Here we report a transcription factor decoy strategy for targeted activation of eight large silent polyketide synthase and non-ribosomal peptide synthetase gene clusters, ranging from 50 to 134 kilobases in multiple streptomycetes, and characterization of a novel oxazole family compound produced by a 98-kb biosynthetic gene cluster. Due to its simplicity and ease, this strategy can be readily scaled up for discovery of natural products in streptomycetes.

Solid lipid nanoparticles for the delivery of anti-microbial oligonucleotides

Novel alternatives to antibiotics are urgently needed for the successful treatment of antimicrobial resistant (AMR) infections. Experimental antibacterial oligonucleotide therapeutics, such as transcription factor decoys (TFD), are a promising approach to circumvent AMR. However, the therapeutic potential of TFD is contingent upon the development of carriers that afford efficient DNA protection against nucleases and delivery of DNA to the target infection site. As a carrier for TFD, here we present three prototypes of anionic solid lipid nanoparticles that were coated with either the cationic bolaamphiphile 12-bis-tetrahydroacridinium or with protamine. Both compounds switched particles zeta potential to positive values, showing efficient complexation with TFD and demonstrable protection from deoxyribonuclease. The effective delivery of TFD into bacteria was confirmed by confocal microscopy while SLN-bacteria interactions were studied by flow cytometry. Antibacterial efficacy was confirmed using a model TFD targeting the Fur iron uptake pathway in E. coli under microaerobic conditions. Biocompatibility of TFD-SLN was assessed using in vitro epithelial cell and in vivo Xenopus laevis embryo models. Taken together these results indicate that TFD-SLN complex can offer preferential accumulation of TFD in bacteria and represent a promising class of carriers for this experimental approach to tackling the worldwide AMR crisis.

Structural Modifications of DPPC Bilayers upon Inclusion of an Antibacterial Cationic Bolaamphiphile.

The emergence of antibiotic-resistant bacterial strains has fostered fundamental research to develop alternative antimicrobial strategies. Among the several systems proposed so far, the association complexes (nanoplexes) formed by transcription factor decoys (TFDs), i.e., short oligonucleotides targeting a crucial bacterial transcription factor, and a bolaform cationic amphiphile, 10,10'-(dodecane-1,12-diyl)-bis-(9-amino-1,2,3,4-tetrahydroacridinium) chloride (12-bis-THA), have demonstrated their potential in vitro and in vivo. The application of these nanoplexes is hampered by a scarce colloidal stability, which can be addressed by including the bolaamphiphile in a liposomal carrier, which is then associated to the TFD. The present study reports an investigation on the effects of 12-bis-THA on the structure of synthetic lipid bilayers to assess the morphology of the mixed assemblies, gain insight into the location of the host within the bilayer, and determine the loading capacity of the carrier. Our results demonstrate that 12-bis-THA promptly inserts within 1,2-dipalmitoyl- sn-glycero-3-phosphocholine (DPPC) bilayers, bending its C-12 spacer chain to adopt a conelike shape and shifting the gel-liquid crystalline transition of the chains to lower temperatures. The host liposomal structure is retained for a bolaamphiphile concentration of up to 3.2% mol to DPPC, whereas higher concentrations lead to the destabilization by means of a detergency-like mechanism, with the simultaneous existence of different lamellar-based structures, such as liposomes, bicelles, and rafts, in which DPPC and 12-bis-THA could be present in different molar ratios. Overall, these results shed light on the interaction of the bolaamphiphile with a lipid bilayer and provide valuable insight to better formulate the antimicrobial amphiphile in liposomal carriers to circumvent the colloidal instability of nanoplexes.

Evaluation of the nuclease resistance of DNA four‐way junctions.

Oligodeoxynucleotides (ODN) offer great promise as therapeutics due to their high binding specificity, low toxicity and ease of synthesis. Hence, it is not surprising that ODNs are being developed as antigene and antisense reagents. ODNs can also control gene expression by targeting proteins. One popular approach uses double stranded DNA as transcription factor decoy sequences that are directed against transcription factors such as NF‐kB and specificity protein 1 (Sp1). More recently, researchers have used a similar approach to target proteins in the extracellular matrix. In these studies, nucleic acids are directed against the damage‐associated molecular pattern (DAMP) molecule, High Mobility Group B1 (HMGB1). We hypothesize that ODNs based on DNA four‐way junctions (4WJs) can be used to target HMGB1. To enhance the ex vivo stability of 4WJs, the helical termini of a DNA junction are modified to generate the ODN, i‐J1. The objective of this study is to investigate the nuclease resistance of i‐J1, a control 4WJ (J1) and the intramolecular 4WJ, J4 against three nucleases. The three nucleases are: DNase I, Exonuclease III (Exo III) and bacteriophage T5 Exonuclease (T5 Exo). DNase I cleaves dsDNA and ssDNA in a largely non‐specific manner to generate mono‐ and oligodeoxyribonucleotides. Exo III has expanded functionally; the enzyme acts as a 3′®5′ exonuclease, 3′‐repair diesterase, 3′‐phosphomoesterase and ribonuclease. Here, we focus on the 3′®5′ exonuclease activity of Exo III. T5 Exo hydrolyzes dsDNA and ssDNA in a 5′®3′ direction to release mono‐, di‐, tri‐nucleotides and oligonucleotides. The nuclease protection data shows that i‐J1 and J4 have significantly higher nuclease than J1 against DNase I and Exo III. We suspect the increase in nuclease resistance in linked to changes in the minor groove dimensions of i‐J1 and J4. A binding model is presented to explain the increase in nuclease resistance of i‐J1 and J4.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Myc Decoy Oligodeoxynucleotide Inhibits Growth and Modulates Differentiation of Mouse Embryonic Stem Cells as a Model of Cancer Stem Cells.

Myc (c-Myc) alone activates the embryonic stem cell-like transcriptional module in both normal and transformed cells. Its dysregulation might lead to increased cancer stem cells (CSCs) population in some tumor cells. In order to investigate the potential of Myc decoy oligodeoxynucleotides for differentiation therapy, mouse embryonic stem cells (mESCs) were used in this study as a model of CSCs. To our best of knowledge this is the first report outlining the application of Myc decoy in transcription factor decoy "TFD" strategy for inducing differentiation in mESCs. A 20-mer double-stranded Myc transcription factor decoy and scrambled oligodeoxynucleotides (ODNs) were designed, analyzed by electrophoretic mobility shift (EMSA) assay and transfected into the mESCs under 2 inhibitors (2i) condition. Further investigations were carried out using fluorescence and confocal microscopy, cell proliferation and apoptosis analysis, alkaline phosphatase and embryoid body formation assay, real-time PCR and western blotting. EMSA data showed that Myc decoy ODNs bound specifically to c-Myc protein. They were found to be localized in both cytoplasm and nucleus of mESCs. Our results revealed the potential capability of Myc decoy ODNs to decrease cell viability by (16.1±2%), to increase the number of cells arrested in G0/G1 phases and apoptosis by (14.2±3.1%) and (12.1±3.2%), respectively regarding the controls. Myc decoy could also modulate differentiation in mESCs despite the presence of 2i/LIF in our medium the presence of 2i/LIF in our medium. The optimized Myc decoy ODNs approach might be considered as a promising alternative strategy for differentiation therapy investigations.

Simultaneous targeted inhibition of Sox2‐Oct4 transcription factors using decoy oligodeoxynucleotides to repress stemness properties in mouse embryonic stem cells

Transcriptional master regulators like Sox2 and Oct4, which are expressed in various human tumors, have been shown to cause tumor growth promotion as well as epithelial dysplasia by means of interfering with progenitor cell differentiation. In order to investigate the potential of Sox2-Oct4 transcription factor decoy (TFD) strategy for differentiation therapy, mouse embryonic stem cells (mESCs) were used in this study as a model of cancer stem cells (CSCs). Sox2-Oct4 complex decoy ODNs (cd-ODNs) were designed according to their elements in the promoter region of Sox2 gene. DNA-protein interactions between decoy ODNs and their corresponding proteins were examined by electrophoretic mobility shift assay (EMSA). Then, decoy and scrambled ODNs were transfected into mESCs with lipofectamine under 2 inhibitors (2i) conditions. Fluorescence and confocal microscopy, cell viability, cell cycle and apoptosis analysis, alkaline phosphatase, embryoid body formation assay, and real-time PCR were used to conduct further investigations. EMSA data showed that Sox2-Oct4 decoy ODNs bound specifically to their recombinant proteins. The results revealed that the synthesized complex decoy can concomitantly target Sox2 and Oct4, which subsequently represses the stemness properties of mESCs compared to controls through decreasing cell viability, arresting cell cycle in G0 /G1 phases, inducing apoptosis, and modulating differentiation in mESCs despite the presence of 2i/LIF in cell culture. While cd-ODN strategy seems to offer great promise for cancer therapy, further studies are still required to put this powerful investigative tool in practice for a wide range of human cancers.

Cationic liposomal vectors incorporating a bolaamphiphile for oligonucleotide antimicrobials

Antibacterial resistance has become a serious crisis for world health over the last few decades, so that new therapeutic approaches are strongly needed to face the threat of resistant infections. Transcription factor decoys (TFD) are a promising new class of antimicrobial oligonucleotides with proven in vivo activity when combined with a bolaamphiphilic cationic molecule, 12-bis-THA. These two molecular species form stable nanoplexes which, however, present very scarce colloidal stability in physiological media, which poses the challenge of drug formulation and delivery. In this work, we reformulated the 12-bis-THA/TFD nanoplexes in a liposomal carrier, which retains the ability to protect the oligonucleotide therapeutic from degradation and deliver it across the bacterial cell wall. We performed a physical-chemical study to investigate how the incorporation of 12-bis-THA and TFD affects the structure of POPC- and POPC/DOPE liposomes. Analysis was performed using dynamic light scattering (DLS), ζ-potential measurements, small-angle x-ray scattering (SAXS), and steady-state fluorescence spectroscopy to better understand the structure of the liposomal formulations containing the 12-bis-THA/TFD complexes. Oligonucleotide delivery to model Escherichia coli bacteria was assessed by means of confocal scanning laser microscopy (CLSM), evidencing the requirement of a fusogenic helper lipid for transfection. Preliminary biological assessments suggested the necessity of further development by modulation of 12-bis-THA concentration in order to optimize its therapeutic index, i.e. the ratio of antibacterial activity to the observed cytotoxicity. In summary, POPC/DOPE/12-bis-THA liposomes appear as promising formulations for TFD delivery.

Antimicrobial Nanoplexes meet Model Bacterial Membranes: the key role of Cardiolipin

Antimicrobial resistance to traditional antibiotics is a crucial challenge of medical research. Oligonucleotide therapeutics, such as antisense or Transcription Factor Decoys (TFDs), have the potential to circumvent current resistance mechanisms by acting on novel targets. However, their full translation into clinical application requires efficient delivery strategies and fundamental comprehension of their interaction with target bacterial cells. To address these points, we employed a novel cationic bolaamphiphile that binds TFDs with high affinity to form self-assembled complexes (nanoplexes). Confocal microscopy revealed that nanoplexes efficiently transfect bacterial cells, consistently with biological efficacy on animal models. To understand the factors affecting the delivery process, liposomes with varying compositions, taken as model synthetic bilayers, were challenged with nanoplexes and investigated with Scattering and Fluorescence techniques. Thanks to the combination of results on bacteria and synthetic membrane models we demonstrate for the first time that the prokaryotic-enriched anionic lipid Cardiolipin (CL) plays a key-role in the TFDs delivery to bacteria. Moreover, we can hypothesize an overall TFD delivery mechanism, where bacterial membrane reorganization with permeability increase and release of the TFD from the nanoplexes are the main factors. These results will be of great benefit to boost the development of oligonucleotides-based antimicrobials of superior efficacy.