Transcription Factor Decoy Oligonucleotides Research Articles

Properties of Double-Stranded Oligonucleotides Modified with Lipophilic Substituents

We have synthesized a series of short, self-complementary oligonucleotide sequences modified at their 5'- and/or 3'- termini with a lipophilic dodecane (C12); these systems serve as models to assess the biophysical properties of double-stranded DNA (dsDNA) equipped with potentially stabilizing lipophilic substituents. Addition of C12 to the 5'-termini of self-complementary 10 nucleotide sequences increased their duplex melting temperatures (T(m)) by approximately 4-8 degrees C over their corresponding unmodified sequences. C12 functionalities added to both the 3'- and 5'-termini increased T(m) values by approximately 10-12 degrees C. The observed increases in T(m) correlated with greater duplex stabilities as determined by the free energy values (DeltaG) derived from T(m) plots. There is a greater degree of stabilization when C12 is positioned with a C.G base pair at the termini, and the stabilizing effect of lipophilic groups far exceeds the effect seen in adding an additional base pair to both ends of DNA. Stable, short dsDNA sequences are of potential interest in the development of transcription factor decoy oligonucleotides as possible therapeutic agents and/or biological tools. These results suggest that the stability of short dsDNA sequences are improved by lipophilic substituents and can be used as the basis for the design of dsDNAs with improved biological stabilities and function under physiological conditions.

Analysis of Promoter Regions Regulating Basal and Interleukin-4-Inducible Expression of the Human CB1 Receptor Gene in T Lymphocytes

The majority of effects of cannabinoids are mediated by the two receptors CB1 and CB2. In addition to neuronal cells, CB1 receptors are expressed in T lymphocytes, in which they are involved in cannabinoid-induced T helper cell biasing. Although basally expressed only weakly in T cells, CB1 receptors are up-regulated in these cells by stimuli such as cannabinoids themselves. This effect is mediated by interleukin-4. In this study, we investigated basal and interleukin-4-inducible expression of the CB1 gene in T lymphocytes. In a promoter analysis, two regions [nucleotides (nts) -3086 to -2490 and nts -1950 to -1653] were identified, which suppress basal transcription of the gene in Jurkat T cells, whereas the region between nts -648 and -559 enhanced basal CB1 transcription. Interleukin-4 markedly induced transcription of CB1 in Jurkat cells and primary human T cells. Experiments using transcription factor decoy oligonucleotides demonstrated that STAT6 mediates regulation of the gene by interleukin-4. Using reporter gene assays and the transcription factor decoy oligonucleotide approach, a binding site for STAT6 was identified at nt -2769 on the human CB1 gene promoter. Interleukin-4 also caused up-regulation of functional CB1 receptor proteins. In interleukin-4 pretreated, but not in naive Jurkat cells, the CB1 agonist R(+)-methanandamide caused a significant inhibition of forskolin-induced cAMP formation. This effect was blocked by the CB1-selective antagonists N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (AM251) and 1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-4-mo rpholinyl-1H-pyrazole-3-carboxamide (AM281). Taken together, these data show that CB1 receptors are expressed and up-regulated by interleukin-4 in T lymphocytes, which enables CB1-mediated communication to cells of other systems, such as neuronal cells.

Induction of Apoptosis of Osteoclasts by Targeting Transcription Factors with Decoy Molecules

We review the effects of two transcription factor decoy oligonucleotides on apoptosis of human osteoclasts (OCs). The first decoy molecule was designed to inhibit nuclear factor kappa-B (NF-kappaB) binding to target sequence, the second to increase estrogen receptor (ER) alpha expression. We found that both decoy molecules are potent inducers of apoptosis of human OCs, associated with increase of caspase 3 activity and decrease of interleukin 6 expression. In addition, we provide evidence indicating that these oligonucleotides are active in vivo in inducing OCs apoptosis. Because OCs are essential for skeletal development and remodeling throughout the life of animal and man, the approach described is of potential clinical importance.

STAT6 transcription factor binding sites with mismatches within the canonical 5′‐TTC…GAA‐3′ motif involved in regulation of δ‐ and µ‐opioid receptors

Opioid receptors are expressed in neuronal and immune cells and regulated in response to immunological processes. Herein, we demonstrate up-regulation of the delta-opioid receptor gene by interleukin-4 in immune cells (primary T and polymorphonuclear leukocytes, Jurkat E6 T cells), and in NG 108-15 neuronal cells. We identified an interleukin-4-responsive element at nt -671 on the murine gene promoter, to which the transcription factor STAT6 binds, as shown by reporter gene analysis and STAT6/DNA interaction studies in living cells with transcription factor decoy oligonucleotides. STAT6 normally binds to palindromic DNA motifs with a 5'-TTC...GAA-3' core. Notably, the delta-opioid receptor STAT6 site (5'-TTC...GGA-3') is an imperfect palindrome with a mismatch within this core sequence. A systematic analysis of possible mismatch 5'-TTC...GAA-3' motifs revealed that STAT6 also binds to the sequence 5'-TTA...GAA-3'. This motif occurs as a polymorphism in the human mu-opioid receptor gene (Kraus et al. 2001 J. Biol. Chem 276, 43901-43908). We show that this mutated element has a significantly reduced STAT6 binding activity which correlates to its reduced interleukin (IL)-4 inducibility. In contrast, the non-canonical STAT6 site of the delta-opioid receptor binds STAT6 with similar high activity as a perfectly palindromic STAT6 site and is strongly inducible by IL-4.

Transcriptional regulation of the human mu-opioid receptor gene by interleukin-6.

Inflammatory pain is counteracted by a number of physiological processes. For example, opioid receptors, which are present on peripheral terminals of sensory neurons, are activated by endogenous opioids, which are released from immune cells migrating to the inflamed tissue. Earlier data demonstrated that interleukin-6 contributes to such inflammation-induced analgesia. In this report, we demonstrated that interleukin-6 strongly induces mu-opioid receptor mRNA in the human neuroblastoma cell line SH SY5Y, whereas delta-opioid receptor mRNA levels are not influenced. The mRNA increase in these cells is followed by an increase in mu-opioid receptor-specific binding. Using transcription factor decoy oligonucleotides, direct evidence was provided that the up-regulation of mu-opioid receptor mRNA in intact cells is dependent on the transcription factors signal transducers and activators of transcription 1 (STAT1) and STAT3, whereas other transcription factors, such as activator protein-1, nuclear factor (NF)-kappaB, or NF-interleukin-6 are not involved. STAT1 and STAT3 bound to a site located at nucleotide -1583 on the promoter of the human mu-opioid receptor gene, as shown by transient transfection experiments, electrophoretic mobility shift assays, and transcription factor decoy oligonucleotides. A mutation analysis of the 5'-TTCATGGAA-3' STAT1/3 element (palindrome underlined) was performed to determine nucleotide residues that are necessary for the binding of STAT1 and STAT3. It suggested that only the palindromic half sides and the two adjacent central nucleotides are required. Neither mutation of the nucleotides outside the palindrome nor mutation of the central nucleotide affected STAT1/3 binding.

Transcription factor decoy oligonucleotides modified with locked nucleic acids: an in vitro study to reconcile biostability with binding affinity

Double-stranded oligonucleotides (ODNs) containing the consensus binding sequence of a transcription factor provide a rationally designed tool to manipulate gene expression at the transcriptional level by the decoy approach. However, modifications introduced into oligonucleotides to increase stability quite often do not guarantee that transcription factor affinity and/or specificity of recognition are retained. We have previously evaluated the use of locked nucleic acids (LNA) in the design of decoy molecules for the transcription factor kappaB. Oligo nucleotides containing LNA substitutions displayed high resistance to exo- and endonucleolytic degradation, with LNA-DNA mix-mers being more stable than LNA-DNA-LNA gap-mers. However, insertion of internal LNA bases resulted in a loss of affinity for the transcription factor. This latter effect apparently depended on positioning of the internal LNA substitutions. Indeed, here we demonstrate that intra- and inter-strand positioning of internal LNAs has to be carefully considered to maintain affinity and achieve high stability, respectively. Unfortunately, our data also indicate that LNA positioning is not the only parameter affecting transcription factor binding, the interference in part being dependent on the intrinsic conformational properties of this nucleotide analog. To circumvent this problem, the successful use of an alpha-L-ribo- configured LNA is demonstrated, indicating LNA-DNA-alpha-L-LNA molecules as promising new decoy agents.

Molecular therapy to inhibit NFkappaB activation by transcription factor decoy oligonucleotides.

Molecular therapy is emerging as a potential strategy for the treatment of various diseases for which few known effective therapies exist. One strategy for combating disease processes has been to target the transcriptional process. Two approaches have been used to accomplish this: the use of antisense complimentary to the mRNA of interest and the use of ribozymes, a unique class of RNA molecules that not only store information but also process catalytic activity. Ribozymes are known to catalytically cleave specific target RNA, leading to its degradation, whereas antisense molecules inhibit translation by binding to mRNA sequences on a stoichiometric basis. More recently, small interfering RNA has been shown to inhibit target gene expression. The application of oligonuclotide technology, such as antisense, to regulate the transcription of disease-related genes in vivo has important therapeutic potential. Transfection of cis-element double-stranded oligodeoxynucleotides has been reported as a powerful tool in a new class of anti-gene strategies for molecular therapy.

Gene therapy with transcription factor decoy oligonucleotides as a potential treatment for cardiovascular diseases.

Cardiovascular diseases including renal diseases are the leading causes of mortality and morbidity in developed countries. Most conventional therapy is inefficient and tends to treat the symptoms rather than the underlying causes of the disorder. Gene therapy based on oligonucleotides (ODN) offers a novel approach for the prevention and treatment of cardiovascular diseases. Gene transfer into somatic cells to interfere with the pathogenesis contributing to cardiovascular disease may provide such a novel approach for better prevention and treatment of cardiovascular disorders. The major development of gene transfer has importantly contributed to intense investigation of the potential of gene therapy in cardiovascular including renal medicine. The amazing advances in molecular biology have provided a dramatic improvement of the technology that is necessary to transfer target genes into somatic cells. Gene transfer methods have been surprisingly improved. In fact, some of them (retroviral vectors, adenoviral vectors or liposome based vectors, etc) have been used in the clinical trials already. Recent progress in molecular biology has provided new techniques to inhibit target gene expression. Especially, application of DNA technology such as an antisense strategy to regulate the transcription of disease-related genes in vivo has important therapeutic potential. Recently, transfection of cis-clement double-stranded ODN (= decoy) has been reported as a new powerful tool in a new class of anti-gene strategies for gene therapy. Transfection of double-stranded ODN corresponding to the cis sequence will result in attenuation of the authentic cis-trans interaction, leading to removal of trans-factors from the endogenous cis-elements with subsequent modulation of gene expression.

Local administration of transcription factor decoy oligonucleotides to nuclear factor-kappaB prevents carrageenin-induced inflammation in rat hind paw.

The transcription factor nuclear factor-kappaB (NF-kappaB) plays a key role in the expression of several genes involved in the inflammatory process. In the present study we investigated in an acute model of inflammation, the carrageenin-induced hind paw edema, the anti-inflammatory effect of double stranded oligodeoxynucleotides (ODN) with consensus nuclear factor-kappaB (NF-kappaB) sequence as transcription factor decoys (TFD) to inhibit NF-kappaB binding to native DNA sites. Local administration of wild-type, but not mutant-ODN decoy, dose-dependently inhibited edema formation induced by carrageenin in rat paw. Molecular analysis performed on soft tissue obtained from inflamed paw demonstrated: (1) an inhibition of NF-kappaB DNA binding activity; (2) a decreased nuclear level of p50 and p65 NF-kappaB subunits; (3) an inhibition of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) protein expression, two inflammatory enzymes transcriptionally controlled by NF-kappaB. Furthermore, SN-50, a cell-permeable peptide capable of inhibiting the nuclear translocation of NF-kappaB complexes, exhibited a similar profile of activity of ODN decoy. Our results indicate for the first time that ODN decoy, acting as an in vivo competitor for the transcription factor's ability to bind to cognate recognition sequence, may represent a novel strategy to modulate acute inflammation.

Transcription factor decoy for NFkappaB inhibits TNF-alpha-induced cytokine and adhesion molecule expression in vivo.

The expression of several cytokines and adhesion molecules is regulated by the transcription factor NFkappaB, which is activated by tumor necrosis factor alpha (TNF-alpha). In this study, we employed a mouse model of nephritis induced by TNF-alpha to examine whether inhibition of NFkappaB activity using transcription factor decoy oligonucleotides (ODN) blocks cytokine and adhesion molecule expression and attenuates the renal inflammatory response. First, we developed a method for delivering FITC-ODN in vivo into mouse kidney glomeruli by employing HVJ-liposome. Then, in order to study the feasibility of decoy strategy in vivo, the reporter gene chloramphenicol acetyltransferase (CAT) driven by three tandemly repeated NFkappaB binding sequences was transfected into the kidney. Intrapenetorial injection of TNF-alpha stimulated CAT expression in vivo, and the increase in CAT expression was completely abolished by NFkappaB decoy ODN, but not scrambled ODN. Therefore, we examined the effect of NFkappaB decoy ODN transfection on TNF-alpha-induced endogenous interleukin (IL)-1alpha, IL-1beta, IL-6, ICAM-1 and VCAM-1 gene expression as assessed by RT-PCR and Northern blotting. Our present data showed that NFkappaB decoy, but not scrambled, ODN abolished TNF-alpha induced gene expression in vivo, as well as glomerular inflammation as assessed by CD45 staining. Taken together, our results suggest the potential utility of NFkappaB decoy strategy for molecular therapy to glomerular inflammatory diseases.

Pressure-mediated oligonucleotide transfection of rat and human cardiovascular tissues.

The application of gene therapy to human disease is currently restricted by the relatively low efficiency and potential hazards of methods of oligonucleotide or gene delivery. Antisense or transcription factor decoy oligonucleotides have been shown to be effective at altering gene expression in cell culture expreriments, but their in vivo application is limited by the efficiency of cellular delivery, the intracellular stability of the compounds, and their duration of activity. We report herein the development of a highly efficient method for naked oligodeoxynucleotide (ODN) transfection into cardiovascular tissues by using controlled, nondistending pressure without the use of viral vectors, lipid formulations, or exposure to other adjunctive, potentially hazardous substances. In this study, we have documented the ability of ex vivo, pressure-mediated transfection to achieve nuclear localization of fluorescent (FITC)-labeled ODN in approximately 90% and 50% of cells in intact human saphenous vein and rat myocardium, respectively. We have further documented that pressure-mediated delivery of antisense ODN can functionally inhibit target gene expression in both of these tissues in a sequence-specific manner at the mRNA and protein levels. This oligonucleotide transfection system may represent a safe means of achieving the intraoperative genetic engineering of failure-resistant human bypass grafts and may provide an avenue for the genetic manipultation of cardiac allograft rejection, allograft vasculopathy, or other transplant diseases.