E2F Decoy Oligodeoxynucleotides Research Articles

Nucleic Acid Drugs for Preventing Restenosis after Coronary Revascularization

Recent progress in molecular and cellular biology has resulted in the development of numerous effective drugs. However, there are still a number of diseases for which no known effective therapy exists, such as ischemic heart disease, vascular bypass graft failure and heart failure. Despite its limitations, oligodeoxynucleotide (ODN)-based therapy is emerging as a potential strategy for the treatment of patients with cardiovascular disease that is resistant to current therapeutic approaches. Indeed, several nucleic acid drugs and delivery methods for heart disease have been developed and their efficacy has been investigated in animal models. Among them, some agents have undergone clinical trials, such as cmyc antisense ODN, E2F and NFκB decoy ODN. However, none of the large randomized placebo-controlled trials has shown conclusive evidence of clinical benefit. Recent experimental studies suggested that siRNA- and miRNA-based strategies have potential as a potent therapeutic approach for the treatment of restenosis. In addition, simultaneous regulation of multiple intracellular signaling pathways is expected to enhance the therapeutic effects. This review focuses on the potential of recent ODN-based gene therapy for the treatment of heart disease, especially restenosis after revascularization.

Nucleic Acid Drugs for Prevention of Cardiac Rejection

Heart transplantation has been broadly performed in humans. However, occurrence of acute and chronic rejection has not yet been resolved. Several inflammatory factors, such as cytokines and adhesion molecules, enhance the rejection. The graft arterial disease (GAD), which is a type of chronic rejection, is characterized by intimal thickening comprised of proliferative smooth muscle cells. Specific treatments that target the attenuation of acute rejection and GAD formation have not been well studied in cardiac transplantation. Recent progress in the nucleic acid drugs, such as antisense oligodeoxynucleotides (ODNs) to regulate the transcription of disease-related genes, has important roles in therapeutic applications. Transfection of cis-element double-stranded DNA, named as “decoy,” has been also reported to be a useful nucleic acid drug. This decoy strategy has been not only a useful method for the experimental studies of gene regulation but also a novel clinical strategy. In this paper, we reviewed the experimental results of NF-κB, E2F, AP-1, and STAT-1 decoy and other ODNs using the experimental heart transplant models.

Molecular engineering of vein bypass grafts

Surgical bypass of arterial occlusions using autogenous vein provides an effective treatment for many patients with advanced coronary or peripheral atherosclerosis. However, the long-term benefit of bypass surgery is limited by the development of de novo occlusive lesions within the vein graft, which occurs in a significant percentage of patients over time. The pathophysiology of vein graft failure involves a complex interplay between an acute vascular injury response and the hemodynamic adaptation of the vein to arterial forces. Cell proliferation, inflammation, and matrix metabolism are critical components of postimplantation remodeling. Conventional pharmacotherapy has had limited impact on graft failure. Vein grafts present a unique and attractive opportunity for molecular engineering, which is defined for purposes of this review as the local application of genomic (eg, gene transfer or gene inhibition) or proteomic interventions designed to alter the healing response. The critical enabling technologies for these strategies are described, with a perspective on preclinical and clinical development for this indication. The recently completed clinical trials of edifoligide (E2F decoy oligodeoxynucleotide) provide important lessons for future studies. A better understanding of the remodeling response of vein grafts in humans is required to design effective molecular therapies and to define the appropriate target populations and surrogate markers for future clinical trials.

E2F decoy oligodeoxynucleotide ameliorates cartilage invasion by infiltrating synovium derived from rheumatoid arthritis

This study examined the ability of E2F decoy oligodeoxynucleotides (ODN) to inhibit proliferation of synovial fibroblasts derived from patients with rheumatoid arthritis (RA). The effect of E2F decoy ODN on cartilage invasion by RA synovium in a murine model of human RA was also investigated. E2F decoy ODN were introduced into synovial tissue and synovial fibroblasts derived from patients with RA using hemagglutinating virus of Japan (HVJ)-liposomes. The effect of E2F decoy ODN on synovial fibroblast proliferation was evaluated by MTT assay and by RT-PCR for the cell cycle regulatory genes proliferating-cell nuclear antigen (PCNA) and cyclin-dependent kinase 2 (cdk2). Changes in production of inflammatory mediators by RA synovial tissue following transfection with E2F decoy ODN were assessed by ELISA. Human cartilage and RA synovial tissue transfected with E2F decoy ODN were co-transplanted in severe combined immunodeficient (SCID) mice. After 4 weeks, the mice were sacrificed and the implants histologically examined for inhibition of cartilage damage by E2F decoy ODN. E2F decoy ODN resulted in significant inhibition of synovial fibroblast proliferation, corresponding with reduced expression of PCNA and cdk2 mRNA in synovial fibroblasts. The production of interleukin-1beta (IL-1beta), IL-6 and matrix metalloproteinase (MMP)-1 by synovial tissue was also significantly inhibited by the introduction of E2F decoy ODN. Further, in an in vivo model, cartilage that was co-implanted with RA synovial tissue transfected with E2F decoy ODN exhibited no invasive and progressive cartilage degradation. These data demonstrate that transfection of E2F decoy ODN prevents cartilage destruction by inhibition of synovial cell proliferation, and suggest that transfection of E2F decoy ODN may provide a useful therapeutic approach for the treatment of joint destruction in arthritis.

E2F decoy oligodeoxynucleotides on neointimal hyperplasia in canine vein graft

Double-stranded DNA with high affinity to E2F as a decoy cis-element blocks the activation of genes mediating the cell cycle, resulting in effective suppression of the smooth muscle cell proliferation that causes intimal hyperplasia. To evaluate the effect of the E2F decoy to suppress neointimal hyperplasia autogenous venous bypass grafts were performed in dogs after incubation with heparin (group 1), with E2F decoy oligodeoxynucleotides (ODN) (groups 2 and 3), or with a random ODN (group 4) using a Japan-liposomeal method based on a hemagglutinating virus. The intimal and medial cross-sectional surface area of the anastomotic site was measured at 4 months after bypass surgery in groups 1, 3, and 4 by computerized planimetry and at 4 weeks in group 2 to compare the intimal/medial (I/M) area ratios. Autogenous vein grafts treated with E2F decoy showed a significant reduction in I/M area ratio (0.26 ± 0.11) compared with the heparin-treated control group (1.49 ± 0.29) or the mismatched ODN-treated group (1.61 ± 0.28; P = .000). There was no difference in the I/M area ratio according to experimental periods (groups 2 vs 3: 0.26 ± 0.11 vs 0.37 ± 0.32; P = .446) or the anastomotic sites (proximal vs distal; P = .934). In conclusion, an E2F decoy can suppress neointimal hyperplasia in autogenous vein grafts, which may prolong patency by reducing graft stenosis.

Local delivery of E2F decoy oligodeoxynucleotides using ultrasound with microbubble agent (Optison) inhibits intimal hyperplasia after balloon injury in rat carotid artery model

Since restenosis after angioplasty still remains a major clinical problems, inhibition of neointimal formation is an important subject. In this study, we focused on the transcription factor, E2F, that plays a pivotal role in the transactivation of cell-cycle regulatory genes, and also we developed a newly delivery system of decoy oligodeoxynucleotides (ODN). We transfected E2F decoy ODN mixed with an echo-contrast microbubble agent (Optison) into rat carotid artery balloon-injured model by using therapeutic ultrasound (US) to inhibit neointimal formation. Two weeks after transfection, the intimal to medial area ratio in E2F decoy + Optison + US group was significantly decreased ( P<0.01). Inhibition of cell growth was also confirmed by PCNA staining. No apparent toxicity such as inflammation could be detected in blood vessels transfected with E2F decoy ODN with Optison and ultrasound. Overall, the present studies demonstrated a novel non-viral ODN transfer method into blood vessels. A novel therapeutic strategy using E2F decoy ODN with Optison using ultrasound may be useful to inhibit restenosis in clinical practice without a viral vector.

E2F decoy oligodeoxynucleotides effectively inhibit growth of human tumor cells

Abnormal cell proliferation, largely dependent upon deregulation of cell-cycle regulatory proteins, is an important feature of several forms of human cancer. The transcription factor, E2F, plays a critical role in the trans-activation of several genes involved in cell-cycle regulation, thereby regulating cell growth. We have demonstrated that E2F decoy oligodeoxynucleotides (ODNs) with a circular dumbbell structure (CD-E2F decoy) corresponding to E2F binding sites effectively inhibit cell proliferation of primary cultured cells. Here we found that the E2F decoy ODNs inhibited serum-induced promoter activity of E2F-dependent genes in a sequence-specific manner in a RB-positive human osteosarcoma, U2OS, as well as in a RB-negative human cervical carcinoma, C33A. This E2F decoy ODN strongly inhibited gene expression of endogenous E2F1 and PCNA and proliferation of these cancer cells. Our results suggest that this decoy ODN strategy could represent a powerful investigative and potentially therapeutic strategy in the prevention and treatment of cancer.

Inhibitory effects of novel E2F decoy oligodeoxynucleotides on mesangial cell proliferation by coexpression of E2F/DP

Proliferation of glomerular mesangial cells (MCs) is an important feature of several forms of glomerulonephritis. The transcription factor E2F coordinately regulates expression of genes required for cell proliferation, thereby mediating cell growth control. Here we investigated the role of E2F1 and E2F4 expression, with or without co-expression of DP1 or DP2, on cell proliferation in transiently transfected primary rat MCs. In transfected cells, cell proliferation induced by over-expression of E2F was significantly enhanced by co-expression of DP proteins. Previous studies showed that the transfection of decoy oligodeoxynucleotides (ODNs) corresponding to E2F binding sites inhibits cell proliferation. Here we have developed a Ring-E2F (R-E2F) decoy ODN with a circular dumbbell structure and compared its effects with those of a phosphorothioated E2F decoy (PS-E2F decoy) ODN. The R-E2F decoy ODN showed enhanced stability in the presence of nucleases and sera, and inhibited E2F/DP-dependent promoter activity of cell cycle genes more effectively than the PS-E2F decoy ODN. Transfection of R-E2F decoy ODN resulted in strong inhibition of cell cycle gene expression and MC proliferation. Our data suggest that E2F/DP complexes play a critical role in the MC proliferation and that the R-E2F decoy ODN may be a powerful tool for inhibiting cell proliferation.

Novel E2F decoy oligodeoxynucleotides inhibit in vitro vascular smooth muscle cell proliferation and in vivo neointimal hyperplasia.

The transcription factor, E2F, plays a critical role in the trans-activation of several genes involved in cell cycle regulation. Previous studies showed that the transfection of cis element double-stranded decoy oligodeoxynucleotides (ODNs) corresponding to E2F binding sites inhibited the proliferation of vascular smooth muscle cells (VSMCs) and neointimal hyperplasia in injured vessels. We have developed a novel E2F decoy ODN with a circular dumbbell structure (CD-E2F) and compared its effects with those of the conventional phosphorothioated E2F decoy (PS-E2F) ODN. CD-E2F ODN was more stable than PS-E2F ODN, largely preserving its structural integrity after incubation in the presence of nucleases and sera. Moreover, CD-E2F ODN inhibited high glucose- and serum-induced transcriptional expression of cell cycle regulatory genes more strongly than PS-E2F ODN. Transfection of CD-E2F ODN resulted in more effective inhibition of VSMC proliferation in vitro and neointimal formation in vivo, compared with PS-E2F ODN. An approximately 40-50% lower dose of CD-E2F ODN than PS-E2F ODN was sufficient to attain similar effects. In conclusion, our results indicate that CD-E2F ODN may be a valuable tool in gene therapy protocols for inhibiting VSMC proliferation and studying transcriptional regulation.

Suppression of Proliferative Cholangitis by E2F Decoy Oligodeoxynucleotide

Background. Proliferative cholangitis (PC) associated with hepatolithiasis results in stricture of the main bile ducts and is a major cause of residual and/or recurrent stones after repeated treatment for hepatolithiasis. The transcription factor E2F controls the expression of several genes involved in cell proliferation. The aim of this study was to inhibit PC using cytostatic gene therapy by transferring fusigenic anionic liposome–hemagglutinating virus of Japan (HVJ–anionic liposome) complexes containing a synthetic double-stranded oligodeoxynucleotide with high affinity for E2F (E2F decoy).Materials and methods. PC was induced by introducing a fine nylon thread into the bile duct in a rat model. HVJ–anionic liposomes containing the E2F decoy were administered directly into the biliary tract. HVJ–anionic liposomes containing a missense oligodeoxynucleotide (scramble decoy) were also given as a control. The count of peribiliary glands in the bile duct, 5′-bromodeoxyuridine (BrdU) labeling index, and immunohistochemical staining for proliferating cell nuclear antigen (PCNA) in the bile duct were compared among untransfected, scramble decoy-transfected, and E2F decoy-transfected rats.Results. E2F decoy-transfected bile ducts showed inhibition of the papillary proliferation of the biliary epithelium and peribiliary gland hyperplasia. BrdU incorporation and PCNA expression in the bile ducts were inhibited in E2F decoy-transfected rats.Conclusion. Our cytostatic gene therapy approach using direct E2F decoy transfer into the biliary tract suppressed PC in a rat model and may offer an effective therapeutic option for reducing recurrence following treatment for hepatolithiasis.

Ex-vivo gene therapy of human vascular bypass grafts with E2F decoy: the PREVENT single-centre, randomised, controlled trial

Cell-cycle blockade by ex-vivo gene therapy of experimental vein grafts inhibits the neointimal hyperplasia and subsequent accelerated atherosclerosis that lead to human bypass-graft failure. In a prospective, randomised, controlled trial, we investigated the safety and biological efficacy of intraoperative gene therapy in patients receiving bypass vein grafts. We studied gene therapy that uses decoy oligodeoxynucleotide, which binds and inactivates the pivotal cell-cycle transcription factor E2F. 41 patients were randomly assigned untreated (16), E2F-decoy-treated (17), or scrambled-oligodeoxynucleotide-treated (eight) human infrainguinal vein grafts. Oligonucleotide was delivered to grafts intraoperatively by ex-vivo pressure-mediated transfection. The primary endpoints were safety and inhibition of target cell-cycle regulatory genes and of DNA synthesis in the grafts. Analysis was by intention to treat. Mean transfection efficiency was 89.0% (SD 1.9). Proliferating-cell nuclear antigen and c-myc mRNA concentrations and bromodeoxyuridine incorporation were decreased in the EF2-decoy group by medians of 73% [IQR 53-84], 70% [50-79], and 74% [56-83], respectively) but not in the scrambled-oligodeoxynucleotide group (p<0.0001). Groups did not differ for postoperative complication rates. At 12 months, fewer graft occlusions, revisions, or critical stenoses were seen in the E2F-decoy group than in the untreated group (hazard ratio 0.34 [95% CI 0.12-0.99]). Intraoperative transfection of human bypass vein grafts with E2F-decoy oligodeoxynucleotide is safe, feasible, and can achieve sequence-specific inhibition of cell-cycle gene expression and DNA replication. Application of this genetic-engineering strategy may lower failure rates of human primary bypass vein grafting.

Intratumoral injection of oligonucleotides to the NF kappa B binding site inhibits cachexia in a mouse tumor model.

Cancer cachexia, characterized by anorexia, weight loss and progressive tissue wasting, has been postulated to be mediated by various cytokines. However, the precise mechanism of cachexia induction is not fully explained. We have developed synthetic double-stranded oligodeoxynucleotides (ODN) as 'decoy' cis-elements that block the binding of nuclear factors to promoter regions of targeted genes, resulting in the inhibition of gene transactivation in vivo as well as in vitro. This novel molecular strategy could be useful for treating a broad range of human diseases including cancer. In this study, we injected decoy ODN targeting the transcriptional factor, NF-kappa B (NF kappa B) binding cis-elements, which are essential for transactivation of gene expression of cytokines, directly into tumors of adenocarcinoma colon26 in mice, in order to examine whether or not cachexia is alleviated by inhibiting the action of cytokines. Tumor growth was not affected by transfection of NF kappa B decoy ODN as compared with scrambled decoy ODN. Nevertheless, transfection of NF kappa B decoy, but not scrambled decoy, ODN resulted in attenuation of the reductions in body weight, epididymal fat, gastrocnemius muscle mass and food intake, which were induced by the tumor presence. Interleukin 6 mRNA in the tumor was also markedly decreased by the transfection of NF kappa B decoy ODN. It is known that the transcriptional factor E2F plays a pivotal role in the coordinated transactivation of cell cycle regulatory genes. Therefore, we hypothesized that the introduction of synthetic double-stranded DNA with high affinity for E2F in vivo as 'decoy' cis-elements might inhibit the tumor growth of colon26, resulting in turn in inhibition of cachexia induction. However, injection of E2F decoy ODN failed to inhibit tumor growth and cachexia induction, as compared with mismatched decoy ODN. Overall, the present study demonstrated that cachexia induced by adenocarcinoma colon26 was inhibited by blocking of NF kappa B, using a novel molecular decoy strategy, without an effect on tumor growth, and also that tumor growth and cachexia induction in the colon26 model were not affected by E2F decoy ODN. These results suggest that cytokines regulated by NF kappa B may play a pivotal role in the induction of cachexia by colon26, providing a new therapeutic strategy for cancer cachexia.

An oligonucleotide decoy for transcription factor E2F inhibits mesangial cell proliferation in vitro.

The transcription factor E2F controls expression of several genes involved in cell proliferation including c-myc, c-myb, proliferating cell nuclear antigen (PCNA), and cdk2 kinase. Having established that both PCNA and cdk2 kinase are induced in rat mesangial cells (MC) by serum stimulation, we attempted to inhibit MC proliferation in vitro by transfecting these cells with cationic liposomes containing a synthetic double-stranded oligodeoxynucleotide (ODN) with high affinity for E2F. Using a gel mobility shift assay, we detected increased specific binding of E2F in MC following serum stimulation. This binding was completely inhibited by preincubation of MC nuclear extracts with the double-stranded ODN with high affinity for E2F but not by preincubation with a missense ODN containing two point mutations. MC were also transfected with a luciferase reporter gene construct containing three E2F binding sites. Luciferase activity was enhanced by serum stimulation of MC, and this effect was specifically abolished by cotransfection of MC with E2F decoy ODN. Furthermore, RT-PCR analysis revealed that serum-induced upregulation of PCNA and cdk2 kinase gene expression was inhibited by E2F decoy ODN transfection but not by transfection of missense ODN. These changes in gene expression were paralleled by a reduction in PCNA and cdk2 kinase protein expression in E2F decoy ODN transfected cells. MC number increased following serum stimulation. This effect was blunted by transfection with E2F decoy ODN but not by transfection of missense ODN. These data suggest that the transcription factor E2F plays a crucial role in the regulation of MC proliferation and that this factor can be successfully targeted to inhibit MC cell cycle progression.

Inhibition of mesangial cell proliferation by E2F decoy oligodeoxynucleotide in vitro and in vivo.

The transcription factor E2F coordinately activates several cell cycle-regulatory genes. We attempted to inhibit the proliferation of mesangial cells in vitro and in vivo by inhibiting E2F activity using a 25-bp decoy oligodeoxynucleotide that contained consensus E2F binding site sequence (E2F-decoy) as a competitive inhibitor. The decoy's effect on human mesangial cell proliferation was evaluated by [3H]thymidine incorporation. The E2F decoy inhibited proliferation in a concentration-dependent manner, whereas a mismatch control oligodeoxynucleotide had little effect. Electrophoretic mobility shift assays demonstrated that the decoy's inhibitory effect was due to the binding of the decoy oligodeoxynucleotide to E2F. The effect of the E2F decoy was then tested in a rat anti-Thy 1.1 glomerulonephritis model. The E2F decoy oligodeoxynucleotide was introduced into the left kidney 36 h after the induction of glomerulonephritis. The administration of E2F decoy suppressed the proliferation of mesangial cells by 71%. Furthermore, treatment with the E2F decoy inhibited the glomerular expression of proliferating cell nuclear antigen at the protein level as well as the mRNA level. These findings indicate that decoy oligonucleotides can suppress the activity of the transcription factor E2F, and may thus have a potential in treating glomerulonephritis.