E2F Decoy Research Articles

Abstract 46: Differential Gene and Transcription Factor Binding Site Expression Between the Intima and Media Drives Intimal Hyperplasia

Background: Vein grafts (VG) fail due to rapid intimal expansion in the weeks to months following implantation. Histologically, this results in a metabolically active intima juxtaposed to a quiescent medial layer. In this study, we leverage this dichotomy to identify the key genes that control intimal growth and the critical transcription factors (TF) that drive their activation. Methods: Microsurgical techniques were used to separate the intima and media of rabbit carotid VGs harvested at 1 (n=4), 3 (n=6), and 6 (n=4) months (m) following implantation. Each layer was independently analyzed for differential mRNA expression using a whole-genome microarray. rVista and TRANSFAC were used to identify TF binding sites within 5000 bp of differentially expressed genes. Results: Early growth of the intima was driven by an enhanced proliferative response and an accumulation of cell mass (Fig). During this early hyperplastic phase, substantial differences in intimal and medial gene expression were observed (197 genes at 1m and 346 at 3m, p<0.001) that resolved by 6m (8 genes). Genes involved in cell cycle regulation and cellular movement dominated the 1 and 3m differences (Fig). TF binding site analysis of up-regulated intimal genes at 1 month revealed enrichment of ETS and HOX, while CREB and bHLH-ZIP were the most over-represented among up-regulated 3m intimal genes. In the medial layer, RXR and zinc finger TF binding sites were most prominent among up-regulated genes at 1 and 3m, respectively. Conclusion: Corresponding to the most active phase of intimal growth, smooth muscle cells demonstrated marked up-regulation of an array of genes that modulate cell proliferation and migration, with these genes under the control of a discrete set of TFs. While previous attempts at modifying TF binding (via an E2F decoy) in the initial days following VG implantation have been unsuccessful, manipulation of a broader set of TFs in the time frame of weeks to months may prove a more promising strategy.

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.

Apoptosis induction by E2F decoy DNA of the prostate cancer cell line

To observe effect of E2F decoy DNA on proliferation and apoptosis of androgen-independent prostate cancer cell line PC-3M the binding specificity of the E2F decoy DNA to the PC-3M nuclear protein was detected by electrophoretic mobility shift assay (EMSA). E2F decoy DNA, ARE decoy DNA and Control decoy DNA were respectively transfected into PC-3M cells with lipofectamine.Their influence on cell proliferative activity was detected by MTT assay.The cell apoptotic rate was determined by flow cytometric(FCM) analysis and chromosome DNA ladder was detected by DNA gel electrophoresis.The change of mRNA expression of C-myc and CyclinD1 were detected by RT-PCR.The change of mRNA expression of C-myc and CyclinD1 were detected by Western-blot. EMSA demonstrated specific binding of the E2F decoy to E2F transcription factor.The PC-3M cell growth was inhibited after transfection. The apoptotic rate was 26.35% and DNA ladder could be observed after transfection.The expression of C-myc and CyclinD1 were inhibited. All these results indicated that E2F decoy DNA induced apoptosis of androgen-independent prostate cancer cell lines PC-3M and inhibited cell proliferation via inhibiting expression of C-myc and CyclinD1.

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.

Inhibition on phenotypic transformation of detrusor smooth muscle cells after bladder outlet obstruction by E2F decoy strategy

To investigate the inhibitory effect of E2F decoy strategy on the phenotypic transformation of detrusor smooth muscle cells (DSMCs) so as to verify the effect of the E2F decoy strategy in improvement of the bladder function after bladder outlet obstruction (BOO). Rat DSMCs were cultured, underwent cyclic mechanical stretch so as to establish BOO model, and then were divided into 3 groups: E2F-ODN decoy group [transfected with E2F-decoy ODN tagged with carboxy-fluorescein (FAM), a at its 3' end], Mis-decoy group (transfected with mismatch E2F-decoy ODN), and control group (without transfection). Inverted fluorescence microscopy was used to detect the green fluorescence of FAM in the successfully transfected cells. The proliferation of the cells was observed by MTT method. RT-PCR was used to examine the mRNA expression of proliferating cell nuclear antigen (PCNA). Western blotting was used to detect the protein expression of PCNA and cdk2 kinase. FAM-labeled E2F-ODN was detected stably in the DSMCs of the E2F-ODN decoy group 24 hours after transfection. The proliferation of the DSMCs of the E2F-ODN decoy group was decreased significantly compared with the mismatch E2F-ODN decoy and control groups (both P < 0.01). The mRNA expression of PCNA, protein expression of PCNA and cdk2 kinase of the E2F-ODN decoy group were all significantly lower than those of the other 2 groups (all P < 0.01). E2F-decoy ODN can be transfected and stably expressed in DSMCs. The phenotypic transformation of DSMCs can be successfully inhibited by E2F decoy strategy, which clarifies the potential role of structural stability-based method on improvement of bladder function recovery after BOO.

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.

New Therapeutic Possibilities for Vein Graft Disease in the Post-Edifoligide Era

Vein graft neointimal hyperplasia involves proliferation and migration of vascular smooth muscle cells into the vessel intima, and ultimately engenders accelerated atherosclerosis and vein graft failure. Since a myriad of stimuli provoke smooth muscle cell proliferation, molecular therapies for vein graft disease have targeted mechanisms fundamental to all cell proliferation - the 'cell-cycle' machinery. Preclinically, the most successful of these therapies has been edifoligide (E2F decoy), a double-stranded oligodeoxynucleotide that binds to the transcription factor known as E2F. Recently, PRoject of Ex vivo vein GRaft Engineering via Transfection (PREVENT) III and IV demonstrated that edifoligide failed to benefit human vein grafts employed to treat lower-extremity ischemia and coronary heart disease, respectively. The clinical failure of edifoligide calls into question previous models of vein graft disease and lends credence to recent animal studies demonstrating that vein graft arterialization substantially involves the immigration into the vein graft of a variety of vascular progenitor cells. Future vein graft disease therapies will likely target not only proliferation of graft-intrinsic cells, but also immigration of graft-extrinsic cells.

Results of PREVENT III: A multicenter, randomized trial of edifoligide for the prevention of vein graft failure in lower extremity bypass surgery

The PREVENT III study was a prospective, randomized, double-blinded, multicenter phase III trial of a novel molecular therapy (edifoligide; E2F decoy) for the prevention of vein graft failure in patients undergoing infrainguinal revascularization for critical limb ischemia (CLI). From November 2001 through October 2003, 1404 patients with CLI were randomized to a single intraoperative ex vivo vein graft treatment with edifoligide or placebo. After surgery, patients underwent graft surveillance by duplex ultrasonography and were followed up for index graft and limb end points to 1 year. A blinded Clinical Events Classification committee reviewed all index graft end points. The primary study end point was the time to nontechnical index graft reintervention or major amputation due to index graft failure. Secondary end points included all-cause graft failure, clinically significant graft stenosis (>70% by angiography or severe stenosis by ultrasonography), amputation/reintervention-free survival, and nontechnical primary graft patency. Event rates were based on Kaplan-Meier estimates. Time-to-event end points were compared by using the log-rank test. Demographics, comorbidities, and procedural details reflected a population with CLI and diffuse atherosclerosis. Tissue loss was the presenting symptom in 75% of patients. High-risk conduits were used in 24% of cases, including an alternative vein in 20% (15% spliced vein and 5% non-great saphenous vein) and 6% less than 3 mm in diameter; 14% of the cases were reoperative bypass grafts. Most (65%) grafts were placed to infrapopliteal targets. Perioperative (30-day) mortality occurred in 2.7% of patients. Major morbidity included myocardial infarction in 4.7% and early graft occlusion in 5.2% of patients. Ex vivo treatment with edifoligide was well tolerated. There was no significant difference between the treatment groups in the primary or secondary trial end points, primary graft patency, or limb salvage. A statistically significant improvement was observed in secondary graft patency (estimated Kaplan-Meier rates were 83% edifoligide and 78% placebo; P = .016) within 1 year. The reduction in secondary patency events was manifest within 30 days of surgery (the relative risk for a 30-day event for edifoligide was 0.45; 95% confidence interval, 0.27-0.76; P = .005). For the overall cohort at 1 year, the estimated Kaplan-Meier rate for survival was 84%, that for primary patency was 61%, that for primary assisted patency was 77%, that for secondary patency was 80%, and that for limb salvage was 88%. In this prospective, randomized, placebo-controlled clinical trial, ex vivo treatment of lower extremity vein grafts with edifoligide did not confer protection from reintervention for graft failure.

Aptamers: An Emerging Class of Therapeutics

Numerous nucleic acid ligands, also termed decoys or aptamers, have been developed during the past 15 years that can inhibit the activity of many pathogenic proteins. Two of them, Macugen and E2F decoy, are in phase III clinical trials. Several properties of aptamers make them an attractive class of therapeutic compounds. Their affinity and specificity for a given protein make it possible to isolate a ligand to virtually any target, and adjusting their bioavailability expands their clinical utility. The ability to develop aptamers that retain activity in multiple organisms facilitates preclinical development. Antidote control of aptamer activity enables safe, tightly controlled therapeutics. Aptamers may prove useful in the treatment of a wide variety of human maladies, including infectious diseases, cancer, and cardiovascular disease. We review the observations that facilitated the development of this emerging class of therapeutics, summarize progress to date, and speculate on the eventual utility of such agents in the clinic.

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.

Design and rationale of the PREVENT III clinical trial: edifoligide for the prevention of infrainguinal vein graft failure.

Surgical bypass of peripheral arterial occlusive disease with autologous vein grafts provides an effective means of restoring blood flow to the lower extremity, and has been a standard therapy for patients with disabling claudication or critical limb ischemia (CLI). However, failure rates may run as high as 50% within 5 years. These graft failures occur as a result of neointimal hyperplasia, a ubiquitous biologic response of blood vessel walls to injury, which is characterized by the migration and proliferation of smooth muscle cells (SMC). The E2F family of transcription factors regulates the expression of genes controlling SMC proliferation. Edifoligide (E2F Decoy) is a novel therapy that inhibits E2F function, thus attenuating neointimal hyperplasia. Its use in conjunction with a patented drug delivery pressurization chamber is under investigation. Using this system, edifoligide is administered to vein grafts in a single, ex vivo treatment following vein harvest and before implantation, resulting in minimal systemic drug exposure and excellent patient compliance. This Phase 3, randomized, double-blind, multicenter clinical trial is designed to evaluate the safety and efficacy of edifoligide in a population of approximately 1400 patients with CLI undergoing infrainguinal bypass for peripheral arterial disease (PAD). The primary outcome measure will be the time to occurrence of non-technical graft failure resulting in either graft revision or major amputation at 12 months after enrollment. A governing Clinical Events Classification committee (CEC) will adjudicate each graft failure to determine its etiology. The PREVENT III trial is the largest multicenter trial ever performed in patients receiving autologous vein bypass grafts for CLI. This landmark study will determine if edifoligide is safe and effective at preventing vein graft failure in patients undergoing lower extremity bypass, but it also provides a unique opportunity to observe current treatment practices in vascular surgery.

Gene therapy with an E2F transcription factor decoy inhibits cell cycle progression in rat anti-Thy 1 glomerulonephritis

Mesangial cell (MC) proliferation is a central feature of many glomerular diseases. Various growth factors and cytokines are known to trigger MC proliferation both in vitro and in vivo. Regardless of the initial stimulus, proliferation is ultimately dependent upon the coordinated activation of cell cycle regulatory genes whose transcription is tightly controlled in mammalian cells. The transcription factor E2F plays an important role in the transactivation of the cell cycle regulatory genes proliferating-cell nuclear antigen (PCNA) and cdk2 kinase. To test whether or not E2F inhibition would blunt glomerular cell cycling in vivo, we treated rats with anti-Thy 1 antibody to induce glomerular injury, and that infused hemagglutinating virus of Japan (HVJ)-liposomes containing synthetic double stranded oligonucleotides (ODN) with high affinity for E2F (E2F decoy) directly into one kidney. First, we confirmed that with HVJ-liposome method fluorescence isothiocynate (FITC)-labeled ODN could be efficiently introduced into rat glomerular cells via renal artery. E2F decoy ODN treatment specifically inhibited mRNA expression of PCNA and cdk2 kinase in kidneys injured with anti-Thy 1 antibody as assessed by RT-PCR. This was associated with a significant decrease in number of glomerular cells in S phase as assessed by 5'-bromo-2'-deoxy-uridine labeling method, and attenuation of glomerular injury assessed histologically. The evidence suggests that intra-renal delivery of E2F decoy ODN by HVJ-liposome method prevents the induction of cell cycle regulatory gene expression and MC proliferation. These data also demonstrate the feasibility and the potential benefit of in vivo gene therapy as a novel strategy in the treatment of glomerular diseases.

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.

NF-&amp;#954;B as a Therapeutic Target for Transcription Factor Decoy Strategy in Inflammatory Diseases

Nuclear factor (NF)-κB is a transcription factor associated with modulation of the immune response. NF-κB is a heterodimer consisting of subunits p50, p52 and p65 (Rel A) or Rel B or cRel (the most common NF-κB heterodimer consist of subunits p50 and p65) that belong to the Rel family of genes, which includes the v-rel oncogene, the c-rel protooncogene and dorsal, a Drosophia morphogen. In its quiesecent state, NF-κB is in the cytoplasm, bound to a family of inhibitory proteins that are collectively called IkB. Several molecules, including interleukin (IL)-1, lipopolysaccharide (LPS), tumor necrosis factor (TNF)-α, reactive oxygen intermediates and viral products activate NF-κB through phosphorylation and subsequent degradation of IkB. Activation by these agents leads to exposure of the nuclear localization signal of NF-κB and entry into the nucleus. Once in the nucleus, NF-κB transcriptionally activates a variety of genes involved in the inflammatory process, including intracellular adhesion molecule (ICAM)-1, vascular cellular adhesion molecule (VCAM)-1, endothelial leukocyte adhesion molecule (ELAM) and IL-8. The family of NF-κB transcription factor is a topic of interest in the biomedical community stemming from the role NF-κB plays in almost every aspect of cell regulation such as stress responses, immune cell activation, apoptosis, proliferation, differentiation and oncogenic transformation. Recently, we have developed a novel molecular strategy termed the decoy approach to inhibit NF-κB activity. Moreover, E2F decoy strategy has already been used in the clinical trials to prevent the vascular disease. Taken together, the decoy strategy is considered to have the potential of clinical application. Keywords: transcription factor, decoy, gene therapy, glomerulonephritis, rheumatoid arthritis, myocardial infarction, transplantation

Transcription factor decoys for the prevention of vein bypass graft failure.

Failure of vein bypass grafts, performed for either coronary or lower extremity arterial occlusions, is a common clinical problem that incurs significant morbidity and mortality. Vein grafts provide a unique opportunity for genetic interventions, since the target tissue is available for manipulation ex vivo prior to implantation, and prior to the onset of the pathophysiologic events that lead to graft disease. Smooth muscle cell proliferation is a hallmark of neointimal hyperplasia in vein grafts and arteries, and is an attractive target for molecular therapy. Gene blockade strategies can be accomplished by delivery of small oligodeoxynucleotides (ODN) that target specific mRNAs ('antisense') or that competitively inhibit transcription factors ('decoys'). Transcription factors are attractive targets for molecular therapy since they influence the expression of a large number of genes involved in a coordinated cellular program. An ODN decoy approach has been developed targeting the transcription factor E2F, which is critically involved in cell cycle progression. Brief (10 minute) incubation of the ODN in solution, using non-distending pressure, results in efficient delivery of the ODN to >80% of cells in the vein wall. Preclinical studies in a rabbit model of vein grafting and hypercholesterolemia demonstrated a marked reduction in neointima formation, as well as prolonged resistance to graft atherosclerosis. Phase I/II studies conducted in lower extremity and coronary bypass patients have demonstrated safety and feasibility, and have also suggested possible efficacy. Large, randomized multicenter, phase III trials are currently under way to evaluate the efficacy of E2F decoy treatment on preventing vein bypass failure in both peripheral and coronary grafting procedures. These studies herald the arrival of a new class of molecular agents into the armamentarium of cardiovascular therapies.

Clinical trials of gene therapy for atherosclerotic cardiovascular disease.

To provide an update on clinical trials of gene therapy for atherosclerotic cardiovascular disease published since 1 August 2001 and summarize the general advantages and potential problems of gene transfer in these disorders. There are two major areas in which gene therapy has entered clinical trials. The first is angiogenesis for coronary and peripheral arterial disease. Two relatively small placebo-controlled trials for coronary disease were reported, one using intramyocardial plasmid VEGF-2 gene, the other using intracoronary adenoviral FGF-4 gene. The VEGF-2 study in no-option patients showed reduced angina, and significant improvement in perfusion and function, whereas the FGF-4 study in less severely affected patients showed promising results in some subsets. In peripheral artery disease two phase 1 studies of adenoviral NV1FGF and VEGF showed some objective improvement in pain, ulcer size and ankle:brachial index in one study and endothelial function in the other. Both adenoviral and plasmid VEGF gene transfer at angioplasty increased vascularity in a phase 2 double-blind study. The other major area is the prevention of graft disease and restenosis using antisense oligodeoxynucleotides. E2F decoy led to a significant reduction in venous graft complications after ex-vivo transfection at the time of coronary bypass surgery, whereas the c-Myc oligodeoxynucleotide was ineffective in preventing in-stent coronary restenosis. There are more reviews of gene therapy for atherosclerosis in the literature than publications with original data or trials, but in the past year the imbalance is being redressed, with some promising results from controlled studies.

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.