Application of Transcription Factor "Decoy" Strategy as Means of Gene Therapy and Study of Gene Expression in Cardiovascular Disease

From the Department of Geriatric Medicine, Osaka University Medical School, Suita, Japan.

Correspondence to Toshio Ogihara, MD, PhD, Professor of Medicine, Department of Geriatric Medicine, Osaka University Medical School, 2-2 Yamada-oka, Suita, Osaka 565, Japan.

Abstract

Abstract—Recent progress in molecular biology has provided new techniques for inhibiting target gene expression. In particular, the application of DNA technology, such as antisense strategy to regulate the transcription of disease-related genes in vivo, has important therapeutic potential. Recently, transfection of cis-element double-stranded oligodeoxynucleotides (ODNs), referred to as "decoy" ODNs, has been reported to be a powerful tool in a new class of anti-gene strategies for gene therapy and in the study of transcriptional regulation. Transfection of double-stranded ODNs corresponding to the cis sequence will result in the attenuation of authentic cis-trans interaction, leading to the removal of trans factors from the endogenous cis elements with subsequent modulation of gene expression. This "decoy" strategy is not only a novel strategy for gene therapy as an anti-gene strategy but also a powerful tool for the study of endogenous gene regulation in vivo as well as in vitro. In this article, we reviewed (1) the mechanisms and (2) the potential applications of decoy strategy.

Key Words: cis-element decoy • restenosis • myocardial infarction • glomerulonephritis • gene regulation

Transfection of cis-element ds ODNs ("decoy" ODNs) has been reported to be a powerful tool that is useful in a new class of anti-gene strategies for gene therapy and in the study of transcriptional regulation.^{1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17} Transfection of ds ODNs corresponding to the cis sequence will result in the attenuation of an authentic cis-trans interaction, leading to the removal of trans factors from the endogenous cis element with subsequent modulation of gene expression (Figure 1). Therefore, the decoy approach may enable us to treat diseases by modulation of endogenous transcriptional regulation. Currently, several studies have reported an application of the decoy ODN strategy as in vivo gene therapy.^{2 15 16 17} The present study provides evidence of an in vivo application of this novel molecular approach as a therapeutic strategy against cardiovascular disease. Alternatively, this strategy also provides a powerful tool by which to study endogenous gene regulation in vitro and in vivo. The decoy approach enables the study of gene regulation in vivo as well as in vitro by modulation of endogenous transcriptional regulation. Previously, many researchers used antisense technology as a "loss-of-function" approach at transcriptional and translational levels.^{18 19 20 21} On the other hand, cis-element decoy strategy is also applicable as a loss-of-function approach at pretranscriptional and transcriptional levels in the study of transcription factors. In this article, we review (1) the mechanisms and (2) potential applications of decoy strategy.

View larger version (26K): [in this window] [in a new window]   Figure 1. Schematic diagram of decoy strategy. a, In the basal state, transcription factor (TF) is bound to cis element (Cis-E), resulting in continuous activation of target gene expression. b, TF decoy cis-element ds ODNs bind to TF, resulting in the prevention of TF interaction and transactivation of TF-promoting target gene expression.

Principles

Correct regulation of gene expression is essential both to normal development and to the correct functioning of the adult organism. Such regulation is usually achieved at the level of DNA transcription, a process that controls which genes are transcribed into RNA by the enzyme RNA polymerase, although posttranscriptional regulation is also important.²² The transcription of specific genes is controlled by regulatory proteins known as transcription factors.²² Transcription factors have been grouped in families on the basis of shared DNA-binding motifs. Other regions of the factors interact with RNA polymerase and its associated proteins to increase or decrease the rate of transcription. The vital role of these factors, together with the fact that a single factor can affect the expression of many genes, suggests that the inactivation of a transcription factor as a result of an inherited mutation is incompatible with survival. Initially, overexpression of TAR-containing sequences (TAR decoys) in a double-copy murine retroviral vector was used to render cells resistant to HIV replication.²³ Currently, TAR decoys, short RNA oligonucleotides corresponding to the HIV TAR sequence, are used to inhibit HIV expression and replication by blocking the binding of the HIV regulatory protein Tat to the authentic TAR region.^{23 24 25} However, such RNA decoys are very difficult to use in vivo. In addition, the regulation of decoy expression is also problematic. To overcome these issues, we hypothesized that synthetic ds DNA with high affinity for transcription factors may be introduced in vivo as a decoy cis element to bind the transcription factors and block the activation of genes mediating such diseases, resulting in an effective therapy for treating diseases, since transfection of ds ODN corresponding to the cis sequence will result in attenuation of the removal of the trans factors from the endogenous cis element with subsequent modulation of gene expression. This approach is particularly attractive for several reasons: (1) the potential drug targets (transcription factors) are plentiful and readily identifiable, (2) the synthesis of the sequence-specific decoy is relatively simple and can be targeted to specific tissues, (3) knowledge of the exact molecular structure of the target transcription factor is unnecessary, and (4) decoy ODNs may be more effective than antisense ODNs in blocking constitutively expressed factors as well as multiple transcription factors that bind to the same cis element. Although the mechanisms of actions of antisense ODNs are still unclear, the principle of the transcription factor decoy approach is simply the reduction of promoter activity due to the inhibition of binding of a transcription factor to a specific sequence in the promoter region (Figure 2). Alternatively, this strategy also provides a powerful tool in the study of endogenous gene regulation in vivo as well as in vitro by modulation of endogenous transcriptional regulation.

View larger version (20K): [in this window] [in a new window]   Figure 2. Target sites for decoy and antisense ODNs. TF indicates transcription factor; decoy, decoy ODN; and antisense, antisense ODN.

Application

Gene Therapy
Potential targets for decoy strategy as gene therapy are summarized in the Table. One important disease potentially amenable to gene therapy based on decoy strategy is restenosis after angioplasty, since the long-term effectiveness of this procedure is limited by the development of restenosis in >40% percent of the patients.^{26 27 28} Intimal hyperplasia after angioplasty develops, in large part, as a result of VSMC proliferation and migration induced by a complex interaction of multiple growth factors that are activated by vascular injury.^{26 27 28} The process of VSMC proliferation is dependent on the coordinated activation of a series of cell cycle regulatory genes, which results in mitosis. A critical element in regulation of cell cycle progression is the complex formed by E2F, cyclin A, and cdk 2.^{29 30} The dissociation of the transcription factor E2F from the retinoblastoma gene product is proposed to play a pivotal role in the regulation of cell proliferation by inducing a coordinated transactivation of genes involved in cell cycle regulation, including c-myc, c-myb, cdc 2, PCNA, and thymidine kinase.^{29 30} Indeed, the antiproliferative effects of the retinoblastoma gene product appear to depend on its capacity to bind to E2F and thereby prevent this transcription factor from binding to the E2F cis element within the promoters of these essential cell cycle regulatory genes.^{29 30} Accordingly, we hypothesized that transfection of VSMCs with a sufficient quantity of the decoy ODN containing the E2F cis element (consensus sequence TTTTCGGCGC) would effectively bind E2F, prevent it from transactivating the gene expression of essential cell cycle regulatory proteins, and thereby inhibit VSMC proliferation and neointimal formation.³ Synthesized 14mer ds ODNs containing the consensus sequence effectively competed with binding of E2F to its binding site, assessed by gel mobility shift assay.³ Transfection of E2F decoy ODNs into rat balloon-injured carotid arteries using the HVJ-liposome method resulted in almost complete inhibition of neointimal formation at 2 weeks after balloon injury, accompanied by a reduction in mRNA of PCNA and cdc 2 kinase, but not ß-actin, whereas mismatched ODNs had no effect on neointimal hyperplasia.³ Of importance, sustained inhibition of neointimal formation by a single administration of E2F decoy ODNs was observed at least up to 8 weeks after the treatment. This is the first successful in vivo transfer of a decoy cis element to bind E2F, modulate gene expression, and consequently inhibit smooth muscle proliferation and vascular lesion formation as gene therapy for restenosis. However, further studies are necessary to enhance cell targeting and minimize the effects on endothelial cell replication at the periphery of the injured transfected area, since reendothelialization of the injured area is critically important.

Similarly, the potential of the transcription factor decoy approach to treat renal diseases, such as glomerulonephritis, has been assessed.^{6 7} Although numerous growth factors, including platelet-derived growth factor and angiotensin II, regulate this process, the proliferation of mesangial cells is also regulated by cell cycle regulatory genes. As discussed above, the transcription factor E2F has been reported to play a pivotal role in the regulation of cell cycle regulatory genes. Indeed, intrarenal arterial perfusion of E2F decoy ODNs inhibited the mesangial cell proliferation induced by anti–Thy 1 antibody, which specifically injures glomerular mesangial cells, resulting in a proliferative glomerular lesion.⁶ Since E2F has been postulated to play an important role in the pathogenesis of numerous diseases, eg, cancer and arthritis, the development of E2F decoy strategy may provide a useful therapeutic tool for treating these proliferative diseases. For the treatment of systemic diseases, tissue-specific inhibition of E2F activity might be important, because replication of cells would be necessary as "wound healing" in certain physiological conditions. Thus, tissue-specific delivery and modifications of the oligonucleotide composition that can prolong decoy stability in vivo will be critical in enhancing the potential therapeutic efficacy.

On the other hand, the transcription factor NF-B also plays a pivotal role in the coordinated transactivation of cytokine and adhesion molecule genes, whose activation has been postulated to be involved in numerous diseases, such as myocardial infarction and glomerulonephritis.^{30 31 32 33 34 35 36 37} These diseases are, importantly, potentially amenable to ODN-based gene therapy, since treatment of these diseases is extremely difficult because of the lack of effective pharmacological agents. The pathophysiology of myocardial infarction and glomerulonephritis is quite complicated.^{31 32 33 34 35 36 37 38} Numerous cytokines, including IL-1, IL-2, IL-6, IL-8, and TNF-, to name a few, regulate this process. However, gene regulation of many cytokines is relatively simple, because the transcription factor NF-B has been reported to upregulate these cytokines.^{39 40 41 42 43} Interestingly, adhesion molecules, such as VCAM and ICAM, are also known to be upregulated by NF-B.^{42 43} Accordingly, we hypothesize that myocardial infarction and glomerulonephritis could be prevented by the blockade of genes regulating cell inflammation—the final common pathway that is induced by NF-B binding. The necessity to block cytokine and adhesion molecule genes at more than one point to achieve maximum inhibitory effects may be due to the redundancy and complexity of the interactions of these genes.

Myocardial reperfusion injury develops, to a large degree, as a result of severe damage of myocytes and endothelial cells, probably induced by the complex interaction of multiple cytokines and adhesion molecules that are activated by reperfusion.^{36 37 38} The process of ischemic reperfusion may be dependent on the coordinated activation of a series of cytokine and adhesion molecule genes that results in the attachment of leukocytes and release of cytotoxic molecules. Importantly, increased NF-B binding activity was confirmed in hearts with myocardial infarction.⁴ Our previous study provided the first evidence of the feasibility of decoy strategy against NF-B in treating myocardial reperfusion injury.⁴ Transfection of NF-B decoy ODNs into rat coronary arteries before left anterior descending coronary artery occlusion markedly reduced the damaged area of myocytes 24 hours after reperfusion, whereas no difference was observed between scrambled decoy ODN-treated and untransfected rats. The therapeutic efficacy of this strategy via intracoronary administration immediately after reperfusion, similar to the clinical situation, was also examined. NF-B decoy ODNs reduced the damage of myocytes due to reperfusion in contrast to rats treated with scrambled control decoy or vehicle. The selectivity of the NF-B decoy ODN effect was confirmed further by the demonstration that reduction of the damaged myocardial area was not observed in rats treated with antisense ODN directed against the rat inducible NO synthase gene. The specificity of the NF-B decoy in the inhibition of cytokine and adhesion molecule expression was also confirmed by in vitro experiments using human and rat coronary artery endothelial cells. Transfection of NF-B decoy ODNs markedly inhibited the protein expression of cytokines (IL-6 and IL-8) and adhesion molecules (VCAM, ICAM, and endothelial leukocyte adhesion molecule) in response to TNF- stimulation in human aortic endothelial cells. In contrast, the control scrambled decoy ODN failed to inhibit the induction of these protein expressions. Cell numbers after transfection were not changed, indicating that the NF-B decoy induces a specific inhibitory effect rather than nonspecific cytotoxicity. Treatment of glomerulonephritis by means of NF-B decoy ODNs is also reported.⁷

Since NF-B has been postulated to play an important role in the pathogenesis of numerous diseases, eg, cancer and arthritis, the development of NF-B decoy strategy may provide a useful therapeutic tool for treating these diseases. Furthermore, modifications of ODN composition to prolong decoy stability in vivo and/or development of a delivery system into the cardiovascular organs/tissues will be critical in the enhancement of potential therapeutic efficacy.^{44 45 46} Despite these limitations, development of this technology offers great promise as a new tool for defining biological processes and treating pathological conditions. Alternatively, the transcription factor NF-B is one of the key regulators promoting nephritis and myocardial infarction after ischemic reperfusion. The development of drugs targeted against NF-B may be provide a novel therapy. We postulate that NF-B binding proteins may be an ideal target for inhibition.

On the other hand, NF-B may also play a pivotal role in the development of atherosclerosis.^{47 48 49} Thus, NF-B decoy strategy might be useful in the treatment of atherosclerosis. However, because atherosclerosis is a chronic systemic disease, it would clearly be necessary to administer NF-B decoy on a long-term basis. Such long-term inhibition of NF-B might be expected to have severe physiological consequences, since the molecule is also required for a variety of acute inflammatory and immune responses. An important concern regarding decoy strategy revolves around the potential inhibition of normal physiological responses. Therefore, the application of decoy strategy as gene therapy may be limited to treatment of acute conditions, namely, "transcription factor–driven diseases." Further studies are necessary to examine these potential side effects. Moreover, the deliver method for decoys is also important. Although direct transfer of "naked" decoy ODNs can be achieved via passive uptake, the transfection efficiency seems to be lower than that with single-stranded antisense ODNs. To enhance the transfection efficiency of decoy ODNs, the cationic liposome method, HVJ-liposome method, or other vector systems are generally used. The majority of ODNs are sequestered and degraded in lysosomes and never reach the nucleus. Because the site of decoy effects is apparently in the nucleus, bypassing the endocytotic pathway and translocation of decoy ODNs from the cytoplasm are extremely important in the practical application of therapeutics. Although the use of endosome-lytic agents or high-mobility group nonhistone protein prebound to the DNA to facilitate nuclear translocation⁵⁰ was examined to overcome this problem, further modification of delivery methods must be necessary. In addition to cellular delivery, systemic administration would likely be needed in the treatment of atherosclerosis. Further modification of delivery systems for decoys would be also important in the treatment of systemic diseases such as atherosclerosis.

Regarding decoy strategy as gene therapy, one of the major concerns is nonspecific effects, particularly those of phosphorothioate-substituted ODNs. This concern is related not only to decoy strategy but also to all ODN-mediated therapy. Non–sequence-specific inhibition may operate through the blockade of cell surface receptor activity or interference with other proteins.⁵¹ At the same time, ODNs containing guanine cytosine dinucleotides may bring about immune activation.⁵² In addition, sequence-specific binding of nontranscriptional factor proteins to ODNs has been reported to result in nonspecific effects of ODN-based gene therapy.⁵² Moreover, Burgess et al⁵³ have reported that the antiproliferative activity of c-myb and c-myc antisense ODNs in VSMCs is caused by a nonantisense mechanism. They have found that a stretch of 4 contiguous guanosine residues is responsible for the sequence-specific but nonantisense antiproliferative effects of c-myb and c-myc. These issues have greatly confused the specificity of effects observed with antisense ODN therapy and will likely also be relevant to ds decoy ODN therapy. To overcome these issues, careful controlled experiments must be performed to eliminate the potential nonspecific effects of ODN-mediated therapy. Scrambled ODNs and mismatched ODNs having several mutations in the consensus sequence are necessary for use as control decoy ODNs.

For gene therapy using ODN-based strategy, the toxicity of phosphorothioate ODN may also be important. Although low-dose administration does not seem to cause any toxicity, bolus infusions may be dangerous. Higher doses over prolonged periods of time may cause kidney damage in animals, as evidenced by proteinuria and leukocytes in the urine.⁵⁴ Liver enzymes may also be increased in most animals treated with moderate to high doses. Several phosphorothioate ODNs have been shown to cause acute hypotensive events in monkeys,^{55 56} probably a result of complement activation.⁵⁷ These effects are transient, if managed appropriately, and relatively uncommon. This toxicity can be avoided by giving intravenous infusions rather than bolus injections. More recently, prolongation of prothrombin, partial thromboplastin, and bleeding times has been reported in monkeys.⁵⁸

Gene Regulation
Another advantage of the decoy strategy is its use as a tool in the study of endogenous gene regulation in vivo. Numerous previous studies have used antisense technology as a loss-of-function approach at the transcriptional and translational levels.^{18 21 59} In addition, the cis-element decoy strategy, serving as a loss-of-function approach at the pretranscriptional and transcriptional levels in the study of transcription factors, was recently used.^{8 12 13 14 15 16 17} A classic approach to define the role of transcription factors in the regulation of genes is the use of chloramphenicol acetyltransferase and luciferase constructs in promoter-reporter gene transfection experiments. This approach is very useful in identifying cis and trans element interactions but has some disadvantages: (1) it is costly and time consuming to make a series of constructs, (2) endogenous gene regulation cannot be analyzed, and (3) it is difficult to identify the specific elements. In contrast, the decoy approach has many advantages: (1) decoys are easily synthesized, (2) endogenous gene regulation and pathophysiological roles can be studied, and (3) specific cis elements can be identified, even if the specific regulatory cis elements have not yet been identified. An example of the utility of the cis-element decoy strategy has been demonstrated by us. The 5'-flanking region of the human angiotensinogen gene has been reported to be a determinant of tissue-specific and cell type–specific expression of the gene in vivo as well as in vitro.^{60 61} In human hepatocytes in vitro, cell type–specific activation of angiotensinogen gene transcription results from the cooperative interaction of a proximal promoter element (AGE 2, from -96 to -52) with a novel cis-acting element termed AGE 3 (from -6 to +22) that resides directly around the transcriptional start site in the core promoter region.^{60 61} However, little is known about the molecular mechanism(s) of angiotensinogen gene regulation in vivo. To examine the gene regulation of angiotensinogen, a decoy approach has been applied. The pivotal role of cis-element AGE 2, rather than AGE 3, in the regulation of hepatic angiotensinogen gene expression was demonstrated by the following evidence: (1) transfection of AGE 2, but not mismatched, decoy ODNs resulted in a transient decrease in high blood pressure, accompanied by reduction of plasma and hepatic angiotensinogen and angiotensin II concentrations, whereas mismatched decoy ODNs showed no effect, and (2) transfection of AGE 3 and mismatched AGE 3 decoy ODNs had no effect on blood pressure.⁸ The present study has also demonstrated the utility of gene transfer and decoy technology for cardiovascular research, especially in evaluating the specific functions of transcription factors of target gene regulation. Similarly, tissue-specific–negative and –positive regulation of the renin gene has also been examined by the decoy approach.¹³

Alternatively, another tool for the loss-of-function approach is transgenic/gene-targeting technology. This technology provides many advantages, such as (1) the ability to study the function of a specific gene in terms of systemic and developmental effects and (2) the ability to study a specific gene function chronically. Nevertheless, this technology has several disadvantages: (1) it is time consuming and costly, 2) the effect of the overexpressed transgene is exerted throughout development, and (3) targeting transgenic expression in a tissue-specific manner is very difficult. If the targeted gene can cause a lethal effect, it is impossible to examine specific functions by transgenic or gene-targeting techniques. Overall, cis-element decoy strategy is useful as a loss-of-function approach in the study of transcription factors at pretranscriptional and transcriptional levels.

Prospectives in Decoy Strategy
The first Food and Drug Administration–approved human gene therapy trial began in 1990 in adenosine deaminase–deficient patients. Five years since the beginning of the first trial, 136 clinical studies of gene therapy were under investigation (as of December 1995). In addition to the antisense strategy, decoy strategy is a powerful tool that is useful in a new class of anti-gene strategies for gene therapy and in the study of transcription regulation. Although few studies have reported the application of the decoy ODN strategy as in vivo gene therapy, this approach is particularly attractive for several reasons: (1) the potential drug targets (transcription factors) are plentiful and readily identifiable, (2) the synthesis of sequence-specific decoys is relatively simple and can be targeted to specific tissues, (3) knowledge of the exact molecular structure of the targeted transcription factor is unnecessary, and (4) decoy ODNs may be more effective than antisense ODNs in blocking constitutively expressed factors as well as multiple transcription factors that bind to the same cis element. Thus, decoy strategy may be useful for treating a broad range of human diseases. In 1996, clinical application of a decoy against E2F was approved by the Food and Drug Administration for the treatment of neointimal hyperplasia in vein bypass grafts, which results in failure in up to 50% of grafts within a period of 10 years.⁶² Although there are still many unresolved issues in the clinical application of decoy strategy, its utility could be widespread for gene therapy in other diseases. On the other hand, in addition to the classic reporter gene method, the utility of decoy strategy for research, especially to evaluate the specific functions of transcription factors of target gene regulation, has been established.

Selected Abbreviations and Acronyms

AGE = angiotensinogen gene–activating element ds = double-stranded HVJ = hemagglutinating virus of Japan (Sendai virus) ICAM = intercellular adhesion molecule IL = interleukin NF-B = nuclear factor-B ODN = oligodeoxynucleotide PCNA = proliferating-cell nuclear antigen TAR = transactivation response region TNF = tumor necrosis factor VCAM = vascular cell adhesion molecule VSMC = vascular smooth muscle cell

Received October 6, 1997; accepted March 16, 1998.

References

1. Isner JM. Oligonucleotide therapeutics: novel cardiovascular targets. Nat Med. 1997;3:834–835.[Medline] [Order article via Infotrieve]
2. Bielinska A, Shivdasani RA, Zhang L, Nabel GJ. Regulation of gene expression with double-stranded phosphorothioate oligonucleotides. Science. 1990;250:997–1000.[Abstract/Free Full Text]
3. Morishita R, Gibbons GH, Horiuchi M, Ellison KE, Nakajima M, Zhang L, Kaneda Y, Ogihara T, Dzau VJ. A novel molecular strategy using cis element "decoy" of E2F binding site inhibits smooth muscle proliferation in vivo. Proc Natl Acad Sci U S A.. 1995;92:5855–5859.[Abstract/Free Full Text]
4. Morishita R, Sugimoto T, Aoki M, Kida I, Tomita N, Moriguchi A, Maeda K, Sawa Y, Kaneda Y, Higaki J, Ogihara T. In vivo transfection of cis element "decoy" against NFkB binding site prevented myocardial infarction as gene therapy. Nat Med. 1997;3:894–899.[Medline] [Order article via Infotrieve]
5. Sawa Y, Morishita R, Suzuki K, Kagisaki K, Kaneda Y, Maeda K, Kadoba K, Matsuda H. A novel strategy for myocardial protection using in vivo transfection of cis element "decoy" against NFkB binding site: evidence for a role of NFkB in ischemic-reperfusion injury. Circulation. 1997;965(suppl II):II-280–II-285.
6. Tomita N, Kim J, Gibbons GH, Baran D, Ogborn M, Stahl RAK, Tomita S, Zhang L, Kaneda Y, Dzau VJ. In vivo gene therapy of anti-Thy 1 nephritis using E2F decoy oligonucleotides [abstract]. J Am Soc Nephrol. 1995;6:887.
7. Tomita N, Morishita R, Higaki J, Kaneda Y, Ogihara T. Potential gene therapy to glomerular disease: application of transcriptional factor decoy approach [abstract]. Nephrology. 1997;3:S33.
8. Morishita R, Higaki J, Tomita N, Aoki M, Moriguchi A, Tamura K, Murakami K, Kaneda Y, Ogihara T. Role of transcriptional cis-elements, angiotensinogen gene–activating elements, of angiotensinogen gene in blood pressure regulation. Hypertension. 1996;27:502–507.[Abstract/Free Full Text]
9. Sharma HW, Perez JR, Higgins-Sochaski K, Hsiao R, Narayanan R. Transcription factor decoy approach to decipher the role of NF-kappa B in oncogenesis. Anticancer Res. 1996;16:61–69.[Medline] [Order article via Infotrieve]
10. Sharma HW, Narayanan R. The NF-kappaB transcription factor in oncogenesis. Anticancer Res. 1996;16:589–596.[Medline] [Order article via Infotrieve]
11. Roshak AK, Jackson JR, McGough K, Chabot-Fletcher M, Mochan E, Marshall LA. Manipulation of distinct NFkappaB proteins alters interleukin-1beta-induced human rheumatoid synovial fibroblast prostaglandin E2 formation. J Biol Chem. 1996;271:31496–31501.[Abstract/Free Full Text]
12. Clusel C, Meguenni S, Elias I, Vasseur M, Blumenfeld M. Inhibition of HSV-1 proliferation by decoy phosphodiester oligonucleotides containing ICP4 recognition sequences. Gene Expr. 1995;4:301–309.[Medline] [Order article via Infotrieve]
13. Yamada T, Horiuchi M, Morishita R, Zhang L, Pratt RE, Dzau VJ. In vivo identification of a negative regulatory element in the mouse renin gene using direct gene transfer. J Clin Invest. 1995;96:1230–1237.
14. di-Fagagna FD, Marzio G, Gutierrez MI, Kang LY, Falaschi A, Giacca M. Molecular and functional interactions of transcription factor USF with the long terminal repeat of human immunodeficiency virus type 1. J Virol. 1995;69:2765–2775.[Abstract]
15. Tanaka H, Vickart P, Bertrand JR, Rayner B, Morvan F, Imbach JL, Paulin D, Malvy C. Sequence-specific interaction of alpha-beta-anomeric double-stranded DNA with the p50 subunit of NF kappa B: application to the decoy approach. Nucleic Acids Res. 1994;22:3069–3074.[Abstract/Free Full Text]
16. Schmedtje JF Jr, Ji YS, Liu WL, DuBois RN, Runge MS. Hypoxia induces cyclooxygenase-2 via the NF-kappaB p65 transcription factor in human vascular endothelial cells. J Biol Chem. 1997;272:601–608.[Abstract/Free Full Text]
17. Gambarotta G, Boccaccio C, Giordano S, Ando M, Stella MC, Comoglio PM. Ets up-regulates MET transcription. Oncogene.. 1996;13:1911–1917.[Medline] [Order article via Infotrieve]
18. Stein CA, Cohen JS. Oligodeoxynucleotides as inhibitors of gene expression: a review. Cancer Res. 1988;48:2659–2668.[Abstract/Free Full Text]
19. Dzau VJ, Horiuchi M. In vivo gene transfer and gene modulation in hypertension research. Hypertension. 1996;28:1132–1137.[Abstract/Free Full Text]
20. Morishita R, Gibbons GH, Ellison KE, Nakajima M, Zhang L, Kaneda Y, Ogihara T, Dzau VJ. Single intraluminal delivery of antisense cdc 2 kinase and PCNA oligonucleotides results in chronic inhibition of neointimal hyperplasia. Proc Natl Acad Sci U S A.. 1993;90:8474–8479.[Abstract/Free Full Text]
21. Tomita N, Morishita R, Higaki J, Aoki M, Nakamura Y, Mikami H, Fukamizu A, Murakami K, Kaneda Y, Ogihara T. Transient decrease in high blood pressure by in vivo transfer of antisense oligodeoxynucleotides against rat angiotensinogen. Hypertension. 1995;26:131–136.[Abstract/Free Full Text]
22. Latchhman DS. Transcription factor mutations and disease. N Engl J Med. 1996;334:28–33.[Free Full Text]
23. Sullenger BA, Gallardo HF, Ungers GE, Giboa E. Overexpression of TAR sequence renders cells resistant to human immunodeficiency virus replication. Cell. 1990;63:601–608.[Medline] [Order article via Infotrieve]
24. Bahner I, Kearns K, Hao QL, Smogorzewska EM, Kohn DB. Transduction of human CD34+ hematopoietic progenitor cells by a retroviral vector expressing an RRE decoy inhibits human immunodeficiency virus type 1 replication in myelomonocytic cells produced in long-term culture. J Virol. 1996;70:4352–4360.[Abstract]
25. Lee SW, Gallardo HF, Gaspar O, Smith C, Gilboa E. Inhibition of HIV-1 in CEM cells by a potent TAR decoy. Gene Ther. 1995;2:377–384.[Medline] [Order article via Infotrieve]
26. Libby P, Schwartz D, Brogi E, Tanaka H, Clinton SK. A cascade model for restenosis: a special case of atherosclerosis progression. Circulation. 1992;86(suppl III):III-47–III-52.
27. Clowes AW, Clowes MM, Fingerle J, Reidy MA. Regulation of smooth muscle cell growth in injured artery. J Cardiovasc Pharmacol. 1989;14:S12–S15.
28. Gibbons GH, Dzau VJ. The emerging concept of vascular remodeling. N Engl J Med. 1994;330:1431–1438.[Free Full Text]
29. Weintraub SJ, Prater CA, Dean DC. Retinoblastoma protein switches the E2F site from positive to negative element. Nature. 1992;358:259–261.[Medline] [Order article via Infotrieve]
30. Pagano M, Draetta G, Durr J. Association of cdk 2 kinase with the transcription factor E2F during S phase. Science. 1992;255:1144–1147.[Abstract/Free Full Text]
31. Chow J, Hartley RB, Jagger C, Dilly SA. ICAM-1 expression in renal disease. J Clin Pathol. 1992;45:880–884.[Abstract/Free Full Text]
32. Floege J, Eng E, Young BA, Johnson R. Factors involved in the regulation of mesangial cell proliferation in vitro and in vivo. Kidney Int. 1993;39:S47–S54.
33. Sterzel RB, Schulze-Lohoff E, Marx M. Cytokines and mesangial cells. Kidney Int. 1993;39:S26–S31.
34. Egido J, Gomez-Chiarri M, Ortiz A, Bustos C, Alonso J, Gomez-Guerrero C, Gomez-Garre D, Lopez-Armada MJ, Plaza J, Gonzalez E. Role of tumor necrosis factor-alpha in the pathogenesis of glomerular diseases. Kidney Int. 1993;39:S59–S64.
35. Herskowitz A, Choi S, Ansari AA, Wesselingh S. Cytokine mRNA expression in postischemic/reperfused myocardium. Am J Pathol. 1995;146:419–428.[Abstract]
36. Kukielka GL, Smith CW, LaRosa GJ, Manning AM, Mendoza LH, Daly TJ, Hughes BJ, Youker KA, Hawkins HK, Michael LH, Rot A, Entman ML. Interleukin-8 gene induction in the myocardium after ischemia and reperfusion in vivo. J Clin Invest. 1995;95:89–103.
37. Kukielka GL, Entman ML. Adhesion molecule-dependent cardiovascular injury. In: Poste G, Metcalf B, eds. Cellular Adhesion: Molecular Definition to Therapeutic Potential. New York, NY: Plenum Publishing Corp; 1995:187–212.
38. Lenardo MJ, Baltimore D. NF-kappa B: a pleiotropic mediator of inducible and tissue-specific gene control. Cell. 1989;58:227–229.[Medline] [Order article via Infotrieve]
39. Libermann TA, Baltimore D. Activation of interleukin-6 gene expression through the NF-kappa B transcription factor. Mol Cell Biol. 1990;10:2327–2334.[Abstract/Free Full Text]
40. Satriano J, Schlondorff. Activation of attenuation of transcriptional factor NF-kB in mouse glomerular cells in response to tumor necrosis factor-, immunoglobulin G, and adenosine 3':5'-cyclic monophosphate. J Clin Invest. 1994;94:1629–1636.
41. Neish AS, Williams AJ, Palmer HJ, Whitley MZ, Collins T. Functional analysis of the human vascular cell adhesion molecule 1 promoter. J Exp Med. 1992;176:1583–1593.[Abstract/Free Full Text]
42. Brennan DC, Jevnikar AM, Takei F, Reubin-Kelley VE. Mesangial cell accessory functions: mediation by intracellular adhesion molecule-1. Kidney Int. 1990;38:1039–1046.[Medline] [Order article via Infotrieve]
43. Morishita R, Gibbons GH, Ellison KE, Nakajima M, Leyen HVL, Zhang L, Kaneda Y, Ogihara T, Dzau VJ. Intimal hyperplasia after vascular injury is inhibited by antisense cdk 2 kinase oligonucleotides. J Clin Invest. 1994;93:1458–1464.
44. Morishita R, Gibbons GH, Kaneda Y, Ogihara T, Dzau VJ. Pharmacokinetics of antisense oligonucleotides (cyclin B1 and cdc 2 kinase) in the vessel wall: enhanced therapeutic utility for restenosis by HVJ-liposome method. Gene. 1994;149:13–19.[Medline] [Order article via Infotrieve]
45. Chu BC, Orgel LE. The stability of different forms of double-stranded decoy DNA in serum and nuclear extracts. Nucleic Acids Res. 1992;20:5857–5858.[Free Full Text]
46. Sedor JR, Konieczkowski M, Huang S, Gronich JH, Nakazato Y, Gordon G, King CH. Cytokines, mesangial cell activation and glomerular injury. Kidney Int. 1993;39:S65–S70.
47. Navab M, Fogelman AM, Berliner JA, Territo MC, Demer LL, Frank JS, Watson AD, Edwards PA, Lusis AJ. Pathogenesis of atherosclerosis. Am J Cardiol. 1995;76:18C–23C.[Medline] [Order article via Infotrieve]
48. Brand K, Page S, Rogler G, Bartsch A, Brandl R, Knuechel R, Page M, Kaltschmidt C, Baeuerle PA, Neumeier D. Activated transcription factor nuclear factor-kappa B is present in the atherosclerotic lesion. J Clin Invest. 1996;97:1715–1722.[Medline] [Order article via Infotrieve]
49. Collins T. Endothelial nuclear factor-kappa B and the initiation of the atherosclerotic lesion. Lab Invest. 1993;68:499–508.[Medline] [Order article via Infotrieve]
50. Kaneda Y, Iwai K, Uchida T. Increased expression of DNA cointroduced with nuclear protein in adult rat liver. Science. 1989;243:375–378.[Abstract/Free Full Text]
51. Gibson I. Antisense approaches to the gene therapy of cancer–"Recnac." Cancer Metastasis Rev. 1996;15:287–299.[Medline] [Order article via Infotrieve]
52. Khaled Z, Benimetskaya L, Zeltser R, Khan T, Sharma HW, Narayanan R, Stein CA. Multiple mechanisms may contribute to the cellular anti-adhesive effects of phosphorothioate oligodeoxynucleotides. Nucleic Acids Res. 1996;24:737–775.[Abstract/Free Full Text]
53. Burgess TL, Fisher EF, Ross SL, Bready JV, Qian YX, Bayewitch LA, Cohen AM, Herrera CJ, Hu SS, Kramer TB, Lott FD, Martin FH, Pierce GF, Simonet L, Farrell CL. The antiproliferative activity of c-myb and c-myc antisense oligonucleotides in smooth muscle cells is caused by a nonantisense mechanism. Proc Natl Acad Sci U S A.. 1995;92:4051–4055.[Abstract/Free Full Text]
54. Srinivasan SK, Iversen P. Review of in vivo pharmacokinetics and toxicology of phosphorothioate oligonucleotides. J Clin Lab Anal. 1995;9:129–137.[Medline] [Order article via Infotrieve]
55. Henry SP, Bolte H, Auletta C, Kornbrust DJ. Evaluation of the toxicity of ISIS 2302, a phosphorothioate oligonucleotide, in a four-week study in cynomolgus monkeys. Toxicology. 1997;120:145–155.[Medline] [Order article via Infotrieve]
56. Cornish KG, Iversen P, Smith L, Arneson M, Bayever E. Cardiovascular effects of a phosphorothioate oligonucleotide with sequence antisense to p53 in the conscious rhesus monkey. Pharmacol Commun. 1993;3:239.
57. Henry SP, Giclas PC, Leeds J, Pangburn M, Auletta C, Levin AA, Kornbrust DJ. Activation of the alternative pathway of complement by a phosphorothioate oligonucleotide: potential mechanism of action. J Pharmacol Exp Ther. 1997;281:810–816.[Abstract/Free Full Text]
58. Crooke ST. Progress in antisense therapeutics. Hematol Pathol. 1995;9:59–72.[Medline] [Order article via Infotrieve]
59. Tomita N, Morishita R, Higaki J, Tomita S, Aoki M, Kaneda Y, Ogihara T. Role of angiotensinogen in blood pressure regulation in normotensive rats: application of a "loss of function" approach. J Hypertens. 1995;131:767–1774.
60. Fukamizu A, Takahashi S, Seo S, Tada M, Tanimoto K, Uehara S, Murakami K. Structure and expression of the human angiotensinogen gene: identification of a unique and highly active promoter. J Biol Chem. 1990;265:7576–7582.[Abstract/Free Full Text]
61. Tamura K, Umehara S, Ishii M, Tanimoto K, Murakami K, Fukamizu A. Molecular mechanism of transcriptional activation of angiotensinogen gene by proximal promoter. J Clin Invest. 1994;93:1370–1379.
62. Mann MJ, Whittemore AD, Donaldson MC, Belkin MA, Orav EJ, Polak J, Dzau VJ. Preliminary clinical experience with genetic engineering of human vein grafts: evidence for target gene inhibition [abstract]. Circulation. 1997;96(suppl I):I-4.