JP2001095573A - Regulation of transcription of gene in vascular cell - Google Patents

Abstract

PROBLEM TO BE SOLVED: To target a transcription factor which is a suitable target for squelching by a double-stranded nucleic acid having a specific sequence for regulating the transcription in a vascular cell, etc., such as an endothelial and a smooth muscle cells and binding the transcription factor. SOLUTION: This method for regulating the transcription of one or more genes in a vascular or a cardiac cell comprises a stage of bringing the cell into contact with a composition comprising one or more double-stranded nucleic acids sequence-specifically bindable to the transcription factor AP-1 and/or CCAAT/enhancer-binding protein (C/EBP) or the related transcription factor. The endothelial cell is a part of a blood vessel or a blood vessel graft. The blood vessel graft is brought into contact with the composition in vivo or ex vivo.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

FIELD OF THE INVENTION The present invention relates to a method for regulating one or more genes in a blood vessel or a heart cell, which sequence-specifically binds the transcription factor AP-1 and / or C / EBP or a related transcription factor. Contacting with a composition comprising one or more double-stranded nucleic acids.

[0002]

BACKGROUND OF THE INVENTION Coronary heart disease (CHD) is a leading cause of death in developed countries. Over the past few decades, aortic coronary vein bypass surgery and percutaneous transluminal angioplasty (PTCA) have been increasingly performed for the treatment of coronary heart disease, resulting in survival rates and the lives of patients with heart disease. The quality was substantially improved.

[0003] Although these methods have been fully established for decades, the likelihood of relapse or complete occlusion of the treated vessel is high, and this complication appears within a few months after surgery. Often. This is a serious medical problem and an economic burden on the insurance system in developed countries.

[0004] One of the main reasons for the complications seen in PTCA and coronary artery bypass surgery appears to be the induction of cell migration and proliferation of the vessel wall. This poor and undesired growth of vascular tissue leads to a remodeling process, which eventually leads to a relapse or reocclusion of the surgically treated blood vessels.

[0005] In order to prevent post-surgical regression or occlusion of the treated vascular segment, a recently used method is to preserve the postoperative vascular shape and diameter after stents or PTCA. Implantation of a designed tubular structure. However, this method is also complicated by mechanical strain on the vessel wall, which results in excessive proliferation and migration of smooth muscle cells, resulting in in-stent restenosis. Furthermore, if these side effects due to the intervention of surgery cannot be suppressed efficiently,
Some clinical studies using various adjuvant medications.

The consequence of a therapeutic failure is the (re) occlusion of the myocardial blood vessels, recurring ischemic disease and, in severe cases, myocardial infarction. This causes massive death of cardiomyocytes in parts of the heart muscle due to insufficient oxygen supply,
Non-contractile scar tissue remains. Thus, inducing vascular growth and / or inducing proliferation of cardiomyocytes in the ischemic portion of the myocardium may provide a possible therapy for the treatment of MI.

[0007] Most cells in the body are in the G ₀ phase of the cell cycle, called a stationary state. Almost all quiescent somatic cells still have the ability to proliferate and can be triggered to re-enter the cell cycle with many stimuli, the most important of which are growth factors and damage.
The growth and remodeling processes are controlled primarily by the level of transcription. For example, due to the physical stress of coronary angiogenesis and stent implantation, a number of genes, most importantly, cyclins, cell cycle specific phosphatases, cell cycle specific transcription factors such as cyclin E, cyclin A, cyclin B, cdc25C , Cdc25A, E
A member of the 2F-class, as well as a number of metabolically important genes,
Alternatively, it induces genes involved in DNA doubling, such as PCNA, histones and dhfr. Although these factors are newly synthesized when entering the cell cycle, many transcription factors involved in the first stage of growth, the so-called immediate-early genes, are already present in the cell and are activated by predetermined stimuli. A known product of this type is AP-1.

[0008] AP-1 is a c-Jun and c-Fos protein (Curra) that interact with each other via a leucine zipper motif.
n and Franza (1988)). Homodimerizes and binds DNA alone
Unlike c-Jun, c-Fos changes its sequence specific for binding to DNA by interaction with c-Jun.
Both proteins are members of a large class of proteins including JunB and JunD (Jun-related) and Fra1 and FosB (Fos-related) (Curran and Vogt (1992) Transcriptional regulation, 797 (McKnight and
Yamamoto) ColdSpring Harbor Laboratory Press). AP
-1 can bind to the consensus sequence TGACTCA motif, but many variants of these sequences can bind strongly by various homo- and heterodimers of this class of members (Franza et al. 1988) Scienc
e 293, 1150; Rauscher et al. (1988) Genes Dev. 2,
1687; Risse et al (1989) EMBO J. 8, 3825; Yang-Yen
et al. (1990) New Biol. 2, 351).

Co-expression of Fos and Jun can dramatically synergistically activate AP-1-dependent transcription (Chiu et a
l. (1988) Cell 54, 541), a substantial degree of experimental variability was observed depending on the type of cells used. Therefore, Fo
s and Jun activities can be expressed specifically in cell types (Baichwal and Tjian (1990) Cell 63, 815), and in each cell type, resident or inducible transcription factors select gene targets. And may be affected by other proteins that may affect the transcriptional activity of hetero- and homo-dimers of the Fos-Jun family. Consistent with the promoter and cell type specificity of AP-1, Fos has c-fos
It has been shown to not activate, but rather repress, promoter transcription (Sassone-Corsi (1988) Cell 54, 553; Luci
bello etal. (1989) Cell 59, 999). Thus, for a given cell type, it is not possible to predict what effect AP-1 will have on a specific promoter.

A method for specifically preventing transcriptional activation by a specific transcription factor is disclosed in WO 95/11687. This is the cis element decoy (cis-eleme
nt decoy), and teaches that treatment of cells with a double-stranded DNA molecule that has a binding aspect for its specific transcription factor can interfere with the activation function of the transcription factor. Due to the external supply of a large number of transcription factor binding sites to cells, preferentially contained in endogenous promoters in the genome, the majority of a given transcription factor is more likely to be in each cis-site than the endogenous target gene. A state of specifically binding to the element decoy is brought about. This method of inhibiting the binding of a transcription factor to its endogenous binding site is also called squelching. Using squelching of transcription with DNA decoy, transcription factor E2
F (Morishita et al. (1995) 92,5855) was successfully used to inhibit cell growth using a DNA fragment that specifically targets.

The fact that about 50% of all human genes are transcription factors is estimated by extrapolating the generally known results of the Human Genome Project. It is known that the complexity and diversity of living organisms is largely caused by the restriction of the expression of certain transcription factors to specific cell types and / or specific stages of development. A transcription factor that has a positive effect on growth in one cell type may have the opposite effect in another cell type or another species.
Thus, the effect of treatment with a given transcription factor decoy is not only between species, but also between various tissues, such as veins and arteries, and the cell type of a given tissue, such as the endothelium of one type of in vivo vascular wall. It can also vary between cells and smooth muscle cells.

[0012]

SUMMARY OF THE INVENTION Accordingly, it is an object of the present invention to regulate transcription in vascular cells such as endothelial and smooth muscle cells, or cardiac cells, and to have specific sequences for binding transcription factors. The targeting of a transcription factor that is a suitable target for decoy (squelching) by the present strand nucleic acid. Preferred transcription factor targets are those transcription factors that are directly or indirectly involved in inducing proliferation and / or remodeling vascular and / or myocardial tissue.

Surprisingly, endothelin-1 (ET-
1) The gene was found to be activated by AP-1 in venous endothelial cells. This activation is caused by extremely extreme mechanical stress. The ET-1 peptide produced by this stimulation is extremely effective in promoting vasoconstriction and growth. ET-1 release was correlated with smooth muscle cell overgrowth as well as remodeling. Therefore, in endothelial cells
It could be shown that activation of the ET-1 gene can be blocked by introducing double-stranded DNA having a binding site for AP-1.

Accordingly, the method of the present invention is directed to the regulation of the transcription of at least one gene in endothelial or cardiac cells, wherein said cells bind sequence-specifically to the transcription factor AP-1 and / or a related transcription factor. Contacting with a composition comprising one or more double-stranded nucleic acids. The method is used in particular on endothelial cells, which are part of a blood vessel or a section of a blood vessel. Typically, this method can be used on vascular sections in vivo, on blood vessels, or before or after transplantation or in vivo or ex vivo before transplantation. Preferably, the heart cells are cardiomyocytes or fibroblasts.

Another unexpected result of the present invention is that CCAAT / enhancer binding protein (C / EBP) is involved in ET-1 activation in cervical aortic smooth muscle cells after mechanical stress. It was found to be doing. Consistent with the aforementioned specific effects of transcription factors in various tissues, AP-1 was not involved in ET-1 gene activation in this cell type. When the C / EBP-specific double-stranded nucleic acid was introduced into smooth muscle cells, the activation of the ET-1 gene could be blocked.

C / EBP is used for normal cell differentiation in various tissues.
And a type of transcription factor important for function (Leks
trom-Himes and Xanthopoulos (1996) J. Biol. Chem.,
 44,28645). Binds only to DNA during dimerization
There are at least six types of C / EBP (Cao et al. (1991) Ge
nes Dev. 5, 1538). C / EBPs, especially in the liver
Are being considered for their role in liver damage.
Involvement in the acute phase response after
It is known (Progress in Liver
 Diseases) (Popper and Schaffner) Vol. 9, 89, W.B.
 Saunders Co., Fey et al. (1990) in Philadelphia
It is described in). C / EBPs are mainly involved in the inflammatory response
It has also been shown to play an important role,
The combined motifs are interleukin-6 (IL-6), I
L-1_β, And tumor necrosis factor _α(INF_α)like
Inflammatory cytokines and IL-8 and IL-12
Found in promoters of other cytokines such as
(Matsusaka et al. (1993) Proc. Natl. Acad. Sc
i. 90, 10193; Plevy et al. (1997) Mol. CellBiol. 1
7, 4572). Promotes vascular tissue growth and remodeling
ET-1, a factor that is known to be
Involvement of C / EBP in other suitable transcription factors in vascular cells
Aiming for target.

Thus, another method of the invention is the regulation of the transcription of one or more genes in vascular or cardiac cells, which binds the cells to the transcription factor C / EBP or related transcription factors in a sequence-specific manner. Contacting with a composition comprising one or more double-stranded nucleic acids.

[0018]

DETAILED DESCRIPTION OF THE INVENTION Individual transcription factors can often independently activate a promoter to a certain extent, but complete activation of this promoter is altered by synergistic activation by two or more transcription factors. It has been known
(Sauer et al. (1995) Science 270,1783). Thus, for stronger promoter regulation than with a double-stranded nucleic acid targeted to a single transcription factor, the method of the present invention may be used to specifically bind to C / EBP and to further regulate the transcription factor AP. It is also conceivable to use a composition comprising one or more sequences which also comprises one or more double-stranded nucleic acids capable of binding sequence-specifically to -1. According to this combination, for example, the transcription of a promoter comprising the AP-1 and C / EBP binding sites can be more strongly regulated or can act independently on two or more promoters simultaneously.

When using the method of the present invention for vascular cells, the preferred targets are smooth muscle cells (SMC) or endothelial cells, and for cardiac cells, the preferred targets are cardiomyocytes or fibroblasts. These cells can be processed directly in situ, for example, in animal or human blood vessels, or can be part of an autologous or xenovascular graft. These grafts, such as arterial or venous bypass grafts, can be treated before transplantation by ex vivo application of the method of the invention or after transplantation by in vivo application of the method of the invention. In a preferred embodiment, the treated vessel is a coronary or peripheral artery or an arteriovenous fistula (eg, a Brescher-Chymino fistula) and the vascular graft is an arterial or venous bypass graft, or a biological or prosthetic arteriovenous shunt. It is.

To prevent occlusions caused by surgical procedures and pathological processes, it is conceivable to specifically regulate the expression of genes involved in vascular cell proliferation or migration. This regulation can be a direct effect on the promoter of the gene by squelching the binding of AP-1, C / EBP or related proteins, or it itself regulates genes involved in vascular cell proliferation or migration It can be an indirect effect by preventing the regulation of transcription factors. Genes involved in growth and migration include genes encoding intracellularly active proteins such as cyclins, cyclin-dependent kinases, apoptosis inhibitors, and extracellular secretions such as metalloproteinases, collagenases, or growth factors And thus genes encoding proteins that are active extracellularly. In particular, growth factors secreted by one vascular cell can stimulate the growth and migration of many other vascular cells. Thus, the regulation of genes acting outside of the targeted cell is of particular importance for the present invention.

The sequence of the nucleic acid used to inhibit the binding of transcription factor AP-1, C / EBP or related transcription factors is 5'-CGC
TTGATGACTCAGCCGGAA-3 '(for AP-1) or 5'-TGCA
A consensus sequence derived from the sequence of the AP-1 or C / EBP site of many promoters, such as GATTGCGCAATTG-3 '(for C / EBP), or specific promoters targeted for transcriptional regulation purposes Or it is the same as the AP-1 or C / EBP site in the enhancer. As noted above, AP-1 and C / EBP are a class of closely related transcription factors that can be hetero- or homo-dimerized (Roman et al. (1990) Genes Dev. Four,
1404; Cao et al. (1991) Genes Dev. 5, 1538). There are many possible heterodimers that appear to recognize different true AP-1 or C / EBP binding sites with different affinities. Thus, slightly different AP-1 or C / EBP binding sites contained in various promoters can be bound only by certain hetero- or homodimers of AP-1, C / EBP or related transcription factors ( Akiro et al. (1990) EMBO J. 9, 1897, R
isse et al. (1989) ibid). Therefore, by using a nucleic acid designed specifically for a promoter, targeted squelching of a transcription factor involved in the control of the specific promoter can be performed. If the promoter contains more than one version of the AP-1 and / or C / EBP binding sites, as is often the case, the nucleic acids representing the sequences of all the binding sites may be used simultaneously. it can. Similarly, one or more double-stranded nucleic acids containing slightly different types of consensus transcription factor binding sites can be used simultaneously.

The affinity of a nucleic acid sequence for binding to AP-1, C / EBP or a related transcription factor can be determined using electrophoretic mobility shift analysis (EMSA) (Sambroo).
k etal. (1989) Molecular clonin
g), Cold Spring Harbor Laboratory Press; Krzesz et
al. (1999) FEBS Lett. 453, 191). This assay is suitable for quality control of nucleic acids for use in the method of the invention or for determining the optimal length of a binding site. This is AP-
1, also used to identify other sequences bound by C / EBP or related transcription factors. The most suitable for such EMSA for the isolation of novel binding sequences is the purification of AP-1, C / EBP or related transcription factors used for several alternating PCR amplifications and selection by EMSA Or recombinant expression (Thiesen and Bach (1990) Nucle
ic Acids Res. 18, 3203).

As mentioned above, genes containing binding sites for AP-1, C / EBP or related transcription factors are suitable targets for the method of the present invention as well as genes that are indirectly affected. Genes encoding proteins involved in vascular tissue growth and / or remodeling and other pathological processes include endothelin genes, macrophage chemotaxis (MCP) genes, and nitric oxide synthase (NOS) genes. Yes, examples of these genes that are particularly important for vascular cells are the prepro-endothelin-1 gene, the MCP-1 gene and the inducible NOS gene.

AP in the promoter or enhancer region
Genes known to contain a -1 binding site, and thus are putative targets for specific squelching according to the methods of the present invention, include, for example, the prepro-endothelin-1 gene,
Endothelin receptor B gene, inducible NOS (iNOS) gene, E-selectin gene, monocyte chemotactic protein-
1 (MCP-1) gene, intercellular adhesion molecule-1 (ICAM)
-1) gene and interleukin-8 (Il-
8) It is a gene.

C / C in the promoter or enhancer region
Genes known to contain an EBP binding site and thus putative targets for specific squelching by the methods of the invention include, for example, the prepro-endothelin-1 gene, the endothelin receptor B gene, the iNOS gene, the ENOS gene,
-Selectin gene, intercellular adhesion molecule-1 (ICAM-
1) Gene, Il-8 gene, and Il-6.

The method of the present invention regulates the transcription of a gene or genes in such a way that the gene or genes are activated. Activation, according to the present invention, means that the transcription rate is increased compared to cells that have not been treated with a composition comprising double-stranded nucleic acid. Such an increase can be detected, for example, by Northern blotting (Sambrook et al. (1989) supra) or RT-PCR (Sambrook et al. (1989) supra). Typically, such an increase is at least 2-fold, more preferably at least 5-fold, at least 20-fold, and most preferably at least 100-fold. For example, activation occurs when AP-1 and / or C / EBP or a related transcription factor acts as a transcriptional repressor for a particular gene and thus squelching of this repressor abolishes repression. Loss of repression is not necessarily equated with activation, but for some promoters, loss of repressor binding is known to be sufficient for activation (Zwi
cker et al. (1995) EMBO J. 14, 4514).

The method of the present invention also regulates the transcription of a gene or genes in such a way that the gene or genes loses or is reactivated. Inhibition, in the context of the present invention, means that the transcription rate is reduced compared to cells that have not been treated with a composition comprising a double-stranded nucleic acid. Such an increase can be achieved, for example, by Northern blotting (Sambrooket).
al. (1989) supra) or RT-PCR (Sambrook
et al. (1989) supra). Typically, such a reduction is at least 2
Fold, more preferably at least 5 fold, at least 20 fold, and most preferably at least 100 fold reduction. For example, if AP-1 and / or C / EBP or a related transcription factor acts as a transcriptional repressor for a particular gene and thus squelching of this activator results in loss or repression of activation, Loss or suppression occurs.

In another embodiment of the invention, the modulation activates one gene or genes while simultaneously losing or suppressing the activation of another gene or genes. Differential effects on individual genes can be determined, for example, by Northern blotting (Sambrook et al. (1989) supra) or RT-PCR (Sambrook et al. (1989) supra) and DNA chip array methods (US Pat. 5,8
37,466).

The double-stranded nucleic acid used in the method of the present invention, in a preferred embodiment, comprises one or more copies of a sequence that specifically binds to AP-1 and / or C / EBP or a related transcription factor. Since synthetic nucleic acids are typically at most 100 bp in length, they can comprise from 1 to 10 complete transcription factor binding sites, depending on the length of the specific transcription factor recognition site selected. Nucleic acids amplified by an enzymatic method such as PCR or grown in a suitable prokaryotic or eukaryotic host
Each transcription factor binding site contains at least about 10 copies, preferably at least about 30 copies, at least about 100 copies, or at least about 300 copies.

The double-stranded nucleic acid used in the method of the present invention is, for example, a vector or an oligonucleotide. In a preferred embodiment, the vector is a plasmid vector, in particular capable of autosomal replication (Wohlgemuth et al.
(1996) Gene Ther. 6, 503-12), which is a plasmid vector capable of increasing the stability of the double-stranded nucleic acid introduced thereby. The length of the double-stranded oligonucleotide is
Typically, it is selected to be about 10-100 bp, preferably about 20-60 bp, most preferably 30-40 bp.

Oligonucleotides are susceptible to rapid degradation by endo and exonucleases, especially DNase and RNase in cells. Therefore, in order to maintain a high concentration of nucleic acid in cells for a long time, the nucleic acid is modified to be stable against degradation. Typically, such stabilization is effected by means of one or more modified internucleotides.
cleotide) by introducing a phosphate residue or by introducing one or more "dephospho" intervening nucleotides.

A well stabilized nucleic acid does not necessarily include such intervening nucleotides at every nucleotide. It is possible to introduce different modifications to the nucleic acid and test the resulting double-stranded nucleic acid for sequence-specific binding to AP-1, C / EBP or related proteins using conventional EMSA assays. This assay can determine the binding constant of the nucleic acid and determine whether the affinity has changed due to the modification. A modified nucleic acid that exhibits sufficient binding can be selected, but sufficient binding means at least about 50%, or at least about 75%, and most preferably about 100%, of the binding of the unmodified nucleic acid.

Nucleic acids that show sufficient binding are tested if they are more stable in cells than unmodified nucleic acids.
In the in vitro assay system used, the modified nucleic acid is introduced into cells using the method of the present invention. The transfected cells were then removed at various time points and analyzed by Northern blot (Sambrook et al. (1989) supra), Southern blot (S
ambrook et al. (1989) supra), PCR (Sambrook et al.
(1989) supra), RT-PCR (Sambrook et al. (1989)
Or the DNA chip array method (US Pat. No. 5,8,8).
37,466), the amount of residual nucleic acid can be analyzed. For a well-modified nucleic acid, the half-life in the cell is at least 48 hours, more preferably at least about 4 days, and most preferably at least about 4 days.
14 days.

A suitable modified intervening nucleotide is Uhlm
Ann and Peyman ((1990) Chem. Rev. 90, 544). Modified intervening nucleotide phosphate residues and / or "dephospho" bridges that may be included in the nucleic acids used in the methods of the present invention are, for example, methylphosphonates, phosphorothioates, phosphorodithioates, phosphoramidates, phosphate esters. , "Dephospho" intervening nucleotide analogs include, for example, siloxane bridges, carbonate bridges, carboxymethyl ester bridges, acetamidodate bridges, and / or
Or containing a thioether bridge. It is also believed that this type of modification increases the shelf life of the composition used in the method of the present invention.

Another aspect of the invention is the stabilization of nucleic acids by introducing structural properties into the nucleic acids that increase the half-life of the nucleic acids. U.S. Pat. No. 5,683,985 discloses structures such as hairpins and dumbbell DNA. It is also conceivable to introduce modified intervening nucleotide phosphate residues and / or "dephospho" bridges simultaneously with these structures. The nucleic acid to be produced is
Binding and stability can be tested in the analytical systems described above.

The composition comprising the double-stranded nucleic acid is contacted with vascular cells or heart cells. The intention of this contact is AP
Transfer of double-stranded nucleic acid that binds to -1 and / or C / EBP or a related transcription factor into a cell, especially into the nucleus of the cell. Thus, nucleic acid modifications and / or additives or auxiliaries known to increase the permeability of membranes are also contemplated for use in the present invention (Uhlmann and Peyman (1
990) Chem Rev. 90, 544).

The composition according to the invention comprises, in a preferred embodiment, essentially only nucleic acids and buffers. This method uses a high concentration of double-stranded nucleic acid to efficiently absorb the nucleic acid into cells, and finally to a high concentration of nucleic acid in the nucleus. A suitable concentration of nucleic acid is at least 1 μM, more preferentially 10 μM, most preferably 50 μM.
μM, to which one or more suitable buffers have been added. One example of such a buffer is 144.3 mmol / l Na ⁺ , 4.0 mmol / l K ⁺ , 138.6 mmol / l
Of Cl ^{-, Ca 2+} 1.7 mmol / l, 1.0 mmol / l ^{Mg 2+} of, HPO ₄ ^2-of 0.4 mmol / l, 19.9 mmol / l of HCO ₃ ^-, including the D- glucose 10.0 mmol / l Tyrode solution.

In another embodiment of the present invention, the composition also comprises at least one additive and / or auxiliary. Additives and / or auxiliaries such as lipids, cationic lipids, polymers, nucleic acid aptamers, peptides and proteins increase the transfer of nucleic acids into cells and target the composition to only a subset of cells It is intended to prevent degradation of the nucleic acid inside the cell, facilitate storage of the nucleic acid composition before application, and / or improve the transfer of the nucleic acid to the nucleus of the cell.

Auxiliaries for increasing the transfer of nucleic acids into cells include, for example, DNA which facilitates transfer of nucleic acids to the nucleus of cells.
Alternatively, it can be a protein or peptide attached to a synthetic peptide-DNA molecule (Schwartiz et al.
al. (1999) Gene Therapy 6, 282; Branden et al. (19
99) Nature Biotechnology 17, 784). As auxiliaries,
Molecules that facilitate release of nucleic acids into the cytoplasm of cells (Planck
et al. (1994) J. Biol. Chem. 269, 12918; Kichler et.
al. (1997) Bioconjug. Chem. 8, 213) or, for example, liposomes (Uhlmann and Peyman (1990) supra).

In order to specifically target the composition to a subset of cells, an auxiliary agent is selected to recognize a protein of the target cell, preferentially a protein or protein domain exposed outside the cell. Can be. Specific targeting can also be described as specific recognition of a cell. There are at least two types of auxiliaries that are suitable for performing this recognition. One type of auxiliaries comprises antibodies or antibody fragments that preferentially recognize proteins or protein domains located on the extracellular part of the cell membrane. Antibodies against such a membrane structure of endothelial cells are described, for example, in Burr.
ows et al. ((1994) Pharmac. Ther. 64, 155), Huges
et al. ((1989) Cancer Res. 49, 6214) and Maruyama
et al. ((1990) Proc. Nat. Aca. Sci. 87, 5744). Antibodies to be used are directed against the membrane structure of smooth muscle, for example-antibody 10F3 (Printseva et al. (1987) Exp. Cell Re
s. 169, 85-Antibody to actin (Desmoliere et al. (1988)
Comptes Reudus des Seances de la Soc. De Biol et d
e ses Filiales 182, 391)-an antibody against angiotensin II receptor (Butch
er et al. (1993) BBRA196, 1280-antibodies against growth factor receptors (Reviews in Me
ndelsohn (1988) Prog. All. 45, 147; Sato et al. (19
89) J. Nat.Canc.Inst. 81, 1600, Hynes et al. (199
4) BBA 1198, 165) or e.g.-EGF receptor (Fan et al. (1993) Cancer Res. 5
3, 4322; Bender et al. (1992) Cancer Res. 52, 121;
Aboud-Pirak et al. (1988) J. Nat.Cancer Inst. 80,
1605)-PDGF receptor (Yu et al. (1994) J. Biol. Chem.
269, 10668; Kelly et al. (1991) 266, 8987)-FGF receptor (Vanhalteswaran et al. (1991) J.
Cell Biol. 115, 418, Zhan et al. (1994) J. Biol. C
269, 20221).

Another class of auxiliaries are ligands which bind with high affinity to cell surface receptors. Such a ligand may be a natural ligand of the receptor,
Alternatively, it can be a modified ligand with higher affinity. Typically, such ligands are peptides known to bind to a given membrane receptor. Furthermore, a novel peptide ligand may be isolated using a receptor of interest in screening a combinatorial peptide library (Lu, Z. et a.
l. (1995) Biotechnol. 13, 366; US Patent No. 5,63.
No. 5,182; Koivunen, E. et al. (1999) J. N.
ucl. Med. 40, 883).

[0042] The membrane receptors expressed on endothelial cells that can be targeted by the ligand, for example PDGF, bFGF, VEGF and TGF _beta and the like (Pusztain et al. (1993) J. Pathol. 169, 19
1). Additionally, the ligand may include an adhesion molecule that binds to proliferating / migrating endothelial cells. This kind of adhesion molecule,
For example, SLex, LFA-1, MAC-1, LECAM
-1 or VLA-4 has already been reported (Augst
ein-Voss et al. (1992) J. Cell Biol. 119, 483; Pau
li et al. (1990) Cancer Metast. Rev. 9, 175; Honn
et al. Cancder Metast. Rev. 11, 353). However, this ligand is not limited to peptide ligands, but can also be a nucleic acid-aptamer that specifically recognizes the cell surface of target cells (Hicke, BJ et al.
al. (1996) J. Clin. Invest. 98, 2688).

[0043] As the membrane receptor expressed in smooth muscle cells that can be targeted by the ligand, for example PDGF, EGF, TGF _alpha, FGF, endothelin A, and _{TGF β (Pusztain et al. (} 1993) J. Pa
thol. 169, 191; Harris (1991) Current Opin. Biotech
nol. 2, 260).

Additives that stabilize nucleic acids in cells are nucleic acid condensing substances such as, for example, cationic polymers, poly-L-lysine or polyethyleneimine.

The composition used in the method of the present invention comprises injection, catheter, trocar, projected pluronic gel (p
rojectiles pluronic gels), drug-release polymers, coated stents, or any other device that provides a localized passageway, and is preferentially applied locally. Ex vivo application of the composition used in the method of the invention also provides a localized passage.

Another embodiment of the present invention is a method of treating a vascular disease, preferably a vascular proliferative disease, wherein said composition comprises vascular cells in an amount sufficient to regulate the transcription of one or more genes. And contacting with the method. Diseases that may be treated using this method include, for example, restenosis, intimal hyperplasia of arterial or venous bypass grafts, graft vasculopathy, and hypertrophy. In addition, this method is, for example, PTCA,
It can also be used as both an adjunct to treatments commonly used in vascular diseases such as PTA, bypass surgery, or the application of arteriovenous shunts. The scope of the present invention includes diseases known to be the result of vascular diseases such as coronary heart disease and myocardial infarction.

The method of the present invention is also intended to be used as an in vitro method. For example, AP-1 and / or C /
An in vitro screening method that can identify genes that are regulated in vascular cells by EBP or related proteins. Such a method comprises combining the cell with one or more double-stranded nucleic acids capable of sequence-specifically binding the transcription factor AP-1 and / or C / EBP or a related protein. including in vitro contact. The transcript levels of thousands of genes were then simultaneously observed, with and without the composition, using, for example, the DNA-chip array method described in US Pat. No. 5,837,466, and compared. We were able to.

Another embodiment of the present invention relates to a transcription factor AP-
1, or SEQ ID NO: 5, or sequence: 5′CTGTTGGTGACTAAT
AACACA3 '(designed AP-1); 5'CTGTGGGTGACTAATCAC
ACA3 '(designed AP-1); 5'GTGCTGACTCAGCAC3' (designed AP-1); 5'CGCTTAGTGACTAAGCG3 '(designed AP-1); 5'TGTGCTGACTCAGCACA3' (designed AP-
1); 5'TTGTGCTGACTCAGCACAA3 '(designed AP-1);
5'TCGCTTAGTGACTAAGCGA3 '(designed AP-1); 5'TGC
TGACTCATGAGTCAGCA3 '(designed AP-1); 5'TGCTGAC
TAATTAGTCAGCA3 '(designed AP-1); 5'GTCGCTTAGTG
ACTAAGCGAC3 '(designed AP-1); 5'CTTGTGCTGACTCA
GCACAAG3 '(designed AP-1); 5'TTGCTGACTCATGAGTC
SEQ ID NOS: 9-20 having AGCAA3 '(designed AP-1)
Is a double-stranded nucleic acid capable of sequence-specifically binding to a related transcription factor having one of the following sequences:

[0049] Another embodiment of the present invention relates to the transcription factor C / EB.
P, or SEQ ID NO: 6, or sequence: GCTTGTGCGGGAATAAA
TAG3 '(designed C / EBP); 5'AGGAATAATGGAATGCCCTG
3 '(designed C / EBP); 5' GACATTGCGCAATGTC3 '(designed C / EBP); 5' AGCATTGGCCAATGCT3 '(designed C / EBP); 5' CGACATTGCGCAATGTCG3 '(designed C
/ EBP); 5'AGGCATTGGCCAATGCCT3 '(designed C / EB
P); 5'ACGACATTGCGCAATGTCGT3 '(designed C / EB
P); 5 'TAGGCATTGGCCAATGCCTA3' (designed C / EB
P); 5'CTGTTGCGCAATTGCGCAACAG3 '(designed C / EB
P); 5'GACTTGCGCAATTGCGCAAGTC3 '(designed C / EB
P) is a double-stranded nucleic acid capable of sequence-specifically binding to a related transcription factor having one of the sequences of SEQ ID NOs: 21-30.

[0050] The double-stranded nucleic acid of the present invention is 12 to 22 nucleotides in length. The optimal length of a given nucleic acid is selected to optimize binding to transcription factors and absorption into cells. Typically, double-stranded nucleic acids shorter than 12 bp are
It binds very weakly to its target protein; those longer than 22 bp bind strongly, but are less efficiently absorbed by cells.
The avidity can be measured by EMSA, and the incorporation of double-stranded nucleic acid is determined by Northern blotting (Sambrook et al.
(1989) supra), Southern blotting (Sambrook e).
t al. (1989) cited reference), PCR (Sambrook et al.
(1989) supra), RT-PCR (Sambrook et a
l. (1989) cited reference), or a DNA chip array (US Pat. No. 5,837,466). The nucleic acids of the invention can be stabilized by the methods described above.

A preferred embodiment of the present invention is a nucleic acid comprising a palindromic binding site, whereby the short double-stranded nucleic acid comprises at least two transcription factor binding sites. The double-stranded nucleic acid designed by this method comprises AP-1, and / or
Or increased statistical probability of binding to C / EBP or related transcription factors. To facilitate the design of the palindromic binding sequence, the palindromic core sequence (AP-1: TGAC
And C / EBP: GCAA) can be tolerated on one side. Preferably, the core sequence can be placed in the center of the nucleic acid to optimize binding to AP-1 and / or C / EBP or a related transcription factor.

The nucleic acid of the present invention is rapidly internalized in cells. Sufficient uptake is characterized by the regulation of genes or genes that can be regulated by AP-1 and / or C / EBP or related transcription factors.
The double-stranded nucleic acid of the present invention can be used after about 2 hours of contact with a cell.
More preferably, after about 1 hour, about 30 minutes, about 10 minutes, most preferably about 2 minutes, the transcription of the gene or genes is regulated. A typical composition used in such an experiment comprises 10 μM / l of double-stranded nucleic acid.

The following figures and examples are merely illustrative and do not limit the scope of the invention.

FIG. 1: (A) Endothelialization on ppET-1 mRNA abundance in isolated sections of rabbit carotid artery (RbCA) or rabbit jugular vein (RbJV) perfused for 6 hours at various intravascular pressures, (B, E) Ro 31
-8220 (0.1 μmol / l), (C, F) Herbimycin A (0.1 μmol
Mol / l), and (D) actinomycin D (0.1 μmol /
l) effect. In a typical RT-PCR analysis, similar findings were obtained in at least four additional experiments using sections from different animals.

FIG. 2: Time course increase during nuclear translocation of (A) AP-1 and (B) C / EBP in endothelium-complete sections of RbJV perfused at 20 mmHg. In a typical EMSA analysis,
Similar results were obtained in at least two additional experiments.

FIG. 3: RbCA perfused for 6 hours at various tube pressures
Or (B, D) C / EBPmut, (C) GATA-2, and (D) C / EB on ppET-1 mRNA abundance in RbJV endothelium-complete sections
Specific decoy-oligodesoxynucleotides for P
(dODN) effect. (E, F) ppET in PAEC (primary culture) incubated or extended for 6 hours under static conditions
-1 mRNA abundance (E) Ro 31-8220 (0.1 μmol / l)
And the effect of Herbimycin A (0.9 μmol / l). In a representative RT-PCR analysis, similar findings were obtained in at least two additional experiments using sections or cells from different animals.

FIG. 4: RbCA perfused for 6 hours at various intraluminal pressures
(C) or (A, C) ppET-1 mRNA in isolated sections of RbJV (A, B)
Effect of specific decoy ODN on abundance (n = 3-5) and (B) intravascular ET-1 peptide (n = 3) on AP-1 and C / EBP alone or in combination. * P <0.05 for 0 or 2 mmHg, P for # 20-160 mmHg
<0.05, P <0.0 for +20 mmHg and C / EBP dODN
Five.

FIG. 5: (A, B) (A) Absence or absence of various dODNs
(B) Various rat ppET-1 promoter-luciferase constructs in cultured PAEC incubated or extended for 6 hours under quiescent conditions in the presence (stars represent -168 bp constructs without AP-1 response element). (Indicated by +; n = 3-7, measured twice). (C) β-galactosidase activity in cultured PAEC co-transfected with the SV40 / β-galactosidase expression vector and various rat ppET-1 promoter-luciferase constructs as a similar indicator of transfection efficiency (n =
3-7, measured twice).

FIG. 6: Schematic representation of the putative transcription factor binding site in the promoter of the rat ppET-1 gene, further indicating the length of various promoter constructs.

[0060]

EXAMPLE 1 In situ model Male New Zealand white rabbit (2.1 ± 0.1 kg body weight, n =
47) with pentobarbital (60 mg / kg iv; Sigma-Aldr
ich, Deisenhofen, Germany) and phlebotomy. The left and right common carotid artery (RbCA) and the external jugular vein (RbJV) were dissected, the adventitia fat and connective tissue were washed and cut in half. Specially designed quartet perfusion chamber (f
It was placed in our-position perfusion chamber and extended to its original length (20.6 ± 0.3 mm) by a movable cannula.
The diameter was continuously observed with a video microscope (Visitron Instruments, Munich, Germany). The tissue baths in the lumen of the section and surrounding tissue baths were prepared with a composition of 144.3 mmol / l Na +, 4.0 mmol / lK +, 138.6 mmol / lC
l-, 1.7 mmol / l Ca2 +, 1.0 mmol / l Mg2
+, 0.4 mmol / l HPO42-, 19.9 mmol / l HC
O3-, 10.0 mmol / l D-glucose heating (37
° C) and oxygenated (lumen: 75% N ₂ , 20% O ₂ , 5
% CO ₂ , P _O2 = 140 mmHg, P _CO2 = 15-20 mmH
g, pH 7.4; bath: 95% CO ₂ , 5% O ₂ , P _{O 2} > 300
_mmHg, and perfused _{P CO2} = _13~38 mmHg, pH7.4) in Tyrode solution, respectively. For perfusion, use an IPC roller pump (Ism
atec, Bertheim, Germany) at a frequency of 1.33 Hz. Adjustable section after 30 min equilibration time
Perfusion was performed at 2,90 or 160 mmHg for 3-12 hours using a suitable afterload device (Hugo Sachs Elektronik, March, Germany).

[0061] 2. Cell culture Endothelial cells were cultured in Hepes-Tyrode solution at 1 U / mL dispase (dispa
se) and isolated from porcine aorta by treatment at 37 ° C. for 7 minutes, 10 U / mL nystatin, 50 U / mL penicillin, 50 μg / mL streptomycin, 5 mmol / L Hepe
s, DME containing 5 mmol / L TES and 20% fetal calf serum
Gelatin coated in M / Ham F12 (1: 1, v / v)
Culture in 60 mm culture dishes (2 mg / mL gelatin in 0.1 M HCl, 30 minutes at ambient temperature). 0.5% trypsin /
The cells were cultured once using 0.2% EDTA (w / v), and this was also coated with gelatin coated BioFlex ^™ Collagen I.
Plated 6-well plates (Flexcell, Hillsborough, North Carolina, USA). This was done using typical cobblestone morphology, positive immunostaining for von Willebrand factor (vWF), and negative immunostaining for smooth muscle α-actin (Krzesz et al. (1999)
453, 191).

3. RT-PCR analysis Frozen sections were minced using a mortar and pestle under liquid nitrogen. After isolation of total RNA using the Quiagen RNeasy kit (Quiagen, Hilden, Germany), up to 3 μg RNA and 200 U Sup in a total volume of 20 μl according to the manufacturer's instructions.
erscriptTM II reverse transcriptase (Gibco Life Technologies,
(Karlsruhe, Germany). Generate cDNA solution 5 for cDNA standardization
μl (about 75 ng cDNA) and the respective primers (Gibc
o) 20 picomoles were used for elongation factor 1 (EF-1) PCR with 1 U Taq DNA polymerase (Gibco) in a total volume of 50 μl. Elongation factor-1 (EF-1) was used as a standard for PCR. PCR products were separated on a 1.5% agarose gel containing 0.1% ethidium bromide, band intensities were measured with a densitometer, CCD-camera equipment and One-Dscan Gel analysis software from Scanaltytics (Billerica, Mass., USA). Was used to adjust the cDNA volume for the next PCR.

All PCR reactions were performed individually for each primer pair in a Hybaid OmnE thermocycler (AWG, Heidelberg, Germany). CD from PAEC
The individual PCR conditions for NA were as follows. ppET-1 product size 432bp, 30 cycles, annealing temperature 53 ° C (forward primer) 5'GGAGCTCCAGAAACAG
CTGTC3 ', (return primer) 5'CTGCTGATAAATACACTTCTTT
CC3 '(rat ppET-1 gene, corresponding to nucleotide sequence 233-664 of GenBank accession number M64711); EF-2 218 bp, 22
Cycle, 58 ° C, 5'GACATCACCAAGGGTGTGCAG3 ', 5'GCGGT
CAGCACAATGGCATA3 '(1990-2207, human EF-2, Z11692).

4. Intravascular ET-1 concentration ET-1 was determined by Hisaki et al. (1994) Am. J. Physiol.
422; Moreau et al. (1997) Circulation 96, 1593
The measurement was performed using a kit (Amersham, Freiburg, Germany).

5. Reporter gene analysis A partial sequence of the rat ppET-1 promoter amplified by PCR was blunt-end cloned into the Sma-1 restriction site of the multiple cloning region of the luciferase expression vector pCMV TK Luc + after removal of the CMV promoter (Paul et
al. (1995) 25,683). The pCMV TK Luc + construct with and without the CMV promoter was used as a control. Site-directed mutagenesis of the AP-1 and GATA sites on the -168 bp construct was performed using TransformerTM Site Directed
Mutagenesis kit (Clontech, Heidelberg, Germany) was used. Co-transfection to estimate transfection efficacy was performed using SV40 / β
-Galactosidase expression vector pUC19 (Paul et al.
(1995) supra).

For transfection, 40% confluent porcine aortic endothelial cells were transfected with 1.5 μg of plasmid DNA and 15 μg of Effectene ™ (Qiagen, Hilden,
(Germany) for 6 hours, the medium was changed and the cells were cultured until they were 80% confluent (usually after 18-24 hours). This was then incubated at rest and exposed to a 20% cyclic strain at 0.5 Hz for 6 hours in a Flexercell FX-3000 computerized stretcher. Luciferase and β in cell lysis products
-Galactosidase activity was measured using the corresponding chemiluminescence and photometric analysis kit (Promega, Mannheim, Germany) and normalized based on its protein content. Transfection efficiency was measured at the light microscopic level by staining cells with the β-galactosidase substrate, o-nitrophenyl-β-D-galactopyranoside (Lim and Cha (1989) Biotechniques 7, 576). .

6. Electrophoretic mobility shift analysis (EMS
A) Preparation of nuclear extracts and [32P] -labeled double-stranded consensus oligonucleotides (Santa Cruz Biotechnology, Heidelberg, Germany), non-denaturing polyacrylamide gel electrophoresis, autoradiography and supershift are described in the literature. (Krzesz et al. (199
9) Reference cited above). Oligonucleotides having the following single-stranded sequence were used (core sequence is underlined): AP-1, 5'CGCTTGA TGACTCA GCCGGAA 3 '; C / EBP, 5'
TGCAGA TTGCGCAA TCTGCA 3 '.

7. Decoy-oligodeoxynucleotide (dODN) technology Double-stranded dODN was prepared from oligodeoxynucleotides (Eurogentec, Cologne, Germany) linked to complementary single-stranded phosphorothioates by literature methods (Krzes
z et al. (1999) supra). Fill the lumen of the carotid artery or jugular vein section with the medium containing double-stranded dODN, or pre-incubate the dODN with cultured PAEC at a concentration of 10 μM for 4 hours, conditions pre-optimized based on EMSA and RT-PCR analysis Had been converted. The dODN-containing medium is then washed with perfusion (section) or fresh medium (PAEC)
Was replaced. The single stranded sequence of dODN was as follows (underlined letters indicate bases linked to phosphorothioate): AP-1,5 ' CGCT TGATGACTCAGCC GGAA
3 '; C / EBP, 5' TGCA GATTGCGCAATC TGCA 3 '; C / EBPmut,
5 ' TGCA GAGACTAGTCTC TGCA 3'; GATA-2, 5 ' CACT TGATAAC
AGAAAGTGATAA CTCT 3 '.

8. Statistical analysis Unless otherwise stated, all data in the figures and text are expressed as mean ± SEM of n experiments with sections or cells from various animals. Statistical evaluation is based on P value <
0.05 was considered statistically significant, and Stud was used for one-sided data.
Performed by ents t-test.

9. Prepro-ET-1 mRNA expression When RbCA is pressurized to 160 mmHg and RbJV is pressurized to 20 mmHg, it expands to the maximum and the average increase in outer diameter is 20% each.
8 and 274%. The mRNA level of the housekeeping reference gene EF-1 was not altered by these changes in perfusion pressure, and no significant loss of EC from perfused sections was observed. Perfusion pressure of 2 or 90 to 160
up to mmHg (RbCA) or from 0 or 5 to 20 mmHg (RbCA
JV) When it was increased, both ppET-1 mRNA and intravascular ET-1 significantly increased (FIGS. 1, 3 and 4). This probably deformation-induced ppET-1 expression was restricted to the endothelium, as it was significantly reduced after section denudation (FIG. 1a). RbJV is a tyrosine kinase inhibitor specific to Srcs (Banes et al. (1995) 73, 349;
rukov et al. (1997) 81, 895) Herbimycin A
(0.1 μmol / l, FIG. 1c), but not protein kinase C (PKC) inhibitor (Wilkinson et al. (1993) B
iochem. J. 295, 335) Ro 31-8220 (0.1 μmol /
1, pretreatment with FIG. 1b) strongly inhibited both basal and pressure-dependent expression of ppET-1. In contrast,
Ro 31-8220 had no effect on RbCA (FIG. 1e) and the deformation-induced increase in ppET-1 expression was virtually abolished after exposing sections to herbimycin A (FIG. 1).
f). Blocking RNA synthesis with actinomycin D (1 μmol / l) abolished the pressure-induced increase in ppET-1 mRNA (FIG. 1d) and eliminated intravascular ET-1 in both types of blood vessels.

Results : The ET-1 gene is de novo expressed in response to pressure-dependent deformation of EC.

10. Activation of transcription factor In EMSA using AP-1 and C / EBP oligo, RbJV
Exposure to an intraluminal pressure of 0 mmHg (n = 3-9 for each transcription factor) resulted in a transient (i.e., 30-60 min) and significant (2- or 3-fold) increase in activity. This nuclear translocation, according to supershift analysis, was mainly composed of β- and δ-isoforms of AP-1 (FIG. 2a)
And in the case of C / EBP (FIG. 2b), the reproducibility was high. Furthermore, the abundance of AP-1 in nuclear extracts was significantly reduced in the presence of high perfusion pressure after pretreatment under basic conditions and RbJV Ro 31-8220 (perfusion for 1 hour at 20 mmHg) 247 ± 3 to 146 ± 13% of later controls). R
In bCA, substantially the same results were obtained for AP-1 and C / EBP, and activation induced by C / EBP β and δ pressure was also blocked by herbimycin A.

Results: After increasing the tube pressure, AP-1 and C / EB
P shows increased binding activity in extracts of RbJV and RbCA.

11. Role of AP-1 and C / EBP AP-1 and C / EBP dODN showed a diffuse but significant effect. For example, in RbJV, AP-1 dODN abolished pressure dependence, but showed that basic ppET-1 mRNA (FIG. 4a) and ET-
One peptide abundance (Figure 4b) did not go away. Meanwhile, C
/ EBP dODN significantly enhanced basal and pressure-dependent ppET-1 expression to the same extent (FIGS. 4a and 4b). In contrast, mutant dODN did not show such an effect and enhanced the specificity of the dODN method (FIG. 3b). The combination of AP-1 with C / EBP dODN markedly reversed the latter strong effect on ppET-1 expression (FIGS. 4a and b).

Unlike in the case of RbJV, C / EBP, but not C / EBPmut dODN, has a pressure-induced pH at RbCA
Increased pET-1 expression was abolished without affecting basal levels (FIG. 4c). In these sections, AP-1 dODN was
GATA-2 dODN and ppET-1 expression in JV was slightly reduced (FIG. 3c).

Results: ppET-1 induction in response to pressure is RbJV
Can be inhibited by AP-1 dODN and in RbCA by C / EBP ODN.

12. Extension reaction of rat ppET-1 promoter PAECs were transiently transfected with various rat ppET-1 promoter-luciferase constructs. These cells bring much higher levels of ppET-1 mRNA and ET-1 peptide into cyclical tone, an effect that is attenuated by both Ro 31-8220 (FIG. 3e) and Herbimycin A (FIG. 3f). These cells were selected for expression in response.
According to β-galactosidase staining and β-galactosidase activity measurement, the transfection effect was about 15%.
Was estimated. All transfected promoter constructs (-98, -168, -350, -590, -729, -1
070, -1250 and -1329 bp) were expressed by PAEC to varying degrees under quiescent conditions, and luciferase activity was significantly increased when exposed to cyclic tension for 6 hours, except for the -98 bp construct (Fig. 5a). The apparent difference in luciferase activity between the individual promoter constructs was due to the difference in β-galactosidase transfection efficiency, as the activity of the enzyme in the double transfected cells appeared to be relatively homogeneous (FIG. 5c). It is not.

The full-length promoter construct was constructed using the extended PAEC
(About 40% of the luciferase activity of the gene obtained by CMV under quiescent conditions).
The construct that produced the shortest extension was a -168 bp fragment, whose expression level was about half that of the full-length promoter construct (Figures 5a and b). Deletion of the single AP-1 binding site at -110 to -100 bp abolished extension responsiveness (Figure 5a). PAEC transfected with this construct
0.1 to AP-1 dODN (63 and 93% inhibition, respectively, FIG. 5b)
Pretreatment with μmol / l Ro 31-8220 to block PKC activity (absence of basal luciferase activity and a 84% reduction in extension-induced luciferase activity) gave a similar effect. In contrast, GATA-2 consensus dODN reduced basal and extension-induced luciferase activity in PAEC transfected with the -168 bp construct by only 28 and 31%, respectively. Furthermore, both basal and stimulatory expression of the full-length promoter construct were clearly inhibited after exposure to AP-1 dODN (58 and 74%, respectively).

C / EBP dODN is -590, -729 or -1250 bp
Although there was no significant effect on luciferase activity in PAECs transfected with the construct, it significantly enhanced extension-dependent luciferase activity in PAECs transfected with the -1070 bp construct (FIG. 5b).

Results : The extension responsiveness of the ppET-1 promoter in PAEC is altered by AP-1. ppET-1 promoter activation can be inhibited by AP-1 dODN, but not by C / EBP dODN.

13. Transfection of intervening vein grafts in rabbits: To determine the effect of AP-1 decoy on neointimal lesion formation after arterial-venous transplantation using rabbit external and common carotid arteries The setting of an in vivo model can be considered. In this experimental model, it is expected that the rate of successful surgery and graft opening will be about 75%. Thus, eight rabbits are used to obtain sufficient data for morphometric analysis, and another set of six are used to demonstrate efficient decoy delivery to venous sections.

After induction of normal anesthesia in New Zealand white rabbits (3-4 kg) intravenously, standard inhalation anesthesia with halothane is performed. A vertical neck incision is made to expose the external jugular vein and the ipsilateral common carotid artery. A 4 cm vein section is obtained using trauma, treated with topical papaverine, and stored in Heparinized Ringer's solution. Transfection of vein sections is performed as follows. Push a small catheter into the vein section and wash the section with water to remove Ringer's solution. After connecting the blood vessels at the base, a decoy solution is injected into the vein to prevent vein dilation. After end control by a second ligation, the decoy solution is incubated with a 30 minute residence time to obtain a sufficient transfection rate. During the incubation period, the ipsilateral carotid artery is dissected and recruited. The arterial section is split with forceps and the transfected vein section is inverted and inserted into the carotid artery end-to-end using standard sutures. After removing the forceps, hemostasis is performed and the wound is closed. The animals in the control group receive a mixed decoy that has no effect on AP-1 activity.

The experiment for examining the efficiency was completed after two days.
Perform electrophoretic mobility shift analysis (EMSA) to demonstrate efficient squelching of the targeted transcription factor (AP-1). The biological effect of the decoy treatment is revealed 4 weeks after transfection by standard computerized morphometry.

14. Suppression of restenosis by transfection of damaged carotid artery Neointima lesion formation in the carotid artery of rabbits after PTA (neoint
In order to determine the effect of C / EBP decoy on imal lesion formation, an in vivo model of restenosis may be set.

A mature New Zealand white rabbit (3-4
kg). Some animals are fed a diet containing 1% cholesterol for two weeks before balloon injury and continue to feed until the transfected vessels are obtained. Balloon injury is performed after anesthesia is induced by the method described in 13. After a vertical neck incision, the right common cervical artery is incised to remove the surrounding tissue and connect the side branches. Rabbits receive systemic heparinization by intravenous injection of heparin (10 U / kg) before clamping the carotid artery. 2 A French Fogarty embolectomy catheter is introduced into the branch of the external carotid artery, inflated in the central portion of the common carotid, and then pulled back to the bifurcation. When this procedure is repeated three times, the endothelium is completely exposed. An infusion catheter filled with normal saline is then pushed into the artery and the carotid artery is washed. After performing base control, inject the decoy solution and incubate with a 30 minute dwell time. Ligation of the side branch used to introduce the catheter (litigatio
After n), blood flow is resumed in the common carotid artery, hemostasis is performed, and the wound is closed.

At the above time intervals, the rabbits were re-anesthetized,
Obtain vascular sections. Experimental animals for which the efficiency of dODN treatment is to be examined are also anesthetized, and vascular sections are obtained. Experiments for efficiency studies were completed two days later, EMSA was performed, and target transcription factors (C / E
Clarify the efficient "decoying" of BP). The biological effect of the decoy treatment is revealed 4 weeks after transfection by standard computerized morphometry.

[0087]

[Sequence List] SEQUENCE LISTING <110> Cardiogene Gentherap. Systeme AG <120> Modulating Transcription of Genes in Vascular Cells <160> 30 <170> Word 6.0, PC-DOS / MS-DOS <210> 1 <211> 21 < 212> DNA <213> Oryctolagus cuniculus <400> 1 ggagctccag aaacagctgt c 21 <210> 2 <211> 24 <212> DNA <213> Oryctolagus cuniculus <400> 2 ctgctgataa atacacttct ttcc 24 <210> 3 <211> 21 < 212> DNA <213> Oryctolagus cuniculus <400> 3 gacatcacca agggtgtgca g 21 <210> 4 <211> 20 <212> DNA <213> Oryctolagus cuniculus <400> 4 gcggtcagca caatggcata 20 <210> 5 <211> 21 <212 > DNA <213> artificial sequence <220> <223> consensus binding site for AP-1 <400> 5 cgcttgatga ctcagccgga a 21 <210> 6 <211> 20 <212> DNA <213> artificial sequence <220> <223 > consensus binding site for C / EBP <400> 6 tgcagattgc gcaatctgca 20 <210> 7 <211> 20 <212> DNA <213> artificial sequence <220> <223> mutated consensus binding site for C / EBP <400> 7 tgcagagact agtctctgca 20 <210> 8 <211> 27 <212> DNA <213> artificial sequence <220> <223> con sensus binding site for GATA-2 <400> 8 cacttgataa cagaaagtga taactct 27 <210> 9 <211> 21 <212> DNA <213> artificial sequence <220> <223> designed AP-1 <400> 9 ctgttggtga ctaataacac a 21 <210> 10 <211> 21 <212> DNA <213> artificial sequence <220> <223> designed AP-1 <400> 10 ctgtgggtga ctaatcacac a 21 <210> 11 <211> 15 <212> DNA <213> artificial sequence <220> <223> designed AP-1 <400> 11 gtgctgactc agcac 15 <210> 12 <211> 17 <212> DNA <213> artificial sequence <220> <223> designed AP-1 <400> 12 cgcttagtga ctaagcg 17 <210> 13 <211> 17 <212> DNA <213> artificial sequence <220> <223> designed AP-1 <400> 13 tgtgctgact cagcaca 17 <210> 14 <211> 19 <212> DNA < 213> artificial sequence <220> <223> designed AP-1 <400> 14 ttgtgctgac tcagcacaa 19 <210> 15 <211> 19 <212> DNA <213> artificial sequence <220> <223> designed AP-1 <400 > 15 tcgcttagtg actaagcga 19 <210> 16 <211> 20 <212> DNA <213> artificial sequence <220> <223> designed AP-1 <400> 16 tgctgactca tgagtcagca 20 <210> 17 <211> 20 <212> DNA <213> artificial sequence <220> <223> designed AP-1 <400> 17 tgctgactaa ttagtcagca 20 <210> 18 <211> 21 <212> DNA <213> artificial sequence <220> <223> designed AP-1 < 400> 18 gtcgcttagt gactaagcga c 21 <210> 19 <211> 21 <212> DNA <213> artificial sequence <220> <223> designed AP-1 <400> 19 cttgtgctga ctcagcacaa g 21 <210> 20 <211> 22 <212> DNA <213> artificial sequence <220> <223> designed AP-1 <400> 20 ttgctgactc atgagtcagc aa 22 <210> 21 <211> 20 <212> DNA <213> artificial sequence <220> <223> designed C / EBP <400> 21 gcttgtgcgg gaataaatag 20 <210> 22 <211> 20 <212> DNA <213> rat PPET-1 <400> 22 aggaataatg gaatgccctg 20 <210> 23 <211> 16 <212> DNA < 213> artificial sequence <220> <223> designed C / EBP <400> 23 gacattgcgc aatgtc 16 <210> 24 <211> 16 <212> DNA <213> artificial sequence <220> <223> designed C / EBP <400 > 24 agcattggcc aatgct 16 <210> 25 <211> 18 <212> DNA <213> artificial sequence <220> <223> designed C / EBP <400> 25 cgacattgcg caatgtcg 18 <210> 26 <211> 18 <212> DNA <213> a rtificial sequence <220> <223> designed C / EBP <400> 26 aggcattggc caatgcct 18 <210> 27 <211> <212> DNA <213> artificial sequence <220> <223> designed C / EBP <400> 27 acgacattgc gcaatgtcgt 20 <210> 28 <211> 20 <212> DNA <213> artificial sequence <220> <223> designed C / EBP <400> 28 taggcattgg ccaatgccta 20 <210> 29 <211> 22 <212> DNA <213 > artificial sequence <220> <223> designed C / EBP <400> 29 ctgttgcgca attgcgcaac ag 22 <210> 30 <211> 22 <212> DNA <213> artificial sequence <220> <223> designed C / EBP <400 > 30 gacttgcgca attgcgcaag tc 22

[Brief description of the drawings]

FIG. 1: Rabbit carotid artery (R) perfused for 6 hours at various intravascular pressures
bCA) or ppET-1 m in isolated sections of rabbit jugular vein (RbJV)
(A) Endothelial removal, (B, E) Ro 31-8220 (0.1
μmol / l), (C, F) herbimycin A (0.1 μmol / l),
And (D) shows the effect of actinomycin D (0.1 μmol / l). In a typical RT-PCR analysis, in at least four additional experiments using sections from different animals,
Similar findings were obtained.

FIG. 2 shows the time course of nuclear translocation of (A) AP-1 and (B) C / EBP in endothelium-complete sections of RbJV perfused at 20 mmHg. In a representative EMSA analysis, at least two additional experiments gave similar results.

FIG. 3: RbCA or RbJV perfused for 6 hours at various intravascular pressures
Endothelium relative to ppET-1 mRNA abundance in intact sections (B,
FIG. 3 shows the effects of specific decoy-oligodesoxynucleotides (dODN) on (D) C / EBPmut, (C) GATA-2, and (D) C / EBP. (E, F) (E) Ro 31-8220 (0.1 μmol / l) and Herbimycin A (0.9%) for ppET-1 mRNA abundance in PAEC (primary culture) incubated or extended for 6 hours under static conditions. μmol / l),
FIG. In a representative RT-PCR analysis, similar findings were obtained in at least two additional experiments using sections or cells from different animals.

FIG. 4: RbCA (C) or R perfused for 6 hours at various intraluminal pressures
(A, C) ppET-1 mRNA abundance in the isolated section of bJV (A, B) (n =
3-5) and (B) Intravascular ET-1 peptide (n = 3)
FIG. 4 shows the effect of specific decoy ODN on AP-1 and C / EBP alone or in combination. *
P <0.05 for 0 or 2 mmHg, P <0.05 for # 20-160 mmHg, +20 mmHg and P for C / EBP dODN
<0.05.

FIG. 5. (A, B) Different rat ppET-1 promoter-luciferase constructs in cultured PAEC incubated or expanded for 6 hours under quiescent conditions in the absence or presence of (A) or (B) various dODNs. (Asterisk indicates no AP-1 response element
Expression of -168 bp construct (indicated by + sign; n)
= 3-7, twice measured). (C) SV40 / β−
Β-galactosidase activity (n = 3-7, measured twice) in cultured PAEC co-transfected with a galactosidase expression vector and various rat ppET-1 promoter-luciferase constructs as a similar transfection efficiency indicator. FIG.

FIG. 6 is a schematic representation of putative transcription factor binding sites in the promoter of the rat ppET-1 gene, further showing various promoter construct lengths.

Continued on the front page (72) Inventor Marx, Hecker Göttingen, Federal Republic of Germany, Henry-Donant-Strasse, 44th (72) Inventor Manfred, Laut Göttingen, Federal Republic of Germany, Porsche Wake, 10 (72) Inventor Andreas, Her. Wagner Germany Göttingen, Stellwan, 11F term (reference) 4B024 AA01 AA11 AA20 BA80 CA01 CA04 CA11 DA02 EA04 FA02 GA11 GA13 GA18 HA17 4B065 AA90X AA91Y AA99Y AB01 AC14 AC20 BA01 BA02 BC01 BD01 BD50 CA24 ACA44 CA47 A47A NA14 ZA36

Claims (21)

[Claims] At least one of the following: endothelial or cardiac cells
A method of regulating the transcription of a gene comprising: contacting said cell with a composition comprising one or more double-stranded nucleic acids capable of sequence-specifically binding to a transcription factor AP-1 or a related transcription factor. A method comprising the steps of: 2. The method of claim 1, wherein the endothelial cells are part of a blood vessel or vascular graft. 3. The method of claim 2, wherein a vascular graft is contacted with said composition in vivo or ex vivo. 4. A method of regulating the transcription of one or more genes in a vascular or heart cell, comprising: binding one or more genes capable of sequence-specifically binding to a transcription factor C / EBP or a related transcription factor. Contacting with a composition comprising the present single-stranded nucleic acid. 5. The method according to claim 4, wherein the composition further comprises a double-stranded nucleic acid capable of sequence-specifically binding to the transcription factor AP-1. 6. The method according to claim 4, wherein the vascular cells are smooth muscle (SMC) or endothelial cells. 7. The method of claim 6, wherein the cells are part of a blood vessel or vascular graft. 8. The method of claim 7, wherein the blood vessel is a coronary or peripheral artery. 9. The method according to claim 1, wherein the gene or genes regulate the growth or migration of the cell. 10. The method according to claim 1, wherein the regulatory gene is selected from the group consisting of an endothelin gene, a macrophage chemotactic protein (MCP) gene, and a nitric oxide synthase (NOS) gene. The described method. (11) the endothelin gene is an endothelin-
11. The method according to claim 10, wherein the MCP gene is one gene, the MCP gene is a MCP-1 gene, and the NOS gene is an inducible NOS gene. 12. The method according to claim 1, wherein the regulation activates the gene or genes. 13. The method according to claim 1, wherein the regulation causes the gene or genes to lose or suppress activation.
The method according to any one of the preceding claims. 14. The method according to claim 1, wherein the double-stranded nucleic acid comprises one or more copies of a sequence that specifically binds to AP-1 or C / EBP. . 15. The method according to claim 1, wherein the double-stranded nucleic acid is a vector or an oligonucleotide. 16. The method according to claim 15, wherein the oligonucleotide or the vector is stabilized. 17. The method according to claim 1, wherein the composition further comprises at least one buffer. 18. The method according to claim 1, wherein the composition further comprises one or more suitable additives or auxiliaries. 19. A method of treating a vascular disease, comprising the steps of: combining vascular cells with the composition of any one of claims 1 to 18 in an amount sufficient to regulate transcription of one or more genes. Contacting. 20. Transcription factor AP-1 or SEQ ID NO: 5 or 9
A double-stranded nucleic acid capable of sequence-specifically binding to a related transcription factor having one of 配 列 20 sequences. 21. A double-stranded nucleic acid capable of sequence-specifically binding to transcription factor C / EBP or a related transcription factor having one of the sequences of SEQ ID NOs: 6 or 21-30.