JP2000501941A - Modified VEGF antisense oligonucleotide - Google Patents

Abstract

  (57) [Summary] Disclosed are oligonucleotides complementary to VEGF-specific nucleic acid sequences that are useful for reducing VEGF expression. Also disclosed are pharmaceutical compositions comprising such oligonucleotides useful for treating various diseases associated with angiogenesis and angiogenesis.

Description

DETAILED DESCRIPTION OF THE INVENTION                 Modified VEGF antisense oligonucleotide Cross-reference of related applications   The present invention was filed on March 2, 1995, and the title of the invention is "human VEGF-specific. No. 08 / 398,945, entitled "Different Oligonucleotides." Partial continuation application, the application of which was filed on January 26, 1995 Is "Inhibition of angiogenesis using VEGF-specific oligonucleotide" No. 08 / 378,860, which is incorporated by reference in its entirety. The title of the invention filed on July 27, 1993 is "anti-expression of vascular endothelial growth factor. Patent Application No. 08 / 098,94, "Sense Oligonucleotide Inhibition" This is a continuation-in-part application of No. 2.Background of the Invention   The present invention relates to angiogenesis and vascular endothelial growth factor. For more information, Ming is an oligonucleotide specific for vascular endothelial growth factor nucleic acid and It relates to the useful treatment of diseases associated with formation.   Retinal neovascular diseases such as diabetic retinopathy, retinopathy of prematurity and age-related macular degeneration Are the leading causes of blindness in the United States and the world, and Biochemical events have not yet been fully elucidated.   Diabetic retinopathy is the leading cause of blindness in working adults (20-64) in the United States (Foster, Harrison's Principles of Inter Harrison's Principles of Internal Medicine (Iselva (Esselbacher et al.) McGraw-Hill, New York (1994) 1994-1995). During the course of diabetes mellitus, the blood vessels in the retina , Not only exudative blood vessels, but also blood vessels that cause retinal hypoxia . The consequences of these complications include bleeding, flocculent vitiligo, retinal infarction, and Retinal neovascularization. If left untreated, 60% see Lose power. The classic therapy for proliferative diabetic retinopathy is panretinal laser photocoagulation (PRP) is there. However, even with panretinal laser photocoagulation, foveal fever, bleeding, and retinal detachment And complications such as choroidal vascular growth can occur. In addition, other benefits of this procedure Poor effects include reduced peripheral vision, reduced night vision, and altered color perception. (Am. J. Ophthalmol. (1976) 81: 383-396; Ophthalmol. (1 991) 98: 741-840). Therefore, a more effective treatment for diabetic retinopathy There is a need for   Retinopathy of prematurity (ROP) is a common cause of blindness in children in the United States (pi). (Pierce et al. (1994) Int. Ophth. Clinics 34: 121-148). immature After birth, the baby is exposed to hyperoxia without supplemental oxygen. Because in the womb This is because the oxygen partial pressure is much lower than when breathing normal room air. This Relative hyperoxia such as is necessary for survival, if at all Things. Retinal blood vessels stop growing into the surrounding retina and, as a result, the growing retina Ischemia and localized hypoxia as the metabolic demand of the drug increases. These Hypoxia stimulates subsequent retinal neovascularization. This angiogenesis usually remits However, irreversible vision loss may occur. At least 10,000 per year There are new cases, with a global estimate of 10 million cases. at present, There is no effective treatment for ROP. Two treatments are available: cryotherapy and laser therapy Are not completely effective, and these methods themselves can cause eye damage. Resulting in reduced vision (Pierce et al. (1994) Int. Ophth. Clinics 3). 4: 121-148). Although many other anti-angiogenic compounds have been tested, No inhibition of angiogenesis has been reported (Smith et al. (1994) Invest. Op. hthalmol.Vis.Sci. 35: 1442; Foley et al. (1994) Invest. phtha 1 mol. Vis. Sci. 35: 1442). Therefore, for effective treatment of ROP There is a need to   Age-related macular degeneration is the leading cause of blindness in older adults in the United States, and Kafka Accounts for up to 30% of all binocular blindness in Caucasian Americans (Author unknown (1994) Prevent Blindness America). This disease Damage to retinal pigment epithelial cells that provides physiological support to light-sensitive photoreceptor cells of the membrane It is characterized by loss of central vision (usually binocular). In most cases, There is no effective treatment at present. Approximately 20% of exudative cases diagnosed early Thus, laser therapy can prevent further loss of vision. However, this The effect is transient (Bressler et al., Principles and Practi ces of Ophthalmology (Albert and Jacobia) c)), Saunders (WB Saunders Co.), Philadelphia, Pencil Veneer (1994) Vol. 2, pp. 834-852). Therefore, age-related macular degeneration There is a need for more effective and permanent treatments.   Macular angiogenesis also includes sickle cell retinopathy, neovascular glaucoma, retinal vein occlusion, It is also the pathology underlying other hypoxia. For these ophthalmic diseases and angiogenesis Other associated pathological conditions (ie, tumor growth, wound healing) are common factors (Knighton et al. (1983) Scie nce 221: 1283-1285; Folkman et al. (1987) Scie. nce 235: 442-446; Klagsbrun et al. (1991) Ann. Rev. Physiol. 53: 217-239; Miller et al. (1993) Principl. es and Practice of Ophthalmology, Saunders, Philadelphia, 760 Aiello et al. (1994) New Eng. J. Med. 331: 1480-1. 487). In addition, retinal neovascularization is released by the retina in response to hypoxia It is hypothesized that this is the result of "vasoformative factor" (Michaelson (1948) Trans. Ophthalmol. Soc. UK. 68: 137-180; and Ashton et al. (1954) Br. J. Opht. halmol. 38: 397-432). Recent experimental data show that vascular endothelial growth factor Shows that there is a high degree of correlation between the current and retinal neovascularization (Aiello et al. (1994) New Eng. J. Med. 331: 1480-1487). In addition, diabetes Elevated levels of vascular endothelial growth factor are found in the vitreous humor of patients (Eyelo et al. , Supra). Therefore, this cytokine / growth factor is It seems to play an important role.   Vascular endothelial growth factor / vascular permeability factor (VEGF / VPF) is endothelial cell specific Mitogen, recently stimulated by hypoxia and required for tumor angiogenesis (Senger et al. (1986) Cancer Res. 46: 5629-5632; Kim et al. (1993) Nature 362: 841-84. 4; Schweiki et al. (1992) Nature 359: 843-845; Plate et al. (1992) Nature 359: 845-848). VEGF / VPF is the dimer dimer of 34-43 kDa (the predominant species is at about 45 kDa). Glycoproteins are sulfide-like linked to various tumor and normal cells. Is synthesized and secreted. In addition, cultured humans such as pigment endothelial cells and perivascular cells Retinal cells secrete VEGF and increase VEGF gene expression in response to hypoxia. (Adamis et al. (1993) Biochem. Biophys. Res. Commun. 193: 631-638; Plouet et al. (1992) Inves. t.Ophthalmol.Vis.Sci. 34: 900; Adamis et al. (1993) Invest.Ophthalm. ol.Vis.Sci. 34: 1440; Aiello et al. (1994) Invest.Ophthalmol.Vis.S ci. 35: 1868; Simorre-Pinatel et al. (1994) Inv. est. Ophthalmol. Vis. Sci. 35: 3393-3400). In contrast, in normal tissue VEGF is at a relatively low level. Therefore, VEGF is associated with angiogenesis Appears to play a major role in many pathological conditions and processes It is. Therefore, VEGF expression in tissues caused by the above various conditions is Control will be the key to treatment or prophylaxis associated with hypoxia.   "Antisense" that can regulate the expression of cellular genes or foreign genes A new chemotherapeutic agent called an oligonucleotide has been developed (Zamekuni (1978) Proc. Natl. Acad. Sci. (USA) 75: 280. 284). Without being bound by any theory or mechanism, antisense The activity of a oligonucleotide is such that the oligonucleotide is a target nucleic acid (eg, Nom region, at least a portion of a gene or mRNA transcript), and thus Target RNA by hybridization inhibition or by RNase H Due to disruption (the ability to activate RNase H when hybridized to RNA) Reliant on disrupting the function of the target.   VEGF-specific antisense oligonucleotides have been developed (C Uchida et al. (1995) Antisense Res. & Dev. 5 (1): 87 (Abst Lacto OP-10); Nomura et al. (1995) Antisense Res. & Dev. 5 (1): 91 (abstract OP-18)), reversing angiogenesis or angiogenesis There is no indication that this should be done. Therefore, VEGF expression can be reduced Oligonus that can finally suppress the development of diseases and abnormalities related to VEGF expression There remains a need for nucleotide development.Summary of the Invention   Cells affected by hypoxia are known to express VEGF. Departure Akira describes a novel synthetic oligonucleotide specific for nucleotides 58-90 of the VEGF gene. Nucleotides induced by hypoxia of VEGF mRNA and protein It also provides that expression can be reduced. The present invention was developed using this knowledge. The invention relates to VEGF-specific oligonucleotides, pharmaceutical compositions, VEGF mRN A and methods for reducing the expression of proteins.   In one aspect, the invention relates to a nucleic acid sequence complementary to a human vascular endothelial growth factor nucleic acid sequence. A synthetic oligonucleotide is provided. Oligonucleotides are shown in SEQ ID NO: An oligonucleotide having the nucleic acid sequence shown in any one of 2 to 16 is provided.   As used herein, the term “synthetic oligonucleotide” refers to at least one 5 Nucleic acid linked covalently via an 'to 3' internucleotide bond Refers to a chemically synthesized polymer composed of otide. In some embodiments, this Oligonucleotide comprises at least one deoxyribonucleotide, ribonucleic acid. Nucleotides or both deoxyribonucleotides and ribonucleotides No. In another embodiment, the synthetic oligonucleotide used in the method of the invention has a length of About 14 to about 28, or about 15 to about 30 nucleotides. Some preferred In embodiments, the oligonucleotides comprise from about 15 to about 25 nucleotides. And in other embodiments comprises about 16-29 nucleotides.   For the purposes of the present invention, "genomic regions or RNA transcripts transcribed therefrom" The term "oligonucleotide sequence complementary to a reotide" means that under physiological conditions, For example, Watson-Crick base pairing (between oligonucleotide and single-stranded nucleic acid) Interaction or Hoogsteen base pairing (oligonucleotide and Or oligonucleotides bind to RNA By other means, including causing pseudoknot formation. Means an oligonucleotide that binds to a nucleic acid sequence. Watson under physiological conditions -Crick or Hoogsteen binding by base pairing is actually It is measured by observing interference with the function of the train.   In some embodiments, a synthetic oligonucleotide of the invention comprises a VEGF mR Without compromising the ability to hybridize to nucleotide sequences contained within NA It is modified in several ways. As used herein, "modified oligonucleotide" At least two of the nucleotides are synthetic bonds, ie, one of the nucleotides Other than a phosphodiester bond between the 5 'end of the nucleotide and the 3' end of the other nucleotide (5 'nucleotide phosphate is replaced by any chemical group) It refers to an oligonucleotide that is more linked. In some preferred embodiments, At least one internucleotide linkage of the oligonucleotide is an alkyl Lephosphonate, phosphorothioate, phosphorodithioate, phosphate ester , Alkylphosphonothioate, phosphoramidate, carbamate, carbonate Acid, phosphate triester, acetamidodate, and / or carboxymethyl Luster.   The term “modified oligonucleotide” also refers to 2′-O-substituted ribonucleotides Have at least one nucleotide with a modified base and / or sugar, such as Oligonucleotides. For the purposes of the present invention, the term "2'-O- "Alkyl" includes 1 to 6 saturated or unsaturated carbon atoms at the 2'-position of the pentose residue. -O-aryl by -O-lower alkyl groups or having 2-6 carbon atoms Or an allyl group, wherein such alkyl, aryl or allyl group is substituted May not be present, or may be, for example, halogen, hydroxy, trifluoro Chill, cyano, nitro, acyl, acyloxy, alkoxy, carboxyl, car Optionally substituted with alkoxy, or hydroxy, amino or Means substitution with a halo group (however, excluding substitution with a 2'-H group) I do. In some embodiments, the oligonucleotide of the invention has a 2′-terminal at the 5 ′ end. O-alkylated 4 or 5 ribonucleotides (ie, 5'2-O- Alkylated ribonucleotides), and / or 2'-O-alkylation at the 3 'end 4 or 5 ribonucleotides (ie, 3'2-O-alkylated ribonucleotides) Nucleotides). In a preferred embodiment, the nucleic acid of the synthetic oligonucleotide Reotide is one or at least one phosphorothioate internucleotide. They are linked by tide bonds. The phosphorothioate linkage is R_PAnd S_PMixing An enantiomer or R_POr S_PStereoregular in either form or It may be substantially stereoregular (Iyer et al. (1995) Tetrahedro n Asymmetry 6: 1051-1054).   In another aspect, the present invention provides a method for suppressing the expression of VEGF. this In a method, a VEGF-specific nucleic acid is contacted with an oligonucleotide of the invention. Let it. As used herein, "nucleic acid" refers to a genomic region or a transcribed therefrom. RNA molecules. In some embodiments, the nucleic acids are mRNA.   Without being bound by any theory or mechanism, it may be used in accordance with the present invention. The activity of the oligonucleotide is such that the oligonucleotide is a target nucleic acid (eg, Genomic region, at least a portion of a gene or mRNA transcript) Relying on disrupting the function of the target. This occurs under physiological conditions. Hybridization is actually achieved by observing interference with the function of the nucleic acid sequence. And measure. Therefore, preferred oligonucleotides used according to the present invention Can be combined with a target nucleic acid and a stable double-stranded (or Hoogsteen base-pairing mechanism) Can form a triplex) and can be targeted by activating RNase H RNA molecules can be effectively destroyed, and nuclease components can be analyzed in vivo. Solution (eg, endonuclease and exonuclease activity) Can show sex. Some of the above modifications of oligonucleotides and the art Other modifications, known in the art, are specific and successfully incorporated into each of these favorable properties. It is complicated.   2. At least one synthetic oligo according to claim 1 in a physiologically acceptable carrier. Pharmaceutical compositions comprising nucleotides are also provided by the present invention.   Another aspect of the invention is the ability to inhibit angiogenesis, and therefore the method of the invention. Pharmaceutical compositions useful for These compositions specifically express vascular endothelial growth factor Of the present invention and physiological and / or pharmacology It contains a chemically acceptable carrier.   The term “pharmacologically acceptable” interferes with the effectiveness of the biological action of the active ingredient Means non-toxic substances that do not. The term "physiologically acceptable" Non-toxic compatible with biological systems, such as cells, cell cultures, tissues, or organisms Substance.   Another aspect of the present invention relates to V-angiogenesis and angiogenesis associated with disease states. To evaluate the role of EGF.BRIEF DESCRIPTION OF THE FIGURES   The above and other objects of the present invention, various features of the present invention, and the present invention itself , Will be more fully understood when the following description is read in conjunction with the accompanying drawings. Attached figure In terms of   FIG. 1 shows VEGFc targeted by the oligonucleotide of the present invention. Schematic showing the regions of the DNA sequence;   FIG. 2 is a graph showing the results of ELSA, induced by cobalt chloride. Shows the ability of oligonucleotides H3-I and H3-J to suppress VEGF expression You;   FIG. 3 is a graph showing the results of ELSA, induced by cobalt chloride. Oligonucleotides H3-D, H3-E and H3-F that suppress VEGF expression Demonstrate the ability of   FIG. 4 is a graph showing the results of ELSA, induced by cobalt chloride. Shows the ability of oligonucleotides H3-G and H3-H to inhibit VEGF expression You;   FIG. 5 is a graph showing the results of ELSA, showing that M21 Oligo that suppresses VEGF expression induced by cobalt chloride in melanoma cells Exhibits the ability of nucleotides H3 and H3-I; and   FIG. 6 is a graph showing the results of ELSA, induced by cobalt chloride. Not showing the ability of the modified H3 oligonucleotide to suppress VEGF expression (H3-K: All 3 -'- O-methylated phosphorothioate ribonucleotides; H3- L: 5 5'2'-O-alkylated phosphorothioate ribonucleotides And the rest are phosphorothioate deoxyribonucleotides; H3-M: three of five '2'-O-alkylated phosphorothioate ribonucleotides; Holothioate deoxyribonucleotides; and H3-N: 5 3'2'-O -Alkylated phosphorothioate ribonucleotides, five 5 ' 2′-O-alkylated phosphorothioate ribonucleotides, the remainder being Holothioate deoxyribonucleotide).Detailed description of preferred embodiments   The patent and scientific literature referred to herein provides insights that are available to those skilled in the art. It is something to establish. Issued U.S. Patents, Allowed Applications, cited herein. , And references cited herein by reference.   The present invention relates to diseases related to angiogenesis and angiogenesis, including retinal neovascularization and VEGF nucleic acid-specific synthetic antisense oligos useful for treating abnormalities Provide nucleotides.   Antisense oligonucleotide method is a new approach to silencing gene expression (Generally Agrawal (1992) Trendsin Biotech. 10: 152; Wagner (1994) Nature 372: 33. 3-335; and Stein et al. (1993) Science 261: 100. 4 to 1012). By binding to a complementary nucleic acid sequence (sense strand), Antisense oligonucleotides suppress RNA splicing and translation be able to. In this way, the antisense oligonucleotide is Protein expression can be suppressed. Antisense oligonucleotides also It has also been shown to bind to genomic DNA to form triple strands and repress transcription. Furthermore, the 17-mer nucleotide sequence occurs statistically only once in the human genome, and Therefore, by using such an antisense oligonucleotide, Extremely accurate targeting is possible.   It has been determined that the region encoding VEGF consists of eight exons ( (1994) J. Biol. Chem. 266: 11947-1. 1954). Three VEGF transcripts 121, 165, and 189 amino acids in length Have been observed, suggesting that an alternative splicing mechanism exists (see Leung et al. (1989) Science 246: 1306-1309; (1991) J. Biol. Chem. 266: 11947-11954). further Recently, a fourth VEGF transcript of length 206 encoding 206 amino acids has been discovered. (Houck et al. (1991) Mol. Endocrinol. 5: 1806-1814). . Transcripts homologous to the 121 and 165 amino acid polypeptides were identified in the bovine system. (Leng et al. (1989) Science 246: 1306-1309), 16 Transcripts corresponding to the five amino acid transcript have also been identified in a mouse system (con ( Conn) et al. (1990) Proc. Natl. Acad. Sci. (USA) 87: 1323-132. 7; Senger et al. (1990) Cancer Res. 50: 1774-1778; (Claffey et al. (1992) J. Biol. Chem. 267: 16317-16322). . Nucleic acid sequences encoding three forms of VEGF have also been reported in humans (te (1991) J. Bio 1. Chem. 266: 11947-11954), Comparison of human VEGF and mouse VEGF reveals more than 85% conservation between species (Crafy et al. (1992) J. Biol. Chem. 267: 16317-1). 6322).   The oligonucleotide of the present invention is useful as a target for suppressing VEGF expression. If it works, it is directed to any part of the VEGF nucleic acid sequence, Is also good. The sequence of the gene encoding VEGF was obtained from mouse (Crafy et al., Supra). And humans (Tisher et al., Supra). VEGF gene The targeting region includes any part of a known exon. In addition, The exon-intron boundary region is potentially potential for antisense suppression of VEGF expression. Is a useful target. One of the useful targets is some representatives specific for human VEGF However, the nucleotide sequence of the oligonucleotide which is not limited is SEQ ID NOs: 2-1. 6.   Oligonucleotides of the present invention include ribonucleotides, deoxyribonucleotides. Or the combination of the two, where the 5 'end of one nucleotide is The 3 'end of the nucleotide is linked by a covalent bond. These oligonucleotides The tides are at least 14 nucleotides in length, but 15 to 30 nucleotides Preferably, it is length, with 15 to 29 mer being most common.   The preparation of these oligonucleotides is based on phosphoramidate chemistry or H-phospho This can be done by art-recognized methods such as phonate chemistry, Can be performed manually or by an automatic synthesizer (Uhlmann et al.). Chem. Rev. (1990) 90: 534-583; and Agrawal (TrendsBi otechnol. (1992) 10: 152-158)).   The oligonucleotides of the present invention also hybridize to VEGF mRNA. Can be modified in many ways without compromising the ability to For example, oligo Nucleotides include the 5 'end of one nucleotide and the 3' end of another nucleotide. Contains at least one internucleotide linkage other than phosphodiester Where the 5 ′ nucleotide phosphodiester linkage is either It may be substituted with a chemical group. Examples of such chemical groups include alkyl phosphone , Phosphorothioate, phosphorodithioate, alkylphosphonothioate G, phosphoramidate, phosphate ester, carbamate, acetamidodate, Carboxymethyl esters, carbonates, and phosphoric acid triesters It is.   For example, US Pat. No. 5,149,797 discloses that the phosphorothioate core region has Flanked by methylphosphonate or phosphoramidate flanking regions Some traditional chimeric oligonucleotides are described. On August 9, 1995 U.S. Patent Application No.47508-559No. is the oligonucleotide phospho One or more flanked by one or more regions of rothioate These nonionic oligonucleotide regions (eg, alkyl phosphonates and And / or phosphoramidate and / or phosphate triester innu "Inverted" chimeric oligonucleotides containing nucleoside linkages are disclosed. ing. Preparation of various oligonucleotides having modified internucleoside linkages It can be manufactured by a known method (for example, Goodchild (1990) Bio). Conjug Chem. 2: 165-167; Agrawar et al. (1988) Proc. Natl. Aca. d.Sci. (USA) 85: 7079-7083; Woolman et al. (1990) Chem. Rev. 90: 534-583; and Agrawal et al. (1992) Trends Biotechno. 10: 152-158).   The phosphorothioate linkage is R_PAnd S_PMay be a mixed enantiomer of Or R_POr S_PStereoregular or substantially stereoregular in any form of (Ayer et al. (1995) Tetrahedron Asymmetry 6: 1051) -1054). Oligonucleotides with phosphorothioate linkages are Luamidite (eg, Agrawar et al. (1988) Proc. Natl. Acad. Sci. SA) 85: 7079-7083) Chemistry and H-phosphonates (e.g. Froehler (1986) Tetrahedron Lett. 27: 5575-55. 78) can be prepared using methods well known in the art, such as chemistry. The method described by Bergot et al. (J. Chromatog. (1992) 5 59: 35-42) can also be used.   Self-stabilized oligonucleotides can also be modified oligonucleotides useful in the methods of the invention. Nucleotides (Tang et al. (1993) NucleicAcids R es. 20: 2729-2735). These oligonucleotides are used for target hybridization. A soybean region; and an antibody complementary to the nucleic acid sequence in the self-stabilizing oligonucleotide. It includes two regions of a self-complementary region having a rigonucleotide sequence.   Other modifications include those inside or at the end of the oligonucleotide molecule. Addition of an internucleoside phosphate linkage to the molecule, eg, an amino group With a variable number of carbon residues between the terminal ribose and deoxyribose Teryl or diamine compounds, and also cleaved or opposite strands or conjugating enzymes Or phosphate modifications that crosslink to other proteins that bind to the genome. So Examples of modified oligonucleotides such as are arabinose instead of ribose Oligonucleotide having a modified base such as a sugar and / or a sugar, or 3 ′ position Chemical groups other than the hydroxyl group (at its 3 'position) and phosphorus at both the 5' and 5 'positions 3 ', 5'-substituted oligos having a sugar attached to a chemical group other than the acid group (at the 5'-position) Nucleotides.   Other examples of sugar modifications include modification at the 2 'position of a ribose residue, including Is by an -O-lower alkyl group containing 1 to 6 saturated or unsaturated carbon atoms, and Is by an -O-aryl or allyl group having 2 to 6 carbon atoms, wherein The -O-alkyl, aryl or allyl group may be unsubstituted, Or optionally substituted (eg, halogen, hydroxy, trifluoro Methyl, cyano, nitro, acyl, acyloxy, alkoxy, carboxy, Rubalkoxy or amino))) or amino or halogen 2′-O-substitution, but is not limited thereto. these Any of the substituents may be the natural 2'-hydroxyl group in the case of ribose, or It is not intended to exclude 2'-H- in the case of xylose. PC T publication WO 94/02498 describes a 2'-flanking DNA core region. Traditional hybrid oligonucleotides having regions of O-substituted ribonucleotides Discloses nucleotides. U.S. Patent Application No. 4 filed August 9, 1995 No. 7508-559 describes 2′-O-substituted (or 2′OH, unsubstituted) RNA A region exists between the two oligodeoxyribonucleotide regions ("traditional" "Reverse" structure compared to "hybrid oligonucleotide") Oligonucleotide Disclosed are "reverse" hybrid oligonucleotides comprising: Especially of the present invention Non-limiting examples of useful oligonucleotides include the 3 'end, 5' end, or 3 ' And 2′-O-alkylated ribonucleotides at both ends of the At least four or five adjacent nucleotides are so modified. Non-limiting examples of 2'-O-alkylated groups include 2'-O-methyl, 2'- O-ethyl, 2'-O-propyl, and 2'-O-butyl.   Other modified oligonucleotides may have nucleotides at their 3 'and / or 5' ends. Capped with bulky substituents that confer lease resistance, or One non-crosslinking oxygen per leotide has substituents. Such modifications are Or any or all of the nucleoside linkages, At either or both ends and / or within the molecule. .   Below are non-limiting examples of useful unmodified and modified oligonucleotides of the present invention. These are listed in Table 1.   The bold nucleotides in the above oligonucleotides are 2'-O-alkyl All nucleotides are non-phosphodithioate such as phosphorothioate Preferably they are linked by an ester internucleotide bond.   The preparation of these modified oligonucleotides is well known in the art (A Grawar et al. (1992) Trends Biotechnol. 10: 152-158; Agrawal. (1995) Curr. Opin. Biotechno 1.6: 12-19). Was For example, phosphoramidate, H-phosphonate chemistry, or methylphosphorua Share nucleotides using art-recognized techniques such as midate chemistry (Eg, Woolman et al. (1990) Chem. R). ev. 90: 543-584; Agrawar et al. (1987) Tetrahedron Lett. 28: (31): 3539-3542; Caruthers et al. (1987) Meth. Enzymol. 154: 287-313; see U.S. Patent No. 5,149,798). Oh Preparation of ligomer phosphorothioate analogs is based on methoxy phosphoramidite (Eg, Agrawar et al. (1988) Proc. Natl. Acad. Sci. (USA) 85 : 7079-7083) or H-phosphonates (e.g., Flairer) (1986) Tetrahedron Lett. 27: 5575-5578). This can be done using methods well known in the art. By Bargot et al. (J. Chromatog. (1992) 559: 35-42). Can also be.   The synthetic antisense oligonucleotides of the invention in the form of a therapeutic composition Diseases, abnormalities and conditions associated with life, such as, but not limited to Are useful for treating retinal neovascularization, tumor growth and wound healing. Take The method comprises administering to the subject a therapeutically effective amount of a vascular endothelial growth factor. The light synthetic oligonucleotide is administered to the cells. These cells are used for cell culture, May be part of a native tissue culture or an animal such as a human or other mammal May be a part or the whole of the body. Administration is as determined by one of skill in the art. Bolus, intermittent or continuous, depending on condition and response . In some preferred embodiments of the above methods of the invention, the oligonucleotide is localized. (Eg, intraocular or between lesions) and / or systemically. `` Topical administration '' Refers to delivery to a defined area or site in the body, whereas `` systemic administration '' Means the organism by ingestion or by intramuscular, intravenous, subcutaneous or intraperitoneal injection. Delivery to the whole body.   Such methods include retinopathy of prematurity (ROP), diabetic retinopathy, age-related macular degeneration, sickle Erythroid retinopathy, neovascular glaucoma, retinal vein occlusion, and other hypoxic diseases Useful for treating.   The synthetic oligonucleotides of the present invention are physiologically and / or pharmacologically acceptable. It can be used as part of a pharmaceutical composition when used with an acceptable carrier. The characteristics of the carrier will depend on the route of administration. Such compositions comprise synthetic oligonucleotides. In addition to otides and carriers, diluents, fillers, salts, buffers, stabilizers, solubilizers , And other materials well known in the art. Pharmaceutical of the present invention The composition may also promote suppression of VEGF expression or reduce angiogenesis. It may also contain other active agents and / or agents of the wax. For example, each Combination of synthetic oligonucleotides directed to different regions of VEGF mRNA Can be used in the pharmaceutical composition of the present invention. The pharmaceutical composition of the present invention further comprises And nucleosides such as azidothymidine, dideoxycytidine and dideoxyinosine It may contain a tide analog. Such additional factors and / or agents may be Synergistic with the synthetic oligonucleotides of the invention, or Minimizes the side effects caused by light synthetic oligonucleotides To Formulated in a pharmaceutical composition. Conversely, certain anti-VEGF or anti-angiogenic factors and And / or minimize these anti-VEGF or anti-vascular Placing the synthetic oligonucleotide of the present invention in a composition of nascent factors and / or drugs Can be combined.   The pharmaceutical compositions of the present invention may comprise, in addition to other pharmacologically acceptable carriers, the synthetic compositions of the present invention. Liposomes containing oligonucleotides combined with amphiphilic drugs such as lipids The amphipathic drug such as the lipid may be a micelle, an insoluble monolayer in an aqueous solution. , Liquid crystal, or in a coherent form as a lamellar layer. For liposome compositions Suitable lipids include, but are not limited to, monoglycerides, jigs Lyserides, sulfatides, lysolecithin, phospholipids, saponins, bile acids, etc. I can do it. One particularly useful lipid carrier is lipofectin. Such liposomes The preparation of the rubber composition is described, for example, in U.S. Pat. No. 4,235,871, U.S. Pat. No. 501,728; US Pat. No. 4,837,028; and US Pat. No. 4,737. 323, is within the skill of the art. Pharmaceutical composition of the present invention The product can also be obtained from oligonucleases as described by Zhao et al. (In press). A compound such as cyclodextrin that facilitates delivery of leotide into cells, or It may further include a slow release polymer.   As used herein, "therapeutically effective amount" refers to a meaningful patient benefit, i.e., Healing of chronic conditions characterized by angiogenesis or reduction or reduction of angiogenesis itself Sufficient of each active ingredient of the pharmaceutical composition or method to show an increase in the rate of healing of the condition Means the total amount. When referring to an individual active ingredient administered alone, this term Refers to only that component. When referring to a combination, the term shall Therapeutic effects, whether administered in combination, in sequence, or all at once It refers to the total amount of active ingredients that show fruit.   In practicing the treatment or use of the present invention, one, two or more of the present inventions may be used. A therapeutically effective amount of a synthetic oligonucleotide of It is given to patients suffering from abnormalities, or to vascularized tissues. Synthetic oligo of the present invention The nucleotides may be used alone or in accordance with other known angiogenesis therapies, according to the methods of the present invention. It can be administered in combination with an agent. With one or more other therapeutic agents When administered to a subject, the synthetic oligonucleotides of the invention may be co-administered with other therapeutic agents. Or can be administered sequentially. If administered sequentially, the attending physician Determine the appropriate order of administering the synthetic oligonucleotides of the invention in combination with the therapeutic agent. Will be determined.   The synthesis of the invention used in the pharmaceutical composition of the invention or for performing the method of the invention Oligonucleotides can be administered intraocularly, orally, by inhalation, or transdermally, subcutaneously, or intramuscularly. It can be carried out by various conventional routes, such as by intravenous or intravenous injection.   When orally administering a therapeutically effective amount of the synthetic oligonucleotide of the present invention, Synthetic oligonucleotides are tablets, capsules, powders, solutions or elixirs It will be in the form of When administered in tablet form, the pharmaceutical composition of the invention further comprises It may contain a solid carrier such as gelatin or an adjuvant. Tablets, capsules And the powder contains about 5-95% of the synthetic oligonucleotide, preferably about 2%. Contains 5 to 90% synthetic oligonucleotides. When administered in liquid form, Liquid carriers such as water, petroleum ether, oils of animal or vegetable origin, e.g. -Nut oil, mineral oil, soybean oil, sesame oil, or synthetic oil can be added. liquid Pharmaceutical compositions in body form can further comprise saline, dextrose or other sugar solutions, or Or glycols such as ethylene glycol, propylene glycol or It may contain ethylene glycol. When administered in liquid form, The composition comprises about 0.5 to 90% by weight of a synthetic oligonucleotide, preferably about 1 to 50%. % By weight of synthetic oligonucleotide.   Intravenous, subcutaneous, intramuscular, When administered by intraocular or intraperitoneal injection, synthetic oligonucleotides It will be in the form of a heat-free parenterally acceptable aqueous solution. pH, isotonic Preparation of such parenterally acceptable solutions, paying due attention to stability, etc. Is within the skill of the art. For intravenous, subcutaneous, intramuscular, intraperitoneal, or intraocular injection A preferred composition for isotonic vehicles, in addition to synthetic oligonucleotides, For example, sodium chloride injection, Ringer's injection, dextrose injection, dext B Or sodium chloride injection, lactated Ringer's injection, or It must contain other known vehicles. The pharmaceutical composition of the present invention is Or stabilizers, preservatives, buffers, antioxidants, or other additives known to those skilled in the art. Agents may be included.   The amount of the synthetic oligonucleotide in the pharmaceutical composition of the invention depends on the condition to be treated. Will depend on the nature and severity of the condition and the nature of the treatment the patient has previously received. U. Ultimately, the attending physician will provide the necessary synthetic oligonucleotides to treat individual patients. Will determine the amount of tide. Initially, your doctor will need low-dose synthetic oligonucleotides The tide will be administered and the patient's response will be observed. Optimum therapeutic effect is obtained in the patient Increase the dose of the synthetic oligonucleotide to be administered until Do not increase the dose further. Various medicaments used to carry out the method of the invention The composition comprises from about 10 μg to about 20 mg of synthetic orifice per kg of body weight or organ weight. It must contain gonucleotides.   The duration of intravenous therapy using the pharmaceutical composition of the present invention depends on the severity of the disease to be treated. Depending on the severity and condition of each individual patient and the potential idiosyncratic response There will be. Ultimately, the attending physician will administer intravenous therapy using the pharmaceutical composition of the present invention. An appropriate period will be determined.   Some diseases are subject to acute treatment, others require long-term therapy And Proliferative retinopathy includes ROP, some cases of diabetic retinopathy, and angiogenesis The threshold can be reached in a few days as seen in glaucoma. Premature babies also Around 35 weeks of pregnancy, several weeks after birth, there is a risk of neovascularization, and the retina becomes vascularized ( will remain at risk for some time until vascularized. Diabetic retinopathy May be acute, but may continue to smoke for quite a long time during the growth phase . Diabetic retinopathy decreases vascular growth signals with neovascularization or retinal destruction As you do, you are finally stopped.   Both acute and long-term interventions for retinal disease are adequate. For VEGF Intravitreal injection of oligonucleotides that cause retinal neovascularization in acute settings It is an effective suppression means. However, in the case of long-term treatment for many years, , Systemic delivery (intraperitoneal) using a carrier such as saline, slow release polymer or liposome , Intramuscular, subcutaneous, intravenous) should also be considered.   Systemic administration of oligonucleotides in some cases of chronic angiogenic disease Is preferred. Because the disease process involves abnormal and leaky blood vessels, Passing through the gate is not a problem. Therefore, systemic delivery is found to be effective. Injection frequency depends on disease process and biological half-life of oligonucleotide Once a month from continuous infusion.   VEGF-specific antisense oligonucleotides are In addition to inhibiting growth, the cytokine in processes involving angiogenesis It is also useful in determining the role of an application. For example, this technique can And in vitro systems that mimic permeability and Useful in in vivo models of angiogenesis.   A mouse model for oxygen-induced retinal neovascularization has been established And this angiogenesis occurs in 100% of the treated animals and is quantitative (Sumi (1994) Invest. Ophthalmol. Vis. Sci. 35: 101-111). This model Using Dell, increased expression of VEGF message and inner nuclear layer and ganglion cell layer ( That is, determine the correlation between the initiation of retinal neovascularization in Müller cells) (Pierce et al. (1995) Proc. Natl. Acad. Sci. (USA) (printing)). this The results were obtained by Northern blotting of the whole retina at various time points during the development of angiogenesis. And in situ hybridization analysis (Pierce et al., Above).   The oligonucleotides of the invention can also be used in methods of reducing VEGF expression. It is also useful. Target VEGF expression may be in vitro, or It may be in cells that express VEGF. In this method, VE Contacting a nucleic acid specific for GF with an oligonucleotide of the invention, the mR The transcription into NA and / or protein is reduced or suppressed.   Oligonucleotides of the invention can suppress VEGF expression at the protein level This means that human VEGF and human glioblastoma (eg, U373ATC C Accession No. HTB17, American Type Culture Collection, B Kuville, Maryland) and human melanoma (eg, SK-MEL-2, ATCC Accession No. HTB68, American Type Culture Collection, VEGF-expressing cell lines such as Kuville, Maryland; or M21) This can be demonstrated using the resulting ELISA. Briefly, human glioblasts Cell line U373 and human melanoma cell line M21 are VEGF-specific according to the invention. When treated with the target oligonucleotide, these cells are transformed into FIGS. VEGF expression is stopped in a sequence-specific manner as shown in FIGS. Figure 6 shows this Show that modification of the oligonucleotide does not reduce its inhibitory activity. Book The oligonucleotides of the invention were also demonstrated by Northern analysis as described in Example 4 below. VEGF mRNA expression was also reduced.   The role of VEGF in in vivo tumorigenesis was demonstrated by injection as an animal model. Can be demonstrated using athymic mice. M21 cells were touched in about 1-1.5 weeks. It is known that diagnosable tumors occur in mice. Alternatively, Enge Engelbreth Holm Swarm (EMS) tumor mat Rix (Matrigel)^TM, Collaborative Research ive Research), Waltham, Mass.) The passed U373 cell line can be used. VEGF-specific orientation of the invention When an oligonucleotide is injected into a mouse, the expression of VEGF is Reduces tumor weight and volume if reduced by otide or pharmaceutical composition Will be done.   VEGF plays a role in retinal neovascularization, which suggests that Shown using a mouse model. VEGF-specific antisense oligonucleotides Otide (JG-3 (SEQ ID NO: 17), JG-4 (SEQ ID NO: 18), and V m (SEQ ID NO: 19)) and the corresponding sense oligonucleotide (V2 (SEQ ID NO: : 20)), three independent experiments were performed. These oligonucleotides , Known nucleic acid sequence of mouse VEGF (Claffee et al. (1992) J . Biol. Chem. 267: 16317-16322). Vm oligo The nucleotide sequence targets the sequence around the translated TGA stop site (TGA). Is what you do. The sequence of JG-4 is the translation initiation site of the mouse VEGF molecule. And targets the sequence on the 5 ′ side of the ATG. J The sequence of G-3 targets the 5 'untranslated region and has a V2 sense The sequence targets a sequence around the translation start site (ATG).   The following examples illustrate preferred embodiments of making and practicing the present invention, Alternative methods can be used to achieve similar results, thus limiting the scope of the invention It is not intended.                                   Example 1                   Preparation of VEGF-specific oligonucleotide   Human VEGF cDNA in vitro eukaryotic cell transcription kit (Stratagene) (La Jolla, CA). This RNA , Sambrook et al. (1989) Molecular Cloning: a Laboratory Manual; Old Spring Harbor Laboratory Press, New York, Vol. 1 Using T-4 polynucleotide kinase as described on page 5.71. hand³²Label with P. The labeled RNA was converted to a randommer 20-mer library and R RNase H (Boehringer Manha), an enzyme that cleaves NA-DNA duplexes , Indianapolis, Indiana). Open Cleavage patterns are analyzed on a 6% polyacrylamide urea gel. Human VEGF sequence Using a ladder, determine the specific position of the cleavage fragment (Sequenase Kit, UNA Ited States Biochemical, Cree Brand, ohio).   Oligonucleotide having a sequence complementary to the VEGF nucleic acid sequence determined as described above Leotidono synthesis was performed by the phosphoramidite method (eg, Ullmann et al. (Chem. Rev. 1990) 90: 534-583; Agrawal (1992), Trends in Biotech.  10: 152-158; Agrawal et al., (1995) Curr. Opin.Biotechnol, 6: 12-19) using Pharmacia Gene Assemble. Performed on a Pharmacia Gene Assembler series synthesizer Was. After assembly and deprotection, the oligonucleotide was ethanol precipitated twice, Dried and suspended at the desired concentration in phosphate buffered saline (PBS).   The purity of these oligonucleotides was determined by capillary gel electrophoresis and ion exchange. Tested by exchange HPLC. Emit endotoxin levels in oligonucleotide preparations Deformed Cell Assay (Bang (1953) Biol. Bull. (Woods Hole) , Mass. 105: 361-362).                                  Example 2                               Culture of human cells   U373 human neuroblastoma cells (American Type Culture Collection) , Rockville, Maryland, ATCC accession number HTB17) with penicillin / Streptomycin (100IU / MI / 100mcg / ml, Mediatech (Mediatech), Washington, DC) supplemented with glucose (4500 mg / m2). 1) and glutamic acid (2 mM). in 'smodified Earls) (DME) media (Mediatech, Washington, DC) Cultured. Cells are grown at 37 ° C with 10% CO_TwoCultured underneath. Cells are cultured in 96-well tissue Play during a dish (Costar Corp., Cambridge, Mass.) And kept as above. Cell anaerobic chamber (BBL) gas pack (Gas Pak), Cockeysville, MD) under anoxic conditions or 2 50 μM CoCl_TwoPlaced in the presence for 18-20 hours.                                  Example 3                     ELISA VEGF protein test   U373 neuroblastoma cells were plated in 96-well tissue culture dishes and 5 μg / Ml of Lipofectin in the presence of various concentrations of antiserum against human VEGF. The oligonucleotide was treated overnight. These cells are renewed after 12 to 15 hours. Reculture using a medium and an oligonucleotide (without lipofectin), Allowed time to recover. Plates in anaerobic chamber (Gas Pack, Cockeysville, Melilla) For 18-20 hours under hypoxic conditions, or 250 pMCoC l_TwoPlaced in the presence for 18-20 hours. Allow cells to serve as uninduced controls It was maintained under normooxic pressure. This medium was used for the above antigen capture ELISA. Analyzed using the assay (about 24 hours after treatment).   Cultures from cells described in Example 2 were prepared for VEGF protein as follows: Was analyzed as follows. 96-well plate (Maxisorb ELISA Nunc A) / S, Canstrap, Denmark) at 4 ° C with 100 μl / well of capture antibody , A monoclonal antibody against human VEGF (R & D Systems) , Minneapolis, Minnesota, 2.5 μg / ml in 1 × PBS) overnight. Using a plate washer (Dynatech, Guernsey Channel Islands) The wells with 1 × PBS / 0.05% Tween-20 (United States (Biochemical, Cleveland, Ohio) three times. Unspecified in the well Differentiated binding sites were obtained by adding 2% normal human serum (100 μl) and incubating the plate at 37 ° C. For 2 hours. Except for this blocking solution 200 μl of conditioned medium containing human VEGF was added to each well and incubated at 37 ° C. Incubate for 2-3 hours or overnight at 4 ° C. Pre The plate was washed as described above. 100 pl primary antibody (618/619; (2 μg / ml in normal human serum) is added to each well and incubated at 37 ° C. for 1-2 hours. I was vate. The primary antibody was affinity-purified rabbit anti-human VEGF polyclonal. It was a nal antibody. Plates were washed as described above. 100 μl test Horseradish peroxidase labeled goat anti-mouse IgG monoclonal Antibodies (1: 10,000) Vector Laboratories , Burlingame, CA) to each well and incubate at 37 ° C for 1 hour. Was added. Plates were washed as described above. TMB Micro Welpe Luoxidase coloring system (Kirkegaard and Perry) Perry), Gaithers Burke, Maryland) (Ceres) 900 Plate Reader (Biotech Instruments) (Bio-Tek Instruments, Inc.), Winston Key, VT). It was quantified at 0 nm. The linear range for this assay is 2 ng to 0.01 ng human VEG. F. Representative results are shown in FIGS.                                  Example 4                           Northern blotting   Inhibition of VEGF expression occurs in cells in the presence of the oligonucleotide of the invention Northern blotting was performed to determine levels. Described in Example 2 above Human U373 cells cultured as described above were plated in a 100 mm tissue culture dish. Oligonucleotides of 0.05 μM, 0.5 μM and 2.0 μM, respectively. C. H-3 (SEQ ID NO: 1) and a sense control (SEQ ID NO: 21) at 0. 05 μM, 0: 5 μM and 2.0 μM are each 5 μg as a lipid carrier. / Ml lipofectin (Gibco-BRL) Gaithersburg, MD) For 12 hours. After 7-8 hours, these cells are It was recultured. Place cells in hypoxia for 18-20 hours or 250 μMC oCl_TwoPlace in the presence for 18-20 hours, choose Chomczynski and And Sacchi (Anal. Biochem. (1987) 162: 1). 56-159) Single Step Acid Guanidinium Thiocyanate-Phenol-Chromate Total RNA was isolated by the chloroform extraction method. Northern blotting, sambu Look et al.'S method (Molecular Cloning: a Laboratory Manual, Cold Spring Ghaber Laboratory Press, New York (1989) Vol. 1, 7.38 ) Or the method of Artera Napan Lirio (Meth. Enz. (1993) 225: 30). 3-328). Phosphor imager for all RNA signals (Phosphorimager) (BioRad, Hercules, CA) ) And a 36B4 cDNA probe (Laborda (1991) N uc1eic Acids Res. 19: 3998). RNA expression is the present invention Decreased in the presence of the VEGF-specific oligonucleotide, but did not Not significantly affected by the presence of the oligonucleotide Was.                                  Example 5                                In vivo test A. Matrigel test   U373 neuroblastoma cells were transfected with 5 μg / ml lipofectin (Gibco-BRL, Gaithersburg, MD) in the presence of 0.5 μM antisense phospho Rothioate oligodeoxynucleotide (H-3, SEQ ID NO: 54) or pair (H3-sense phosphorothioate oligonucleotide; SEQ ID NO: 74) Treated for 7 hours. 1 × 10⁶250 μl of the cells treated with Trigel^TM(Collaborative Research, Waltham, Mass .; 10 ~ 12 mg / ml), and athymic mice (about 20 g) 6-8 weeks old (chars) Char1es River Laboratories, Wilmint (Massachusetts, Mass.). These cells Expressed increased levels of VEGF in response to hypoxia. Keep the mouse unlimited And sacrificed 8 days after injection. The skin was incised to expose the Matrigel pellet. Surrounding Rough photography of the blood vessels was performed using a Zeiss microscope. Matrigel Plugs were removed and formalin-fixed for paraffin embedding and histological analysis . Tissue sections are stained with hematoxylin and eosin and placed in Matrigel plugs. Vascular growth was quantified.   Injection of Matrigel alone resulted in a clear plug with no apparent angiogenesis. won. Matrigel plugs combined with U373 neuroblastoma cells are visible Bleeding was included. In addition, the capillary bed around the plug stains darker, The tube was more winding. With cells treated with antisense oligonucleotides Athymic mice, both injected with Matrigel plugs, were untreated with Matrigel plugs Injected with cells or cells pretreated with control oligonucleotide Angiogenesis was less than that of. Matrigelp containing antisense treated cells The lag also had less visible bleeding. These results were It is suggested that nucleotide treatment suppresses VEGF-induced angiogenesis. B. Tumor testing   Six-week-old athymic mice (about 20 g) were collected from Charles River Laboratories To buy. Human melanoma M2 placed through athymic mice in the presence of Matrigel One cell or human neuroblastoma U373 cell is injected subcutaneously into the flanks of athymic mice (2-20 × 10⁶). Palpable tumors form in 1-2 weeks. Antise Subcutaneous injection of a sense or sense control oligonucleotide one day after tumor cell injection To start. Once the oligonucleotide concentration was determined, it was in the range of 5-50 mg / kg. It is an enclosure. The animals are then injected for 3 weeks. Then the animals were slaughtered, Remove the tumor. First, the tumors are analyzed for weight and volume. Further analysis Include anti-human VEGF / VPF monoclonal antibodies (R & D Systems, Minnea Police, Minnesota) for VEGF / VPF protein or in Cut VEGF / VPF RNA using in situ hybridization Includes stripping and staining. Antisense Oligonucleotide of the Invention Mice injected with the drug have smaller tumors than mice injected with the vehicle or control. It is expected to have ulcers.                                  Example 6                          Animal model of retinal neovascularization A. Preparation of oligonucleotide   The following oligonucleotides: JG-3 (SEQ ID NO: 17), JG-4 (SEQ ID NO: 17) : 18), Vm (SEQ ID NO: 19), and V2 (SEQ ID NO: 20) Performed as described in Example 1. B. Preparation of animal model   7 day old mice (P7, C57b1 / 6J, Children's Hospital Leading Facilities (Children's Hospital BreedingFacilities) , Boston, Mass.) With a Bird 3-M oxygen blender (flow) Speed: 1.5 liters / minute; Bird, Palm Springs, CA) 5 days of oxygen overload (75 ± 2) in a sealed sealed incubator %) Exposure to oxygen. The oxygen concentration can be measured with an oxygen analyzer (Beckman, (Model D2, Irvine, CA). 5 days later (P12 ), The mice were returned to room air. 5 days after returning to room air (P17) Maximum retinal neovascularization was observed. After P12, the level of retinal neovascularization is just remission Was about to start. C. treatment   After removing the mouse from oxygen, a Hamilton syringe and a 33 gauge needle (Hami1ton Company, Reno, Nevada) The chisense oligonucleotide was injected into the vitreous. Abel animals for treatment Anesthesia was achieved by intraperitoneal administration of tin. Antisense oligonucleotide at P12 in mice A single injection of otide (or a sense or non-sense control) The final concentration was about 30 pM. Animals were injected intraperitoneally with tribromoethanol at P17 (0.1 ml / g body weight) and sacrificed by cervical dislocation. D. Microscope observation   The eyes are enucleated and fixed in 4% paraformaldehyde, and the gene, Embedded. Serial sections of all eyes were sagittal cut through the cornea and parallel to the optic nerve. these Sections were stained with hematoxylin and periodate-Schiff (PAS) staining. place The degree of angiogenesis in the ocular placement is determined by the endothelial cells that extend into the vitreous past the inner limiting membrane. Determined by counting vesicle nuclei. Nuclear and vascular profiles from new vessels Wheels can be distinguished from other structures in the retina and counted in cross-sections using an optical microscope did. Another eye section was also prepared and the in situ hybridizer to the VEGF probe Investigated by isisation.   To examine the retinal vascular network using fluorescein-dextran, four mice were High molecular weight fluorescein-dextran (%) Perfusion with 50 mg / ml solution of Chemical Company, St. Louis, MO did. Remove eyes, fix in paraformaldehyde, glycerol-gelatin A flat-mounted slide was prepared by using. Olympus B X60 fluorescence microscope (Olympus America Corp.) Observation and photographing by fluorescence microscopy using Lingham, Mass. Shadowed.Equivalent   Those skilled in the art will recognize many equivalents to the specific embodiments of the invention described herein. It can be ascertained using only or routine experimentation. So And the like are intended to be encompassed by the following claims.

────────────────────────────────────────────────── ─── Continuation of front page    (81) Designated countries EP (AT, BE, CH, DE, DK, ES, FI, FR, GB, GR, IE, IT, L U, MC, NL, PT, SE), OA (BF, BJ, CF) , CG, CI, CM, GA, GN, ML, MR, NE, SN, TD, TG), AP (KE, LS, MW, SD, S Z, UG), UA (AM, AZ, BY, KG, KZ, MD , RU, TJ, TM), AL, AM, AT, AU, AZ , BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, FI, GB, GE, H U, IL, IS, JP, KE, KG, KP, KR, KZ , LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, P T, RO, RU, SD, SE, SG, SI, SK, TJ , TM, TR, TT, UA, UG, UZ, VN

Claims (1)

[Claims]   1. SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 and And a human selected from the group consisting of oligonucleotides having SEQ ID NO: 16 Synthetic oligonucleotides complementary to nucleic acid sequences specific for vascular endothelial growth factor.   2. 2. The oligonucleotide according to claim 1, having SEQ ID NO: 2.   3. 2. The oligonucleotide according to claim 1, having SEQ ID NO: 3.   4. 2. The oligonucleotide according to claim 1, having SEQ ID NO: 4.   5. 2. The oligonucleotide according to claim 1, having SEQ ID NO: 5.   6. 2. The oligonucleotide according to claim 1, having SEQ ID NO: 6.   7. 2. The oligonucleotide according to claim 1, having SEQ ID NO: 7.   8. 2. The oligonucleotide according to claim 1, having SEQ ID NO: 8.   9. 2. The oligonucleotide according to claim 1, having SEQ ID NO: 9.   10. 2. The oligonucleotide according to claim 1, having SEQ ID NO: 10.   11. 2. The oligonucleotide according to claim 1, having SEQ ID NO: 11.   12. 2. The oligonucleotide according to claim 1, having SEQ ID NO: 12.   13. 2. The oligonucleotide according to claim 1, having SEQ ID NO: 13.   14． 2. The oligonucleotide according to claim 1, having SEQ ID NO: 14.   15. 2. The oligonucleotide according to claim 1, having SEQ ID NO: 15.   16. 2. The oligonucleotide according to claim 1, having SEQ ID NO: 16.   17． Alkyl phosphonates, phosphorothioates, phosphorodithioates, Phosphate esters, alkyl phosphonothioates, phosphoramidates, carbamates Salts, carbonates, phosphate triesters, acetamidodates, and carboxylic acids The group consisting of cimethyl ester internucleotide linkages and combinations thereof The oligonucleotide of claim 1 having a modification selected from:   18. Has at least one phosphorothioate internucleotide linkage The oligonucleotide of claim 17, wherein   19. 19. The method according to claim 18, which has a phosphorothioate internucleotide linkage. The oligonucleotide as described.   20. 2. The method according to claim 1, which consists essentially of 2'-O-alkylated ribonucleotides. The oligonucleotide as described.   21.4 or 5 5'2'-O-alkylated ribonucleotides. Item 6. The oligonucleotide according to Item 1.   22.4 or 5 comprising 3'2'-O-alkylated ribonucleotides. Item 6. The oligonucleotide according to Item 1.   Claims comprising 23.4 or 5 3'2'-O-alkylated ribonucleotides. Item 22. The oligonucleotide according to Item 21,   Claims comprising 24.4 or 5 5'2'-O-alkylated ribonucleotides. Item 19. The oligonucleotide according to Item 18.   25. VEGF-specific nucleic acid sequence and oligonucleotide according to claim 1 A method for suppressing VEGF expression, comprising a step of contacting.   26. 18. A nucleic acid sequence specific for VEGF and a synthetic oligonucleotide according to claim 17. A method for suppressing VEGF expression, comprising a step of contacting a tide.   27. 2. At least one synthesis according to claim 1, in a physiologically acceptable carrier. A pharmaceutical composition comprising the oligonucleotide.   28. 18. At least one combination according to claim 17 in a physiologically acceptable carrier. A pharmaceutical composition comprising a synthetic oligonucleotide.