JP2009000106A - Nucleic acid-based modulation of female reproductive disease and condition - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for treating diseases or conditions associated with the levels of expressions of vascular endothelial growth factor (VEGF), and to provide vascular endothelial growth factor receptor. <P>SOLUTION: The nucleic acid molecule includes, for example, a dsRNA, siRNA, antisense, 2,5-A chimera, aptamer and enzymatic nucleic acid molecule such as hammerhead ribozyme, DNAzyme and allozyme, modulates the expression of vascular endothelial growth factor (VEGF) and/or vascular endothelial growth factor receptor (VEGFr) gene. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

This application is filed in US Patent Application 60 / 334,461 to Sandberg et al. (Filed Nov. 30, 2001, entitled “Method and Reagent for the Mod”).
ulation of Female Reproductive Diseases and Conditions "), and Pavco et al., US Patent Application 10/138,
674 (filed on May 3, 2002), the latter being a continuation-in-part of Pavco et al. US patent application 09 / 870,161, which is a Pavco et al. US patent application 09 / 708,690 (filed November 7, 2000), which is a continuation-in-part of Pavco et al., US patent application 09 / 371,722 (filed August 10, 1999). Yes, this is a continuation-in-part of Pavco et al., US patent application 08 / 584,040 (filed January 11, 1996), which is a Pavco et al. US patent application 60 / 005,974 (October 1995). Claims priority based on 26th application. The titles of these earlier applications are “Method and Reagent for Treatmen”
t of Diseases or Conditions Related to Levels of Vessel Endothelial Growth Factor Receptor. Each of these applications is hereby incorporated by reference in its entirety, including the drawings and tables.

TECHNICAL FIELD This invention relates to methods and reagents for the treatment of diseases or conditions associated with levels of expression of vascular endothelial growth factor (VEGF) and vascular endothelial growth factor receptor. In particular, the invention relates to diseases and conditions associated with angiogenesis, such as, but not limited to, cancer, tumor angiogenesis, or ophthalmic indications such as diabetic retinopathy, or age-related muscularity. Degeneration, proliferative diabetic retinopathy, hypoxia-induced angiogenesis, rheumatoid arthritis, psoriasis, wound healing, endometriosis, endometrial carcinoma, gynecological bleeding disorder, irregular menstrual cycle, ovulation, Modulates the expression of vascular endothelial growth factor and / or vascular endothelial growth factor receptor, eg, VEGFR1 and / or VEGFR2, useful for the prevention, treatment, management and / or diagnosis of premenstrual syndrome (PMS) and menopausal dysfunction Featuring nucleic acid-based molecules and methods.

  The following is a description of the related art, and none of these are admitted to be prior art to the present invention.

VEGF, also called vascular permeability factor (VPF) and vasculotropin, is a potent and highly specific mitogen for vascular endothelial cells (for review see Ferrara, 1993 Trends Cardiovas. Med. 3). , 244; Neufeld
et al. , 1994, Prog. Growth Factor Res. 5, 89). VEGF-induced neovascularization can result in a variety of pathogenic conditions such as tumor angiogenesis or ocular indications such as diabetic retinopathy or age-related muscular degeneration, proliferative diabetic retinopathy, It has been implicated in hypoxia-induced angiogenesis, rheumatoid arthritis, psoriasis, wound healing and others.

VEGF is an endothelial cell-specific mitogenic factor and is a 34-45 kDa glycoprotein having a wide range of activities such as promoting angiogenesis, enhancing vascular permeability and other activities. VEGF belongs to the platelet-derived growth factor (PDGF) family of growth factors and has about 18% homology at the amino acid level with the A and B chains of PDGF. In addition, VEGF contains eight conserved cysteine residues common to all growth factors belonging to the PDGF family (Neufeld et al., Supra). The VEGF protein is thought to exist primarily as a disulfide bond homodimer, and VEGF monomers have been shown to be inactive (Plouet et al., 1989 EMBO J. 8, 3801).

VEGF exerts its effect on vascular endothelial cells by binding to specific high affinity cell surface receptors. Covalent cross-linking experiments using ¹²⁵ I-labeled VEGF protein identified three high molecular weight complexes of 225, 195 and 175 kDa that were presumed to be VEGF and VEGF receptor complexes (Vaisman et al., 1990 J. Biol. Biol.Chem.265, 19461). Based on these studies, VEGF-specific receptors with molecular weights of 180, 150 and 130 kDa were predicted. 150 and 130 kDa receptors have been identified in endothelial cells. VEGF receptors belong to a superfamily of receptor tyrosine kinases (RTKs) characterized by a conserved cytoplasmic catalytic kinase domain and hydrophilic kinase sequences. The extracellular domain of the VEGF receptor is composed of seven immunoglobulin-like domains that are thought to be involved in VEGF binding function.

The two most abundant and high affinity receptors for VEGF are Shibuya et al. (1990).
Flt-1 (VEGFR1) (fms-like tyrosine kinase) cloned by Oncogene 5,519), and Terman et al. (1991 Oncogene).
6, 1677) KDR (VEGFR2) (kinase insert domain containing receptor). The murine homologue of KDR cloned by Mathews et al. (1991, Proc. Natl. Acad. Sci., USA, 88, 9026) has 85% amino acid homology with KDR and flk-1 (fetal liver kinase-1 ). High affinity binding of VEGF to its receptor is regulated by cell surface associated heparin and heparin-like molecules (Gitay-Goren et al., 1992 J. Biol. Chem. 267, 6093).

  VEGF expression has been associated with several pathological conditions, such as tumor angiogenesis, some forms of blindness, rheumatoid arthritis, psoriasis and others. Furthermore, many studies have shown that VEGF is necessary and sufficient for neovascularization. Takashita et al. (1995 J. Clin. Invest. 93, 662) show that a single dose of VEGF increased collateral vessel development in a rabbit model of ischemia. VEGF can also induce neovascularization when injected into the cornea. Expression of the VEGF gene in CHO cells is sufficient to confer oncogenic potential to the cells. Kim et al. (Supra) and Millauer et al. (Supra) inhibited tumor-induced neovascularization using monoclonal antibodies to dominant negative forms of VEGF or VEGFR2 receptor.

  During development, VEGF and its receptor are associated with areas of new blood vessel growth (Millauer et al., 1993 Cell 72, 835; Shalaby et al., 1993 J. Clin. Invest. 91, 2235). Furthermore, transgenic mice lacking any of the VEGF receptors are incompletely vascularized and these mice do not survive. VEGFR2 appears to be required for endothelial cell differentiation, and VEGFR1 appears to be required at a later stage of angiogenesis (Shalaby et al., 1995 Nature 376, 62; Fung et al., 1995 Nature 376). 66). That is, these receptors appear to be necessary to properly signal endothelial cells or their precursors to respond to pro-angiogenic stimuli.

  Increasing evidence suggests that the VEGF family may also be involved in both the pathogenesis and maintenance of peritoneal endometriosis. Peritoneal endometriosis is a highly debilitating gynecological disorder with a high prevalence. Currently, it is generally accepted that the pathogenesis of peritoneal endometriosis involves removal of the detached endometrium. Maintenance of exfoliated endometrial tissue depends on the production and maintenance of a large blood supply both within and around the ectopic tissue.

  Endometriosis is an estimated disease affecting 77 million women and teenagers worldwide. Endometriosis is the leading cause of infertility, chronic pelvic pain and hysterectomy. Endometriosis can be characterized by the fact that endometrial tissue (tissue inside the uterus that is made up every month and detaches during menstruation) is found in another area outside the uterus of the body. Endometrial tissue can degrade and bleed in response to hormonal instructions every month. However, unlike the endometrium, these tissue deposits have no way of leaving the body. The result is internal bleeding, blood degeneration and sequestration from tissue growth, inflammation of the surrounding area, expression of stimulating enzymes and formation of scar tissue. In addition, depending on the location of growth, interference with other areas of the intestine, bladder, small intestine and pelvic cavity can occur. Endometrial tissue has been found to remain in the skin and other extrapelvic locations, such as the arms, feet, and even the brain.

  At present, the presence of endometriosis can only be confirmed by surgery, such as laparoscopic examination, but can be inferred based on symptoms, physiological findings and diagnostic tests. Endometriosis can be treated by a variety of methods, both surgical and drug. Most commonly, surgery is done to remove by removal, removal, electrical destruction, cauterization or other methods, and the adhesions are also removed. Surgery includes, but is not limited to, laparoscopy; laparotomy; anterior and sacral sacrum and various levels of hysterectomy to remove some or all of the reproductive organs. Often, this method reduces only the symptoms associated with growth in the reproductive organs, not the intestines or kidneys and associated areas where endometriosis may be present.

  There are several drugs that are used either alone or in combination with surgery to treat endometriosis. These include contraceptives, GnRH agonists, and / or synthetic hormones. GnRH agonists are commonly used in women at all stages of the disease and sometimes cause severe side effects. GnRH (gonadotropin releasing hormone) analogs can be divided into two groups: agonists and antagonists. Agonists are commonly used in the treatment of endometriosis by inhibiting the production of follicle stimulating hormone (FSH) and luteinizing hormone (LH), which are common hormones required for ovulation. If they are not secreted, the body enters "pseudomenopause" and stops further transplant growth. However, even in this case, there are only temporary measures that can be used only for short-term intervals. Once the body returns to its normal state, endometriosis begins to transplant itself again.

Angiogenesis appears to be involved in the pathogenesis of endometriosis. According to the transplantation theory, when exfoliated endometrium adheres to the peritoneal layer, the establishment of a new blood supply is essential for the survival of endometrial transplantation and the development of endometriosis (Donnez et al. 1998, Hum.Reprod., 13, 1686-1690). Postmenstrual endometrial growth and repair are closely associated with angiogenesis. Abnormalities in these processes result in excessive or unpredictable bleeding patterns, which are common for many women. Therefore, it is important to understand which factors regulate normal endometrial angiogenesis. Vascular endothelial growth factor (VEGF) is an endothelial cell-specific mitogen that plays an important role in normal and diseased angiogenesis (Fasciani et al., 2000, Mol. Hum. Reprod., 6). Sharkey et al., 2000, J. Clin. Endocrinol. Metab., 85, 402-409). Origins of this factor include ectopic endometrium, ectopic endometrial tissue and peritoneal fluid macrophages. What is important about this etiology is the proper peritoneal environment in which the detached endometrium is inoculated and transplanted. Secondly, established ectopic tissue depends on the peritoneal environment, ie, the environment that supports angiogenesis, for its survival. Increased knowledge about the involvement of the VEGF family in endometrial angiogenesis creates the potential for new methods for its medical management, with a particular focus on anti-angiogenesis control of VEGF action. (McLaren, 2001, Hum. Reprod. Update, 6, 45-55).

  Pavco et al. (International Publication No. WO 97/15662) describe methods and reagents for treating diseases or conditions associated with levels of vascular endothelial growth factor receptor.

  Robinson (International Publication No. WO 95/04142) describes the inhibition of VEGF expression using certain antisense oligonucleotides that target VEGF RNA.

  Jellinek et al. (1994 Biochemistry 33, 10450) describe the use of specific VEGF-specific high affinity RNA aptamers to inhibit the binding of VEGF to its receptor.

  Rockwell and Goldstein (International Publication WO 95/21868) describe the use of certain anti-VEGF receptor monoclonal antibodies to neutralize the effects of VEGF on endothelial cells.

  Pappa (International Publication No. WO 01/32920) describes specific genes such as cathepsin D, AEBP-1, stromelysin-3, cystatin B, protease inhibitor 1, sFRP4, gelsolin, IGFBP-3, bispecific phosphatase 1, PAEP, Ig gamma chain, ferritin, complement component 3, pro-alpha-1 type III collagen, proline 4-hydroxylase, alpha-2 type I collagen, claudin-4, melanoma adhesion protein, procollagen C-endo Peptidase enhancer, nascent polypeptide-related complex alpha polypeptide, elongation factor 1 alpha (EF-1-alpha), vitamin D3 25 hydroxylase, CSRP-1, steroid-induced acute regulatory protein, apolipoprotein E, transcobalami II, prosaposin, early growth response 1 (EGR1), ribosomal protein S6, adenosine deaminase RNA-specific protein, RAD21, guanine nucleotide binding protein beta polypeptide 2-like 1 (RACK1) and podocalyxin gene (all of which are endometrium Inhibitors, such as certain ribozymes and antisense nucleic acid molecules that are expressed differently in tissues in individual patients with the disease.

  Laberbera et al. (International Publication WO 00/73416) describe specific antisense nucleic acid molecules that target the follicle stimulating hormone receptor.

  Storella et al. (International Publication No. WO 99/63116) describe modulators of prothymosin gene products, such as certain ribozymes and antisense nucleic acid molecules, for the treatment of endometriosis.

SUMMARY OF THE INVENTION The present invention relates to nucleic acid based molecules such as enzymatic nucleic acid molecules, allozymes, antisense nucleic acids s, 2-5A antisense chimeras, triplex-forming oligonucleotides, decoy RNAs, dsRNAs, siRNAs, aptamers, and nucleic acids. Features antisense nucleic acids containing a cleaved chemical group, and methods for modulating vascular endothelial growth factor (VEGF) and / or vascular endothelial growth factor receptor (VEGFr) gene expression. Non-limiting examples of genes encoding the vascular endothelial growth factor receptor of the present invention include VEGFR1, VEGFR2 or combinations thereof. In particular, the invention relates to angiogenesis-related diseases and conditions, such as, but not limited to, tumor angiogenesis, cancers such as breast cancer, lung cancer, colorectal cancer, kidney cancer, pancreatic cancer, or melanoma, or eye Sexual indications such as diabetic retinopathy, or age-related muscular degeneration, and female reproductive diseases and conditions such as, but not limited to, endometriosis, endometrial carcinoma, gynecological bleeding disorders, irregular menstruation Vascular endothelial growth factor and / or vascular endothelial growth factor receptor (eg, VEGFR1 and / or useful for prevention, treatment, management, and / or diagnosis of cycle, ovulation, premenstrual syndrome (PMS), and menopausal dysfunction Features nucleic acid-based molecules and methods that regulate the expression of VEGFR2).

  In one aspect, the invention features molecules and methods based on one or more nucleic acids that modulate the expression of a gene encoding a vascular endothelial growth factor receptor, independently or in combination. In particular, the present invention relates to tumor angiogenesis, cancer such as breast cancer, lung cancer, colorectal cancer, kidney cancer, pancreatic cancer, or melanoma, or ophthalmic indications such as diabetic retinopathy or addition. Age-related muscular degeneration, and female reproductive diseases and conditions such as, but not limited to, endometriosis, endometrial carcinoma, gynecological bleeding disorders, irregular menstrual cycle, ovulation, premenstrual syndrome (PMS), and VEGF (eg, Genbank accession number NM_003376), VEGFR1 receptor (eg, Genbank accession number NM_002019), and VEGFR2 receptor (eg, Genbank accession number NM_002253) useful for the prevention, treatment, management and / or diagnosis of menopausal dysfunction Characterized by a nucleic acid molecule that regulates the expression of.

In one embodiment, the present invention provides compounds of formula I: (SEQ ID NO: 5977)
5'-u _s a _s c _s a _s au uc U GAu Gag gcg aaa gcc Gaa Aag aca aB-3 '
[Wherein each a is a 2′-O-methyladenosine nucleotide, each g is a 2′-O-methylguanosine nucleotide, each c is a 2′-O-methylcytidine nucleotide, and each u is 2 '-O-methyluridine nucleotides, each A is adenosine, each G is guanosine, each s independently represents a phosphorothioate internucleotide linkage, U is 2'-deoxy-2'-C -Allyluridine, B is an inverted deoxy abasic component]
Characterized by a compound having This compound is also referred to as ANGIOZYME ™ ribozyme.

In another aspect, the present invention provides:
Formula II: (SEQ ID NO: 5978)
5'-g _s a _s g _s u _s ugc U GAuGagg ccgaaa ggccGaaAgucugB-3 '
[Wherein each a is a 2′-O-methyladenosine nucleotide, each g is a 2′-O-methylguanosine nucleotide, each c is a 2′-O-methylcytidine nucleotide, and each u is 2 '-O-methyluridine nucleotides, each A is adenosine, each G is guanosine, each s independently represents a phosphorothioate internucleotide linkage, U is 2'-deoxy-2'-C -Allyluridine, B is an inverted deoxy abasic component]
Characterized by a compound having

  In one aspect, the invention features a composition comprising a nucleic acid molecule of the invention in a pharmaceutically acceptable carrier. In another aspect, the invention features a composition comprising a compound of Formula I and / or Formula II in a pharmaceutically acceptable carrier or diluent.

  In one aspect, the invention features a method of administering a nucleic acid molecule of the invention to a cell, eg, a mammalian cell (eg, a human cell), the method comprising, for example, delivery under conditions suitable for administration. Contacting the cell with a nucleic acid molecule in the presence of a reagent (eg, lipid, cationic lipid, phospholipid, or liposome). In another aspect, the invention features a method of administering a compound of Formula I and / or Formula II to a cell, eg, a mammalian cell (eg, a human cell), under conditions suitable for administration. And, for example, contacting the cell with the compound in the presence of a delivery reagent (eg, lipid, cationic lipid, phospholipid, or liposome).

  In one aspect, the invention features a mammalian cell comprising a nucleic acid molecule of the invention, where the mammalian cell is, for example, a human cell. In another embodiment, the invention also features a mammalian cell comprising a compound of formula I and / or formula II, wherein the mammalian cell is a human cell, for example.

  In one embodiment, the present invention provides an angiogenesis, eg, tumor angiogenesis, or an ocular indication, eg, diabetic retinopathy, or age-related muscular degeneration, or intrauterine in a subject. Characterized by a method of inhibiting membrane neovascularization, the method comprises contacting a subject with a nucleic acid molecule of the invention under conditions suitable for inhibition. In another aspect, the present invention relates to angiogenesis, such as tumor angiogenesis, or ocular indications such as diabetic retinopathy, or age-related muscular degeneration, or endometrial neoplasia in a subject. Characterized by a method of inhibiting angiogenesis, the method comprises contacting a subject with a compound of formula I and / or formula II under conditions suitable for inhibition.

  In another aspect, the invention features a method of treating a subject having an ophthalmic condition associated with elevated levels of VEGF receptor, such as diabetic retinopathy, or age-related muscle degeneration. Contacting the subject's cells with a nucleic acid molecule, eg, an enzymatic nucleic acid molecule that targets VEGF receptor RNA, eg, a molecule of formula I and / or II, under conditions suitable for treatment.

  In another aspect, the invention relates to conditions associated with elevated levels of VEGR and / or VEGF receptors, such as tumor angiogenesis, cancer such as breast cancer, lung cancer, colorectal cancer, kidney cancer, pancreatic cancer, or Melanoma, ophthalmic disease or indication, such as diabetic retinopathy, or age-related muscular degeneration, rheumatoid arthritis, psoriatic endometriosis, endometrial carcinoma, gynecological bleeding disorder, irregular menstruation Characterized by a method of treating a subject having a cycle, ovulation, premenstrual syndrome (PMS), or menopausal dysfunction, wherein the method comprises subjecting a subject's cells to a nucleic acid molecule of the invention, such as, for example, Contacting with a compound of formula I and / or formula II.

  In yet another embodiment, the treatment methods of the invention further comprise using one or more drug therapies under conditions suitable for treatment. Non-limiting examples of other drug therapies that can be used in combination with the nucleic acid molecules of the invention include 5-fluorouridine, leucovorin, irinotecan (CAMPTOSAR® or CPT-11 or camptothecin-11 or campto), Paclitaxel, or carboplatin, GnRH (gonadotropin-releasing hormone) agonist, leupron depo (leuprolide acetate), cinaler (naferaline acetate), zolodex (gosererum acetate), suprefact (buserelem acetate), donazole, or oral contraceptives, for example This includes, but is not limited to, depoprovera or provera (medroxyprogesterone acetate), or any other estrogen / progesterone contraceptive.

In one aspect, the invention features a method of administering a nucleic acid molecule of the invention to a mammal, eg, a human, under conditions suitable for administration, eg, a delivery reagent (eg, lipid , Cationic lipids, phospholipids, or liposomes) in the presence of a mammal. In another aspect, the invention features a method of administering a compound of Formula I and / or Formula II to a mammal, eg, a human, the method under conditions suitable for administration, eg, , Contacting with a compound in the presence of a delivery reagent (eg, lipid, cationic lipid, phospholipid, or liposome).

  In one aspect, the invention features a nucleic acid molecule that downregulates expression of a vascular endothelial growth factor (VEGF) and / or vascular endothelial growth factor receptor (VEGFr) gene, wherein the VEGFr gene comprises: For example, VEGFR1 or VEGFR2 or any combination thereof is included.

  In one embodiment, the nucleic acid molecules of the invention, eg, enzymatic nucleic acid molecules, antisense nucleic acid molecules, 2-5A antisense chimeras, triplex-forming oligonucleotides, decoy RNAs, dsRNAs, siRNAs, aptamers, or nucleic acid cleavage chemistry An antisense nucleic acid containing a group is present in tumor angiogenesis, cancer such as breast cancer, lung cancer, colorectal cancer, kidney cancer, pancreatic cancer, or melanoma, ophthalmic disease or ophthalmic indication, such as diabetic retina Or age-related muscular degeneration, rheumatoid arthritis, psoriasis endometriosis, endometrial carcinoma, gynecological bleeding disorders, irregular menstrual cycle, ovulation, premenstrual syndrome (PMS), or menopausal dysfunction It is suitable for treatment, management and / or diagnosis.

  Such nucleic acid molecules are also found in diabetic retinopathy, muscular degeneration, neovascular glaucoma, myopic degeneration, vulgaris, nodular sclerosis adenofibroma, flaming nevus, Sturge-Weber syndrome, Klipel It is also useful for the prevention of diseases and conditions such as Tornonnay-Weber syndrome, Osler-Weber-Rendu syndrome, and other diseases or conditions associated with the level of VEGFR1 or VEGFR2 in cells or tissues.

  In another aspect, the invention features a composition comprising a nucleic acid molecule of the invention in a pharmaceutically acceptable carrier or diluent.

  In another embodiment, an enzymatic nucleic acid molecule, antisense nucleic acid molecule, 2-5A antisense chimera, triplex-forming oligonucleotide, decoy RNA, dsRNA, siRNA, aptamer, or nucleic acid cleavage chemical group of the invention. Sense nucleic acids are adapted for fertility regulation.

  In one embodiment, the enzymatic nucleic acid molecule of the present invention is a hammerhead, inozyme, tinzyme, DNAzyme, amberzyme, or G-cleaving agent configuration.

  In one embodiment, the enzymatic nucleic acid molecule of the invention comprises 8-100 bases complementary to the RNA of the VEGFR1 and / or VEGFR2 gene. In another embodiment, the enzymatic nucleic acid molecule of the invention comprises 14-24 bases complementary to the RNA of the VEGFR1 and / or VEGFR2 gene.

In one embodiment, the siRNA molecules of the invention comprise double stranded RNA, wherein one strand of RNA is complementary to the RNA of the VEGFR1 and / or VEGFR2 gene. In another embodiment, the siRNA molecules of the invention comprise double stranded RNA, wherein one strand of RNA comprises a portion of the sequence of RNA having a VEGFR1 and / or VEGFR2 sequence. In yet another embodiment, the siRNA molecules of the invention comprise double stranded RNA, wherein both strands of the RNA are joined by a non-nucleotide linker. Alternatively, siRNA molecules of the invention comprise double stranded RNA in which both strands of RNA are connected by a nucleotide linker, such as a loop or stem-loop structure.

  In one embodiment, the single-stranded component of the siRNA molecule of the invention is about 14 to about 50 nucleotides in length, and in another embodiment, the single-stranded component of the siRNA molecule of the invention is about 14 , 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides in length. In yet another embodiment, one of the siRNA molecules of the present invention The strand component is about 23 nucleotides in length, and in one embodiment the siRNA molecule of the invention is about 28 to about 56 nucleotides in length, and in another embodiment the siRNA molecule of the invention is about 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, or 52 nucleotides in length. In yet another embodiment, the siRNA molecules of the invention are about 46 nucleotides in length.

  In one embodiment, an antisense nucleic acid molecule, antisense nucleic acid molecule, 2-5A antisense chimera, triplex-forming oligonucleotide, decoy RNA, dsRNA, siRNA, aptamer, or nucleic acid cleavage chemical group of the invention. Nucleic acids are chemically synthesized.

  In another embodiment, an antisense nucleic acid molecule, antisense nucleic acid molecule, 2-5A antisense chimera, triplex-forming oligonucleotide, decoy RNA, dsRNA, siRNA, aptamer, or nucleic acid cleavage chemical group of the invention. The nucleic acid contains at least one 2'-sugar modification.

  In another embodiment, the enzymatic, nucleic acid molecule, antisense nucleic acid molecule, 2-5A antisense chimera, triplex-forming oligonucleotide, decoy RNA, dsRNA, siRNA, aptamer, or nucleic acid cleavage chemical group of the invention. The sense nucleic acid contains at least one nucleobase modification.

  In another embodiment, an antisense nucleic acid molecule, antisense nucleic acid molecule, 2-5A antisense chimera, triplex-forming oligonucleotide, decoy RNA, dsRNA, siRNA, aptamer, or nucleic acid cleavage chemical group of the invention. The nucleic acid contains at least one phosphate backbone modification.

  In one aspect, the invention features a mammalian cell, eg, a human cell, comprising a nucleic acid molecule of the invention.

  In another aspect, the invention features a method of reducing the expression or activity of VEGF and / or VEGFr, eg, VEGFR1 and / or VEGFR2, in a cell, wherein the method is performed under conditions suitable for the reduction. Contacting with a nucleic acid molecule of the invention that modulates the expression and / or activity of VEGF and / or VEGFr.

In another aspect, the invention features a method of treating a subject having a condition associated with levels of VEGF and / or VEGFr, eg, VEGFR1 and / or VEGFR2, wherein the method is suitable for treatment Further comprising using one or more drug therapies under certain conditions. In one embodiment, the present invention relates to tumor angiogenesis, tumor angiogenesis, cancer, such as, but not limited to, tumor and cancer types, ophthalmic diseases or eyes indicated in the diagnosis section of Table III. Sexual indications such as diabetic retinopathy or age-related muscular degeneration, rheumatoid arthritis, psoriasis and / or endometriosis, endometrial carcinoma, gynecological bleeding disorders, irregular menstrual cycle, ovulation, menstruation Featuring a method of treating a subject with pre-syndrome (PMS), or menopausal dysfunction, the method modulates VEGF and / or VEGFr expression and / or activity in a subject under conditions suitable for treatment Administering a nucleic acid molecule of the invention.

  In another aspect, the invention features a method of modulating conception in a subject, the method modulating the expression and / or activity of VEGF and / or VEGFr in a subject under conditions suitable for treatment. Administering a nucleic acid molecule.

In another aspect, the invention features a method of cleaving RNA encoded by a VEGF, VEGFR1 and / or VEGFR2 gene, the method comprising, for example, a divalent cation (eg, a divalent cation) Contacting the enzymatic nucleic acid molecule of the invention having endonuclease activity with RNA encoded by the VEGFR1 and / or VEGFR2 gene under conditions in which cleavage occurs in the presence of Mg ²⁺ ).

  In one embodiment, the nucleic acid molecule of the invention comprises a cap structure, such as a 3 ′, 3′-linked or 5 ′, 5′-linked deoxyabasic ribose derivative, wherein the cap structure is enzymatically The nucleic acid molecule may be present at the 5 ′ end, or at the 3 ′ end, or at both the 5 ′ end and the 3 ′ end.

  In another embodiment, the nucleic acid molecule of the invention comprises a cap structure, eg, a 3 ′, 3′-linked or 5 ′, 5′-linked deoxyabasic ribose derivative, wherein the cap structure is antisense It may be present at the 5 ′ end, or the 3 ′ end, or both the 5 ′ end and the 3 ′ end of the nucleic acid molecule.

  In one aspect, the invention features an expression vector that includes a nucleic acid sequence encoding at least one nucleic acid molecule of the invention so as to allow the vector to express the nucleic acid molecule.

  In another aspect, the invention features a mammalian cell, eg, a human cell, comprising an expression vector of the invention.

  In yet another embodiment, the expression vector of the present invention further comprises a sequence of nucleic acid molecules complementary to RNA encoded by VEGF and / or VEGFr, eg, VEGFR1 and / or VEGFR2 gene.

  In one embodiment, the expression vector of the present invention comprises nucleic acid sequences encoding two or more nucleic acid molecules of the present invention, which may be the same or different.

In another aspect, the invention provides tumor angiogenesis, cancer such as breast cancer, lung cancer, colorectal cancer, kidney cancer, pancreatic cancer, or melanoma, or ophthalmic indications such as diabetic retinopathy, or Treat or manage age-related muscular degeneration and / or endometriosis, endometrial carcinoma, gynecological bleeding disorders, irregular menstrual cycle, ovulation, premenstrual syndrome (PMS), or menopausal dysfunction Characterized in that the method comprises, under conditions suitable for treatment, eg one or more other therapies such as 5-fluorouridine, leucovorin, irinotecan (CAMPTOSAR® or CPT-11 or camptothecin- 11 or campto), paclitaxel, or carboplatin, GnRH (gonadotropin releasing hormone) agonist, leupron depot (leupro acetate) ), Cinaler (naferaline acetate), zolodex (goserelin acetate), sprefact (buserelin acetate), donazole, or oral contraceptives, such as, but not limited to, depo-provera or probera (medroxyprogesterone acetate), or others A nucleic acid molecule of the present invention that modulates the expression and / or activity of VEGF and / or VEGFr under conditions suitable for administration to a subject of any of the estrogen / progesterone contraceptives, such as an enzymatic nucleic acid of the present invention Molecule, antisense nucleic acid molecule, 2
-5A comprising administering to a subject an antisense nucleic acid comprising an antisense chimera, triplex-forming oligonucleotide, decoy RNA, dsRNA, siRNA, aptamer, or nucleic acid cleavage chemical group.

  In one embodiment, the method of treatment comprises at least 5 ribose residues, at least 10 2′-O-methyl modifications, and 3′-terminal modifications, eg, 3′-3 ′ inverted abasic components. Features a nucleic acid molecule of the invention (eg, an enzymatic nucleic acid or an antisense nucleic acid molecule). In another embodiment, the nucleic acid molecule of the invention further comprises a phosphorothioate linkage at at least 3 of the 5 'terminal nucleotides.

  In another aspect, the present invention provides a mammal, eg, a human, with an enzymatic nucleic acid molecule, antisense nucleic acid molecule, 2-5A antisense chimera, triplex-forming oligonucleotide, decoy RNA, dsRNA, siRNA, It features a method of administering an aptamer, or an antisense nucleic acid containing a nucleic acid cleavage chemical group, which comprises, for example, a delivery reagent (eg, lipid, cationic lipid, phospholipid, or liposome) under conditions suitable for administration. ) In the presence of a nucleic acid molecule.

  In yet another aspect, the invention provides an enzymatic nucleic acid molecule, antisense nucleic acid molecule, 2-5A antisense chimera, triplex-forming oligonucleotide, decoy RNA, dsRNA, siRNA, aptamer, or nucleic acid cleavage chemistry of the invention. Characterized in that an antisense nucleic acid comprising a group is administered to a mammal along with other therapies, the method comprising contacting the mammal, eg, a human, with the nucleic acid molecule and other therapies under conditions suitable for administration. Including.

  In another embodiment, other therapies contemplated by the present invention that can be used with the nucleic acid molecules of the present invention include, but are not limited to, 5-fluorouridine, leucovorin, irinotecan (CAMPTOSAR®) or CPT- 11 or camptothecin-11 or campto), paclitaxel, or carboplatin, GnRH (gonadotropin releasing hormone) agonist, leuprondepo (leuprolide acetate), cinaler (naferaline acetate), zolodex (goserelin acetate), suprefact (buserelin acetate), Donazol or oral contraceptives such as, but not limited to, depot-provera or probera (medroxyprogesterone acetate), or other estrogen / progesterone contraceptives.

  In one embodiment, the invention relates to tumor angiogenesis, cancer such as breast cancer, lung cancer, colorectal cancer, kidney cancer, pancreatic cancer, or melanoma, or ophthalmic indications such as diabetic retinopathy, Or treatment or management of age-related muscular degeneration and / or endometriosis, endometrial carcinoma, gynecological bleeding disorders, irregular menstrual cycle, ovulation, premenstrual syndrome (PMS), or menopausal dysfunction Wherein the expression of the VEGFR1 and / or VEGFR2 gene is down-regulated using an enzymatic nucleic acid molecule. Such enzymatic nucleic acid molecules can be of the motif of hammerhead, NCH, G-cleaving agent, amberzyme, tinzyme, and / or DNAzyme.

  In another aspect, the invention features, as a method of fertility regulation, down-regulating the expression of VEGF and / or VEGFr, eg, VEGFR1 and / or VEGFR2 genes, using enzymatic nucleic acid molecules . Such enzymatic nucleic acid molecules can be of the motif of hammerhead, NCH, G-cleaving agent, amberzyme, tinzyme, and / or DNAzyme. In one embodiment, the nucleic acid molecules of the invention are complementary to the substrate sequences of Tables V and VI. Examples of enzymatic nucleic acid molecules of the invention are shown in Tables V and VI. Examples of such enzymatic nucleic acid molecules consist essentially of the sequences defined in these tables.

  “Inhibit,” “down-regulate,” or “decrease” means expression of a gene, or the level of a nucleic acid encoding one or more proteins or protein subunits or equivalent nucleic acids, or one or more It means that the activity of the above protein or protein subunit, for example, VEGFR1, VEGFR2 and / or flk-1, is reduced compared to that observed in the absence of the nucleic acid molecule of the present invention. In one embodiment, inhibition, down-regulation or reduction by an enzymatic nucleic acid molecule is preferably an enzymatically inactive or attenuated molecule that can bind to the same site of the target nucleic acid but cannot cleave the nucleic acid. Below the level observed in the presence of. In another embodiment, inhibition, downregulation, or reduction by antisense oligonucleotides is preferably below the level observed in the presence of, for example, a scrambled sequence or mismatched oligonucleotide. In another embodiment, the inhibition, downregulation, or reduction of VEGF and / or VEGFr, eg, VEGFR1 and / or VBGFR2, by the nucleic acid molecule of the invention is greater in the presence of the nucleic acid molecule and in its absence.

  “Upregulation” refers to the expression of a gene, or the level of a nucleic acid encoding one or more proteins or protein subunits or equivalent nucleic acids, or one or more proteins or protein subunits such as VEGFR1 and Means that the activity of VEGFR2 is higher than observed in the absence of the nucleic acid molecules of the invention. Increase expression of, for example, VEGF and / or VEGFr genes (eg, VEGFR1 and / or VEGFR2 genes) to treat, prevent, reduce or modify pathogenic conditions caused or exacerbated by the absence or low level of gene expression Can be made.

  “Modulate” means expression, level of gene expression, the level of a nucleic acid encoding one or more proteins or protein subunits or equivalent nucleic acid, or one or more activities of a protein or protein subunit Or upregulated or downregulated so that the activity is higher or lower than observed in the absence of the nucleic acid molecules of the invention.

“Enzymatic nucleic acid molecule” means a nucleic acid molecule having complementarity in a substrate binding region of a specific gene target and having an enzymatic activity that specifically cleaves the target nucleic acid. That is, enzymatic nucleic acid molecules can cleave nucleic acids between molecules, thereby inactivating the target nucleic acid molecule. Such complementary regions allow the enzymatic nucleic acid molecule to fully hybridize to the target nucleic acid and thus allow cleavage to occur. Although 100% complementarity is preferred, complementarity as low as 50-75% is also useful in the present invention (eg Werner and Uhlenbeck, 1995, Nucleic Acids Research, 23, 2092-2096; Hammann et al., 1999, see Aiasisense and Nucleic Acid Drug Dev., 9, 25-31). The nucleic acid may be modified with a base, sugar and / or phosphate group. The term enzymatic nucleic acid is: ribozyme, catalytic RNA, enzymatic RNA, catalytic DNA, aptazyme or aptamer binding ribozyme, controllable ribozyme, catalytic oligonucleotide, nucleozyme, DNAzyme, RNA enzyme, endoribonuclease, endo Used interchangeably with phrases such as nuclease, minizyme, leadzyme, oligozyme or DNA enzyme. All these terms describe nucleic acid molecules with enzymatic activity. The particular enzymatic nucleic acid molecules described in this application are not limiting in the present invention. Those skilled in the art will recognize that in the enzymatic nucleic acid molecules of the present invention, the substrate is a nucleotide sequence that has a specific substrate binding site complementary to one or more target nucleic acid regions and that provides nucleic acid cleavage activity to the molecule. It will be appreciated that it is in or around the binding site (Cech et al., US Pat. No. 4,987,071, Cech et al., 1988, JAM).
A 260: 20 3030).

  Currently, at least some variants of naturally occurring enzymatic nucleic acids are known. Each can catalyze the hydrolysis of nucleic acid phosphodiester bonds in trans under physiological conditions (thus cleaving other nucleic acid molecules). Table I summarizes some characteristics of these ribozymes. In general, enzymatic nucleic acids act by first binding to a target nucleic acid. Such binding is caused by the target binding portion of the enzymatic nucleic acid held in close proximity to the enzymatic portion of the molecule that acts to cleave the target nucleic acid. That is, the enzymatic nucleic acid first recognizes the target nucleic acid, then binds to it by complementary base pairing, and once it binds to the correct site, it acts enzymatically to cleave the target nucleic acid. Such strategic cleavage of the target nucleic acid will destroy its ability to direct the synthesis of the encoded protein. An enzymatic nucleic acid can bind and cleave its nucleic acid target, then search for another target away from the nucleic acid and repeatedly cleave it by binding to a new target. That is, one ribozyme can cleave many molecules of the target nucleic acid. Furthermore, ribozymes are highly specific inhibitors of gene expression, and the specificity of the inhibition depends not only on the base pairing mechanism of binding to the target nucleic acid but also on the mechanism of target nucleic acid cleavage. One mismatch or base substitution near the site of cleavage can completely eliminate the catalytic activity of the ribozyme.

In one of the preferred embodiments of the invention described herein, the enzymatic nucleic acid molecule of the invention is formed with a hammerhead or hairpin motif, but hepatitis delta virus, group I intron, group II intron or RNaseP RNA (with RNA guide sequence), Neurospora VS RNA, DNAzyme, NCH cleavage motif, or G cleavage agent motif. Examples of such hammerhead motifs are described by Dreyfus, (supra), Rossi et al. , 1992, AIDS Research and Human Retroviruses 8,183. Examples of hairpin motifs are described in Hampel et al. , EP 0360257, Hampel and Tritz, 1989 Biochemistry 28, 4929, Feldstein et al. 1989, Gene 82, 53, Haseloff and Gerlach, 1989, Gene, 82, 43, Hampel et al. 1990 Nucleic Acids Res. 18, 299, Chowira & McSwigen, US Pat. No. 5,631,359. Examples of hepatitis delta virus motifs are according to Perrotta and Bean, 1992 Biochemistry 31, 16; examples of RNaseP motifs are described in Guerrier-Takada et al. 1983 Cell 35, 849; Forster and Altman, 1990, Science 249, 783; Li and Altman, 1996, Nucleic Acids Res. 24,835. Examples of Neurospora VS RNA ribozyme motifs can be found in Collins (Saville and Collins, 1990 Cell 61, 685-696; Saville and Collins, 1991 Proc. Natl. Acad. Sci. USA 88, 8826-893; , 2795-2799; Guo and Collins, 1995, EMBO, J. 14, 363). Examples of group II introns can be found in Griffin et al. , 1995, Chem. Biol. 2, 761; Michels and Pyle, 1995, Biochemistry 34, 2965; Pyle et al. , International Publication WO 96/22689. Examples of group I introns are described in Cech et al. U.S. Pat. No. 4,987,071. Examples of DNAzymes are described in Usman et al. , International Publication WO 95/11304; Chartrand et al. , 1995, NAR 23, 4092; Breaker et al. , 1995, Chem. Bio
. 2,655; Santoro et al. 1997, PNAS 94, 4262 and Beigelman et al. , International Publication WO99 / 55857. Examples of NCH cleavage motifs are described in Ludwig & Sproat, International Publication No. WO 98/58058, and examples of G cleavage agents are described in Kore et al. 1998, Nucleic Acids Research 26, 4116-4120, and Eckstein et al. , International Publication WO99 / 16871. Further motifs include aptazyme (Breaker et al., WO 98/43993), amberzyme (Beigelman et al., US patent application 09 / 301,511) and tinzyme (FIG. 7) (Beigelman. Et al., USA). Patent application 09 / 918,728). All of these documents, including drawings, are hereby incorporated by reference in their entirety as part of this specification and may be used in the present invention. These particular motifs or configurations are not limiting in the present invention, and those skilled in the art will recognize that in the enzymatic nucleic acid molecules of the present invention it is complementary to one or more of the RNA regions of the target gene. Will recognize that it only has a specific substrate binding site and has a nucleotide sequence that confers RNA-cleaving activity to the molecule in or around that substrate binding site (Cech et al, U.S. Pat. No. 4,987,071).

  As used herein, “nucleic acid molecule” means a molecule having nucleotides. Nucleic acids can be single stranded, double stranded, or multiple stranded, and can include modified or unmodified nucleotides or non-nucleotides or various mixtures and combinations thereof.

  By “enzymatic moiety” or “catalytic domain” is meant a portion / region essential for cleavage of the nucleic acid substrate of the enzymatic nucleic acid molecule (see, eg, FIG. 6).

By “substrate binding arm” or “substrate binding domain” is meant a portion / region of an enzymatic nucleic acid that can interact (ie, form base pairs) with a portion of the substrate, such as by complementarity. Preferably, such complementarity is 100%, but may be less if desired. For example, about 10 out of 14 can form base pairs (eg, Wemer and
Uhlenbeck, 1995, Nucleic Acids Research, 23, 2092-2096; Hammann et al. , 1999, Antisense and Nucleic Acid Drug Dev. , 9, 25-31). An example of such an arm is generally shown in FIGS. 6-8. That is, these arms contain sequences in the enzymatic nucleic acid that are intended to bring the enzymatic and target nucleic acids together through complementary base-pairing interactions. The enzymatic nucleic acids of the invention may have binding arms of various lengths, continuous or discontinuous. The length of the binding arm is preferably 4 nucleotides or more and is long enough to interact stably with the target nucleic acid, preferably 12-100 nucleotides; more preferably 14-24 nucleotides (See, eg, Werner and Uhlenbeck, supra, Hamman et al., Supra; Hampel et al., EP 0360257; Berzal-Herrance et al., 1993, EMBO J., 12, 2567-73) or 8 -14 nucleotides in length. If two binding arms are selected, the length of the binding arms is symmetrical (ie, each binding arm is the same length; eg, 5 and 5 nucleotides, 6 and 6 nucleotides or 7 and 7 nucleotides long) Or asymmetric (ie, the binding arms are of different lengths; eg, 3 and 5 nucleotides; 6 and 3 nucleotides; 3 and 6 nucleotides long; 4 and 5 nucleotides long; 4 and 6 nucleotides long; 4 and 7 nucleotides) Designed to be long).

“Inozyme” or “NCH” motifs or configurations are described in Ludwig et al. , International Publication WO 98/58058 and US Patent Application 08 / 878,6.
40 refers to an enzymatic nucleic acid molecule comprising a motif commonly described as NCH Rz. Inozyme is an endonuclease activity that cleaves a nucleic acid substrate having a truncated triplet NCH /, where N is a nucleotide, C is a cytidine, H is adenosine, uridine or cytidine, and / represents a cleavage site. Have Inozymes also have endonuclease activity that cleaves nucleic acid substrates having a truncated triplet NCN /, where N is a nucleotide, C is a cytidine, and / represents a cleavage site. “I” in FIG. 6 represents an inosine nucleotide, preferably riboinosine or xyleucine nucleoside.

The “G-cleaving agent” motif or configuration is shown in FIG. 6 and Eckstein
et al. , US Pat. No. 6,127,173 refers to an enzymatic nucleic acid molecule comprising a motif generally described as a G-cleaving agent Rz. The G-cleaving agent has an endonuclease activity that cleaves a nucleic acid substrate having a cleavage triplet NYN /, where N is a nucleotide, Y is a uridine or cytidine, and / represents a cleavage site. The G-cleaving agent can be chemically modified, which is generally shown in FIG.

  “Amberzyme” motifs or configurations are described by Beigelman et al. , International Publication No. WO 99/55857 and US Patent Application 09 / 476,387. Amberzyme has an endonuclease activity that cleaves a nucleic acid substrate having a cleaved triplet NG / N, where N is a nucleotide, G is a guanosine, and / represents a cleavage site. Amberzymes can be chemically modified by substitution with modified nucleotides to enhance nuclease stability. In addition, different nucleoside and / or non-nucleoside linkers can be used to replace the 5'-gaaa-3 'loop shown in the figure. An amberzyme is a non-limiting example of an enzymatic nucleic acid molecule that does not require a ribonucleotide (2'-OH) group in its own nucleic acid sequence for activity.

  “Chinzyme” motifs or configurations are illustrated in FIG. 7 and Beigelman et al. , International Publication No. WO 99/55857 and US Patent Application 09 / 918,728. A tinzyme is an endonuclease activity that cleaves an RNA substrate having a cleavage triplet, such as, but not limited to, YG / Y, where Y is uridine or cytidine, G is guanosine, and / represents a cleavage site. Have Tinzymes can be chemically modified as shown generally in FIG. 7 to enhance nuclease stability by replacing guanosine nucleotides with various substitutions, eg, 2′-O-methyl guanosine nucleotides. . In addition, different nucleotide and / or non-nucleotide linkers can be used to replace the 5'-gaaa-2 'loop shown in the figure. A tinzyme is a non-limiting example of an enzymatic nucleic acid molecule that does not require a ribonucleotide (2'-OH) group in its own nucleic acid sequence for activity.

By “DNAzyme” is meant an enzymatic nucleic acid molecule that does not require the presence of a 2′-OH group in its own nucleic acid sequence for activity. In certain embodiments, an enzymatic nucleic acid molecule can have a linked linker or other linked or associated group, component, or chain that includes one or more nucleotides having 2′-OH groups. DNAzymes may be synthesized chemically or expressed externally in vivo with single stranded DNA vectors or equivalents. A non-limiting example of a DNAzyme is shown in FIG. 8, and Usman et al. U.S. Pat. No. 6,159,714; Chartrand et al. , 1995, NAR
23, 4092; Breaker et al. , 1995, Chem. Bio. 2,655; Santoro et al. , 1997, PNAS 94, 4262; Breaker, 1999, Nature Biotechnology, 17, 422-42.
3; and Santroet. al. , 2000, J; Am. Chem. Soc. 122, 2433-39. The “10-23” DNAzyme motif is one particular type of DNAzyme that has been evolved using in vitro selection and is described in Santoro et al. (Supra) and Joyce et al. U.S. Pat. No. 5,807,718. Alternative DNAzyme motifs can be selected using techniques similar to those described in these references and are therefore within the scope of the present invention.

  “Sufficient length” means that the nucleic acid molecule of the invention is of sufficient length to provide the intended function under the expected conditions. For example, the nucleic acid molecules of the present invention need to be “sufficiently long” to provide a stable interaction with the target nucleic acid molecule under the expected binding conditions and circumstances. In another non-limiting example, “enough length” for a binding arm of an enzymatic nucleic acid is sufficient to provide stable binding to the target site under the reaction conditions and circumstances in which the binding arm sequence is expected. Means long. The binding arm is not long enough to prevent useful turnover of the nucleic acid molecule.

  “Stablely interacting” is the interaction between an oligonucleotide and a target nucleic acid sufficient for the intended purpose (eg, enzymatic cleavage of the target nucleic acid) (eg, complementary in the target under physiological conditions). By forming hydrogen bonds with nucleotides).

  RNA equivalent to VEGF, VEGFR1 and / or VEGFR2 encodes VEGF, VEGFR1 and / or VEGFR2 protein, or various organisms such as humans, rodents, primates, rabbits, pigs, protozoa Including nucleic acid molecules having homology (partial or complete) with nucleic acids encoding proteins having functions similar to VEGF, VEGFR1 and / or VEGFR2 proteins in animals, fungi, plants, and other microorganisms and parasites means. Equivalent nucleic acid sequences include, for example, regions such as 5'-untranslated region, 3'-untranslated region, intron, intron-exon junction, etc., in addition to the coding region.

  “Homology” means that the nucleotide sequences of two or more nucleic acid molecules are partially or completely identical.

"Antisense nucleic acid" refers to RNA-RNA or RNA-DNA or RNA-PNA (protein nucleic acid; Egmol el al., 1993 Nature 365, 566) interaction that binds to the target nucleic acid and alters the activity of the target nucleic acid (See Stein and Cheng, 1993 Science 261, 1004 and Arrow et al., US Pat. No. 5,849,902 for a review). Typically, the antisense molecule is complementary to the target sequence along one contiguous sequence of the antisense molecule. However, in some embodiments, the antisense molecule can bind to the substrate such that the substrate molecule forms a loop, and / or the antisense molecule can bind so that the antisense molecule forms a loop. it can. That is, an antisense molecule may be complementary to two (or more) non-contiguous substrate sequences, or two (or more) non-contiguous sequence portions of an antisense molecule may be They may be complementary or both. For an overview of current antisense strategies, see Schmajuk et al. 1999, J. MoI. Biol. Chem. , 274, 2183-21789, Delihas et al. , 1997, Nature, 15, 751-753, Stein et al. , 1997, Antisense N .; A. Drug Dev. 7, 151, Crooke, 2000, Methods Enzymol. , 313, 3-45, Crooke, 1998, Biotech. Genet. Eng Rev. 15, 121-157, Crooke, 1997, Ad. Pharmac
ol. 40, 1-49. In addition, antisense DNA can be used to target nucleic acids by DNA-RNA interaction, thereby activating RNase H, which can digest target nucleic acids in the duplex. Antisense oligonucleotides can include one or more RNAseH activation regions that can activate RNAseH cleavage of the target nucleic acid. Antisense DNA may be chemically synthesized or expressed using a single-stranded DNA expression vector or equivalent thereof.

  “RNase H activation region” refers to a region of a nucleic acid molecule that can bind to a target nucleic acid to form a non-covalent complex that is recognized by a cellular RNase H enzyme (generally 4-25 nucleotides in length, Preferably 5-11 nucleotides long) (see, eg, Arrow et al., US Pat. No. 5,849,902; Arrow et al., US Pat. No. 5,989,912). The RNase H enzyme binds to the nucleic acid molecule-target nucleic acid complex and cleaves the target nucleic acid sequence. An RNase H activation region can be, for example, a phosphodiester, a phosphorothioate (eg, at least 4 of the nucleotides are phosphorothioate substitutions; more specifically, 4-11 of the nucleotides are phosphorothioate substitutions), phosphorodithioate, 5 Including '-thiophosphate, or methylphosphonate backbone chemistry or combinations thereof. In addition to one or more of the above-described backbone chemistry, the RNase H activation region can also include various sugar chemistry. For example, the RNase H activation region can include nucleotide sugar chemistry of deoxyribose, arabino, fluoroarabino, or combinations thereof. Those skilled in the art will appreciate that the above description is a non-limiting example and that any combination of phosphate, sugar and base chemistry of the nucleic acid that supports the activity of the RNase H enzyme is within the definition of the RNase H activation region and within the scope of the invention You will understand that.

  By “2-5A antisense chimera” is meant an antisense oligonucleotide comprising a 5 ′ phosphorylated 2′-5 ′ linked adenylate moiety. These chimeras bind to the target nucleic acid in a sequence-specific manner and activate cellular 2-5A-dependent ribonuclease, which in turn cleaves the target nucleic acid (Torrance et al., 1993 Proc. Natl. Acad. Sci. USA90. Silverman et al., 2000, Methods Enzymol., 313, 522-533; Player and Torrance, 1998, Pharmacol. Ther., 78, 55-113).

  "Triplex-forming oligonucleotide" means a double-stranded polynucleotide, eg, an oligonucleotide that can bind to DNA in a sequence specific manner to form a triple helix. The formation of such triple helix structures has been shown to inhibit transcription of targeted genes (Duval-Valctin et al., 1992 Proc. Natl. Acad. Sci. USA 89, 504; Fox, 2000 Curr. Med. Chem., 7, 17-37; Praseth et al., 2000, Biochim. Biophys. Acta, 1489, 181-206).

  “Gene” means a nucleic acid sequence that encodes RNA, such as, but not limited to, a structural gene that encodes a polypeptide.

As used herein, the term “complementarity” means that a nucleic acid forms a hydrogen bond with another nucleic acid sequence, either by traditional Watson-Crick or other non-traditional types. Means you can. With respect to the nucleic acid molecules of the present invention, the free energy of binding between the nucleic acid molecule and its target or complementary sequence is sufficient for the relevant function of the nucleic acid to proceed, such as enzymatic nucleic acid cleavage, antisense or triple helix inhibition. . Determination of binding free energy for nucleic acid molecules is well known in the art (eg, Turner et al., 1987, CSH Symp. Quant. Biol. LII pp. 123-133; Frier et al, 1986, Proc Nat.Acad.Sc
i. USA 83: 9373-9377; Turner et al. , 1987, J. et al. Am. Chem. Soc. 109: 3783-3785). The percentage of complementarity indicates the percentage of consecutive residues in a nucleic acid molecule that can form hydrogen bonds (eg, Watson-Crick base pairing) with a second nucleic acid sequence (eg, 5 in 10 bases). 6,7.8,9,10 bases are 50%, 60%, 70%, 80%, 90% and 100% complementarity). “Complete complementarity” means that all consecutive residues of a nucleic acid sequence will hydrogen bond with the same number of consecutive residues in a second nucleic acid sequence.

“RNA” means a molecule comprising at least one ribonucleotide component. “Ribonucleotide” or “2′-OH” means a nucleotide having a hydroxyl group at the 2 ′ position of the β-D-ribofuranose component.

  As used herein, “nucleic acid decoy molecule”, or “decoy” means a nucleic acid molecule that mimics the natural binding domain for a ligand. Thus, decoys compete with naturally occurring binding targets for binding to specific ligands. For example, overexpression of HIV transactivation response (TAR) RNA acts as a “decoy” and effectively binds to HIV tat protein, thereby preventing it from binding to the TAR sequence encoded in HIV RNA. (Sullanger et al., 1990, Cell, 63, 601-608).

  As used herein, “aptamer” or “nucleic acid aptamer” means a nucleic acid molecule that specifically binds to a target molecule, where the nucleic acid molecule is recognized by the target molecule in its natural setting. It has a different sequence from the sequence. Alternatively, the aptamer may be a nucleic acid molecule that binds to the target molecule, where the target molecule does not naturally bind to the nucleic acid. The target molecule can be any molecule of interest. For example, aptamers can be used to bind to the ligand binding domain of a protein, thereby preventing naturally occurring interaction between the ligand and the protein. Similarly, the nucleic acid molecules of the invention can bind to the VEGFR1 or VEGFR2 receptor and interfere with the activity of the receptor. This is a non-limiting example, and those skilled in the art will appreciate that other aspects can be readily generated using techniques generally known in the art. For example, Gold et al. U.S. Pat. Nos. 5,475,096 and 5,270,163; Gold et al. , 1995, Anne. Rev. Biochem. , 64, 763; Brody and Gold, 2000, J. MoI. Biotechnol. 74, 5; Sun, 2000, Curr. Opin. Mol. Ther. , 2, 100; Kusser, 2000, J. MoI. Biotechnol. 74, 27; Hermann and Patel, 2000, Science, 287, 820; and Jayasena, 1999, Clinical Chemistry, 45, 1628.

  As used herein, the term “double stranded RNA” or “dsRNA” refers to a double stranded RNA molecule capable of performing RNA interference “RNAi”, eg, short interfering RNA “siRNA”. See, for example, Bass, 2001, Nature, 411, 428-429; Elbashir et al. , 2001, Nature, 411, 494-498; and Kreutzer et al. , International Publication WO 00/44895; Zernica-Goetz et al. , International Publication WO 01/36646; Fire, International Publication WO 99/32619; Plaetinck et al. , International Publication WO 00/01846; Melo and Fire, International Publication WO 01/29058; Deschamps Depallette, International Publication WO 99/07409; and Li et al. , See International Publication WO 00/44914.

As used herein, “nucleic acid sensor molecule” or “allozyme” means a nucleic acid molecule comprising an enzymatic domain and a sensor domain, wherein the ability of the enzymatic nucleic acid domain to catalyze a chemical reaction is defined as a target Depends on interaction with a signaling molecule, such as a nucleic acid, polynucleotide, oligonucleotide, peptide, polypeptide, or protein, such as VEGF, VEGFR1 and / or VEGFR2. By introducing chemical modifications, additional functional groups and / or linkers into the nucleic acid sensor molecule, enhancing the catalytic activity of the nucleic acid sensor molecule, increasing the binding affinity of the sensor domain to the target nucleic acid, and / or the nucleic acid sensor molecule Nuclease / chemical stability improvements are possible and are therefore within the scope of the present invention (eg, Usman et al., US Patent Application 09 / 877,526, George et al., US Pat. No. 5,834). , 186 and 5,741,679, Shih et al., US Pat. No. 5,589,332, Nathan et al., US Pat. No. 5,871,914, Nathan and Ellington, International Publication WO 00/24931, Breaker et al., International publication WO00 / 26226 and 98/27104, and Sullenger et al., See patent application 09 / 205,520 US).

As used herein, a “sensor component” or “sensor domain” of a nucleic acid sensor molecule refers to a target signaling molecule, eg, a nucleic acid sequence in one or more regions of a target nucleic acid molecule or two or more target nucleic acid molecules. Means a nucleic acid sequence (eg, RNA or DNA or analog thereof) that interacts with and causes the enzymatic nucleic acid component of the nucleic acid sensor molecule to catalyze or stop catalyzing the reaction To do. In the presence of a target signaling molecule of the invention, such as VEGF, VEGFR1 and / or VEGFR2, the ability of the sensor component, eg, the ability to modulate the catalytic activity of the nucleic acid sensor molecule, is altered or decreased. The sensor component can include recognition properties associated with chemical or physical signals that can modulate the nucleic acid sensor molecule by chemical or physical changes to the structure of the nucleic acid sensor molecule. Sensor components can be derived from naturally occurring nucleic acid binding sequences, eg, RNA that binds to other nucleic acid sequences in vivo. Alternatively, the sensor component may be derived from a nucleic acid molecule (aptamer) that has evolved to bind to a nucleic acid sequence in the target nucleic acid molecule (eg, Gold et al., US Pat. No. 5,475,096 and 5, 270, 163). The sensor component may be covalently bound to the nucleic acid sensor molecule or non-covalently associated. Those skilled in the art will understand that all that is necessary is that the sensor component can selectively inhibit the activity of the nucleic acid sensor molecule to catalyze the reaction.

  By “target molecule” or “target signaling molecule” is meant a molecule that can interact with a nucleic acid sensor molecule, particularly the sensor domain of a nucleic acid sensor molecule, such that the nucleic acid sensor molecule becomes active or inactive. The chemical structure of the molecule so that the interaction of the signaling agent with the nucleic acid sensor molecule results in the activity of the enzymatic nucleic acid component of the nucleic acid sensor molecule being modulated, eg, it is activated or deactivated, The enzymatic nucleic acid component of the nucleic acid sensor molecule can be modified by physical, topological, or conformational changes. Signaling agents include target signaling molecules such as macromolecules, ligands, small molecules, metals and ions, nucleic acid molecules (but not limited to RNA and DNA or analogs thereof), proteins, peptides, antibodies, polysaccharides, lipids, sugars , Microorganisms or cellular metabolites, pharmaceuticals, and purified or purified forms of organic and inorganic molecules such as VEGF, VEGFR1 and / or VEGFR2.

As used herein, the term “triplex-forming oligonucleotide” means an oligonucleotide that can bind to double-stranded DNA in a sequence-specific manner to form a triple helix. The formation of such triple helix structures has been shown to inhibit transcription of targeted genes (Duval-Valctin et al., 1992).
Proc. Natl. Acad. Sci. USA89,504; Fox, 2000, C
urr. Med. Chem. , 7, 17-37; Praseth et al. , 2000, Biochim. Biophys. Acta, 1489, 181-206).

  Nucleic acid molecules that modulate the expression of VEGF and / or VEGFr, such as VEGFR1 and / or VEGFR2-specific nucleic acids, are various angiogenesis-related diseases and conditions, such as, but not limited to, tumor angiogenesis, cancer, such as Breast cancer, lung cancer, colorectal cancer, kidney cancer, pancreatic cancer, or melanoma, or ocular indications such as diabetic retinopathy, or age-related muscular degeneration, and / or endometriosis, endometrium It is a novel treatment method for treating or managing carcinomas, gynecological bleeding disorders, irregular menstrual cycles, ovulation, premenstrual syndrome (PMS), and / or menopausal dysfunction. Nucleic acid molecules that modulate the expression of VEGF and / or VEGFr, eg, VEGFR1 and / or VEGFR2 specific nucleic acids, are also novel methods of controlling ovulation or embryo implantation and thus provide a new means of fertility regulation.

  In one embodiment of the invention, the nucleic acid molecule of the invention can be 12-100 nucleotides in length. Exemplary enzymatic nucleic acid molecules of the invention are shown as Formula I and / or Formula II. For example, the enzymatic nucleic acid molecule of the present invention is preferably 15-50 nucleotides in length, more preferably 25-40 nucleotides in length, eg, 34, 36, or 38 nucleotides in length ( See, for example, Jarvis et al., 1996, J. Biol. Chem., 271, 29107-29112). Exemplary DNAzymes of the invention are preferably 15-40 nucleotides in length, more preferably 25-35 nucleotides in length, such as 29, 30, 31, or 32 nucleotides in length (eg, , Santoro et al., 1998, Biochemistry, 37, 13330-13342; Chartrand et al., 1995, Nucleic Acids Research, 23, 4092-4096). Exemplary antisense molecules of the invention are preferably 15-75 nucleotides in length, more preferably 20-35 nucleotides in length, for example 25, 26, 27, or 28 nucleotides in length ( For example, see Woolf et al., 1992, PNAS., 89, 7305-7309; Mimer et al., 1997, Nature Biotechnology, 15, 537-541). Exemplary triplex-forming oligonucleotide molecules of the present invention are preferably 10-40 nucleotides in length, more preferably 12-25 nucleotides in length, eg, 18, 19, 20, or 21 nucleotides in length. (See, eg, Maher et al., 1990, Biochemistry, 29, 8S20-8826; Strobel and Dervan, 1990, Science, 249, 73-75). One skilled in the art understands that the nucleic acid molecule need only be of a length and conformation sufficient and appropriate for the nucleic acid molecule to catalyze the reactions contemplated herein. Will. The length of the nucleic acid molecules of the invention is not limited by the general limitations described.

  In a preferred embodiment, a nucleic acid molecule that regulates, eg, downregulates, replication or expression of VEGF and / or VEGFr, eg, VEGFR1 and / or VEGFR2, comprises 8-100 bases complementary to the VEGFR1 and / or VEGFR2 nucleic acid molecule. Including. More preferably, the nucleic acid molecule that regulates replication or expression of VEGF and / or VEGFr, eg, VEGFR1 and / or VEGFR2, comprises 14-24 bases complementary to the nucleic acid molecule of VEGFR1 and / or VEGFR2.

The present invention provides a method for producing a group of gene regulators based on nucleic acids that exhibit a high degree of specificity for a desired target nucleic acid. For example, the nucleic acid molecules of the present invention are preferably VEGF and / or VEGFr, such as VEGFR1 and / or VEGFR2, such that one or several nucleic acid molecules of the present invention provide specific treatment of a disease or condition. Target a highly conserved sequence region of the target nucleic acid encoding (especially the VEGF, VEGFR1 and / or VEGFR2 gene). Such nucleic acid molecules are delivered externally to specific tissues or cellular targets as needed. Alternatively, nucleic acid molecules may be expressed from DNA and / or RNA vectors delivered to specific cells.

  As used herein, “cell” is used in its normal biological sense and does not refer to an entire multicellular organism. The cells are e.g. in vitro in cell cultures or in multicellular organisms, e.g. in birds, plants and mammals, e.g. Can exist in The cell can be prokaryotic (eg, a bacterial cell) or eukaryotic (eg, a mammalian or plant cell).

  "VEGFR1 and / or VEGFR2 protein" means a protein receptor having vascular endothelial growth factor receptor activity, for example, having the ability to bind to vascular endothelial growth factor and / or having tyrosine kinase activity, or a mutant protein thereof Derivative means.

  “Highly conserved sequence region” means that the nucleotide sequence of one or more regions in a target gene is one generation and another generation, or one biological system and another biological It means that there is no significant difference with the system.

  “Angiogenesis” refers to the formation of new blood vessels, an essential process in reproduction, development and wound healing. “Tumor angiogenesis” refers to the induction of blood vessel growth from surrounding tissue to a solid tumor. Tumor growth and tumor metastasis depend on angiogenesis (for review, Folkman, 1985 (supra), Followan 1990 J. Natl. Cancer Inst., 82, 4; Folkman and Shing, 1992 J. Biol. Chem. 267, 10931).

  Angiogenesis plays an important role in other diseases, such as arthritis where new blood vessels invade joints and destroy cartilage (Folkman and Shing, supra).

  “Retinopathy” refers to inflammation of the retina and / or degenerative state of the retina, which can lead to closure of the retina and ultimately blindness. In “diabetic retinopathy”, angiogenesis causes the capillaries in the retina to enter the vitreous, resulting in bleeding and blindness, which is also seen in neonatal retinopathy (for review see Folkman, 1985 (supra), Folkman 1990 (supra); Folkman and Shing, 1992 (supra)).

  Nucleic acid-based inhibitors of expression of VEGF and / or VEGFr, eg, VEGFR1 and / or VEGFR2, alone or in combination with other therapies, include angiogenesis-related diseases and conditions such as, but not limited to, tumors Angiogenesis, cancer, such as breast cancer, lung cancer, colorectal cancer, kidney cancer, pancreatic cancer, or melanoma, or ocular indications, such as diabetic retinopathy, or age-related muscular degeneration, and / or Endometritis, endometrial carcinoma, gynecological bleeding disorders, irregular menstrual cycle, ovulation, premenstrual syndrome (PMS), menopausal dysfunction, and levels of VEGF, VEGFR1 and / or VEGFR2 in cells or tissues Useful for the prevention, treatment, and / or management of other diseases or conditions that are related or responsive. Decreased expression of VEGF and / or VEGFr, such as VEGFR1 and / or VEGFR2 (especially RNA levels of VEGF, VEGFR1 and / or VEGFR2 genes), and thus a decrease in the level of the respective protein, to some extent a symptom of the disease or condition Reduce. Nucleic acid-based inhibitors of VEGF and / or VEGFr, eg, VEGFR1 and / or VEGFR2 expression are also useful as fertility regulators, eg, by inhibiting ovulation or embryonic uterine implantation.

  The nucleic acid molecules of the invention can be added directly or can be complexed with cationic lipids, encapsulated in liposomes, or delivered to target cells or tissues by other methods. The acid complex can be administered locally to the relevant tissue ex vivo or in vivo using an injection, infusion pump or stent, with or without incorporation into the biopolymer. In a preferred embodiment, the nucleic acid inhibitor comprises a sequence that is complementary to a polynucleotide (eg, DNA and RNA) having VEGF and / or VEGFr, eg, VEGFR1 and / or VEGFR2 sequences.

  The triplex molecules of the present invention can be provided to target a DNA target region and include a DNA equivalent of a sequence complementary to a target sequence or a specific target (substrate) sequence. . Typically, the antisense molecule is complementary to the target sequence along one contiguous sequence of the antisense molecule. However, in some embodiments, the antisense molecule can bind to the substrate such that the substrate molecule forms a loop, and / or the antisense molecule can bind so that the antisense molecule forms a loop. it can. That is, an antisense molecule may be complementary to two (or more) non-contiguous substrate sequences, or two (or more) non-contiguous sequence portions of an antisense molecule may be They may be complementary or both.

“Consisting essentially of” means that the active nucleic acid molecule of the present invention, eg, an enzymatic nucleic acid molecule, has the same enzyme center or It is meant to include a core and a binding arm that can bind to a nucleic acid. There may be other sequences that do not significantly interfere with such cleavage. That is, the core region may include, for example, one or more loops, stem-loop structures, or linkers that do not interfere with enzymatic activity. That is, a particular region of the nucleic acid molecule of the present invention may be such a loop, stem-loop, nucleotide linker, and / or non-nucleotide linker and is generally represented as the sequence “X”. That is, the core region may include, for example, one or more loops or stem-loop structures that do not interfere with enzymatic activity. For example, the core sequence of a hammerhead enzymatic nucleic acid is a conserved sequence such as 5′-CUGAUGAG-3 ′ and 5′-CGAA-3 ′ (X is 5′- GCCCGUUAGCC- 3 ′ ( SEQ ID NO: 5979), or other stem II regions known in the art, or nucleotide and / or non-nucleotide linkers). Similarly, other nucleic acid molecules of the present invention, such as inozymes, G-cleaving agents, amberzymes, tinzymes, DNAzymes, antisenses, 2-5A antisenses, triplex-forming nucleic acids, aptamers, decoy nucleic acids, dsRNA or siRNA Other sequences that do not interfere with the function of the nucleic acid molecule or non-nucleotide linkers may be present.

The sequence X may be a linker with a length of 2 nucleotides or more, preferably 3,4,5,6,7,8,9,10,15,20,26,30 nucleotides, where , The nucleotides may preferably base pair internally to form a stem of 2 base pairs or more. Alternatively or additionally, sequence X may be a non-nucleotide linker. In yet another embodiment, the nucleotide linker X may be a nucleic acid aptamer, such as ATP aptamer, HIV Rev aptamer (RRE), HIV Tat aptamer (TAR), and others (for review see Gold et al., 1995). , Annu. Rev. Biochem., 64, 763; and Szostak & Ellington, 1993, The RNA World, ed. Gesteland and Atkins, pp. 511, CSH Laboratory Press). Nucleic acid aptamers include nucleic acid sequences that can interact with a ligand. The ligand can be any natural or synthetic molecule, such as, but not limited to, resins, metabolites, nucleosides,
Nucleotides, drugs, toxins, transition state analogs, peptides, lipids, proteins, amino acids, nucleic acid molecules, hormones, carbohydrates, receptors, cells, viruses, bacteria and others.

  In yet another embodiment, the non-nucleotide linker X is as defined herein. As used herein, the term “non-nucleotide” includes abasic nucleotides, polyethers, polyamines, polyamides, peptides, carbohydrates, lipids, or polyhydrocarbon compounds. Specific examples include Seela and Kaiser, Nucleic Acids Res. 1990, 18: 6353 and Nucleic Acids Res. 1987, 15: 3113; Cload and Schepartz, J. Am. Am. Chem. Soc. 1991, 113: 6324; Richardson and Schepartz, J. Am. Am. Chem. Soc. 1991, 113: 5109; Ma et al. , Nucleic Acids Res. 1993, 21: 2585 and Biochemistry 1993, 32: 1751; Durand et al. , Nucleic Acids Res. 1990, 18: 6353; McCurdy et al. , Nucleosides & Nucleotides 1991, 10: 287; Jschke et al. Tetrahedron Lett. 1993, 34: 301; Ono et al. Biochemistry 1991, 30: 9914; Arnold et al. , International Publication WO 89/02439; Usman et al. , International Publication WO 95/06731; Dudycz et al. , International Publication WO 95/11910 and Ferentz and Verdine, J.A. Am. Chem. Soc. 1991, 113: 4000 (all cited herein as part of this specification).

  The term “non-nucleotide” can also be introduced into a nucleic acid strand in place of one or more nucleotide units, including either sugar and / or phosphate substitutions, with the remaining base being its enzymatic By any group or compound that is capable of exerting activity. A group or compound is abasic if it does not contain a commonly recognized nucleotide base, such as adenosine, guanine, cytosine, uracil or thymine. Thus, in one aspect, the invention features an enzymatic nucleic acid molecule having one or more non-nucleotide components and having enzymatic activity to cleave RNA or DNA molecules.

  In another aspect of the invention, a nucleic acid that interacts with a target nucleic acid molecule to down-regulate VEGF and / or VEGFr, eg, VEGFR1 and / or VEGFR2 (especially VEGF, VEGFR1 and / or VEGFR2 genes) activity. Molecules are expressed from transcription units inserted into DNA or RNA vectors. The recombinant vector is preferably a DNA plasmid or a viral vector. Enzymatic nucleic acid molecules or antisense expressing viral vectors can be constructed based on, but not limited to, adeno-associated virus, retrovirus, adenovirus or alphavirus. Recombinant vectors capable of expressing enzymatic nucleic acid molecules or antisense are delivered as described above and remain in the target cells. Alternatively, viral vectors that provide for transient expression of enzymatic nucleic acid molecules or antisense can be used. Such vectors can be repeatedly administered as necessary. Once expressed, the enzymatic nucleic acid molecule or antisense binds to the target nucleic acid and down regulates its function or expression. Delivery of enzymatic nucleic acid molecules or vectors expressing antisense can be delivered systemically (eg, by intravenous or intramuscular administration) to target cells that have been explanted from the patient and then reintroduced into the patient, Alternatively, it can be performed by any other means that allows introduction into the desired target cells. Antisense DNA can be expressed using single-stranded DNA intracellular expression vectors.

  By “vector” is meant any nucleic acid and / or virus-based procedure used to deliver a desired nucleic acid.

  “Subject” or “patient” means an organism or cell itself that is a donor or recipient of explanted cells. A “subject” or “patient” also refers to an organism that can be administered a nucleic acid molecule of the invention. Preferably, the subject or patient is a mammal or a mammalian cell. More preferably, the subject or patient is a human or human cell.

  “Enhanced enzymatic activity” is meant to include activity measured in cells and / or in vivo, where the activity reflects both the catalytic activity and stability of the nucleic acid molecule of the present invention. Is. In the present invention, the product of these properties can be increased in vivo compared to total RNA enzymatic nucleic acids or total DNA enzymes. In some cases, the activity or stability of the nucleic acid molecule is reduced (ie, 10 or more times lower), but the overall activity of the nucleic acid molecule may be enhanced in vivo.

  The nucleic acid molecules of the present invention can be used independently or in combination with or together with other agents to treat the diseases or conditions described above. For example, to treat a disease or condition associated with the levels of VEGFR1 and / or VEGFR2, as will be apparent to those skilled in the art, under conditions suitable for treatment, either independently or one or more agents In combination, the patient can be treated or other suitable cells can be treated.

  In yet another embodiment, the molecules of the invention described herein can be used in combination with other known therapeutic agents to treat the conditions or diseases described above. For example, the molecules described can be used in combination with one or more known therapeutic agents to treat angiogenesis-related diseases and conditions such as, but not limited to, tumor angiogenesis, cancer, eg, breast cancer, Lung cancer, colorectal cancer, kidney cancer, pancreatic cancer, or melanoma, or ocular indications such as diabetic retinopathy or age-related muscular degeneration, and / or endometriosis, fertility regulation, endometrium Tumors, gynecological bleeding disorders, irregular menstrual cycles, ovulation, premenstrual syndrome (PMS), menopausal dysfunction, endometrial carcinoma, and / or VEGF and / or VEGFr, eg, VEGFR1 and / or VEGFR2 expression Other diseases or conditions that respond to modulation can be treated.

  Other features and advantages of the invention will be apparent from the following description of preferred embodiments of the invention and from the claims.

BRIEF DESCRIPTION OF THE FIGURES FIG. 1 shows a secondary structure model of ANGIOZYME ™ ribozyme bound to its RNA target.

  FIG. 2 shows the time course of inhibition of primary tumor growth after systemic administration of ANGIOZYME ™ in an LLC mouse model.

  FIG. 3 shows the inhibition of primary tumor growth after systemic administration of ANGIOZYME ™ according to a dosing regimen in an LLC mouse model.

  FIG. 4 shows dose-dependent inhibition of tumor metastasis after systemic administration of ANGIOZYME ™ in a mouse colorectal model.

FIG. 5 is a graph showing the plasma concentration profile of ANGIOZYME ™ after a single subcutaneous administration (SC) at a dose of 10, 30, 100 or 300 mg / m ² .

FIG. 6 shows an example of a chemically stabilized ribozyme motif. HH Rz represents the hammerhead ribozyme motif (Usman et al., 1996, Curr. Op. Struct. Bio., 1,527); NCH Rz represents the NCH ribozyme motif (Ludwig & Sproat, WO 98/58058 and US Patent) Application 08 / 878,640); G-cleaving agent represents G-cleaving ribozyme motif (Kore et
al. 1998, Nucleic Acids Research, 26, 4116-4120, Eckstein et al. , US Pat. No. 6,127,173). N or n independently represents a nucleotide, which may be the same or different and may be complementary to each other; rI represents a ribo-inosine nucleotide; the arrow represents a cleavage in the target Represents a site. Although position 4 of HH Rz and NCH Rz is shown as having a 2′-C-allyl modification, those skilled in the art will recognize this position in the art unless the modification significantly inhibits the activity of the ribozyme. It will be appreciated that other well-known modifications can be made.

  FIG. 7 shows an example of a chemically stabilized tinzyme A ribozyme motif (see, eg, Beigemanet al., WO 99/55857 and US patent application 09 / 918,728).

  FIG. 8 shows the results of Santoro et al. 1997, PNAS, 94, 4262 and Joyce et al. U.S. Pat. No. 5,807,718. Shows examples of DNAzyme motifs described by.

  FIG. 9 shows data showing the inhibition of soluble VEGFR1 in clinical trials with ANGIOZYME (SEQ ID NO: 5977).

  FIG. 10 shows a generalized outline for a mouse model of proliferative retinopathy, showing the time of ribozyme administration.

  FIG. 11 shows a graph showing the effectiveness of enzymatic nucleic acid molecules targeting the VEGF-receptor in a mouse model of proliferative retinopathy.

Detailed Description of the Invention
Nucleic acid molecules and mechanism of action
Enzymatic nucleic acids : Currently, several variants of naturally occurring enzymatic nucleic acids are known. In addition, several in vitro selection (evolution) strategies (Orgel, 1979, Proc. R. Soc. London, B205, 435) have been used to develop new nucleic acid catalysts that can catalyze the cleavage and ligation of phosphodiester bonds. (Joyce, 1989, Gene, 82, 83-87; Beaudry et al., 1992, Science 257, 635-641; Joyce, 1992, Scientific American 267, 90-97; Breaker et al., 1994, TIBTECH. 268; Bartel et al., 1993, Science 261: 1411-1418; Szostak, 1993, TIBS 17, 89-93; Kumar
et al. 1995, FASEB J. et al. , 9, 1183; Breaker, 1996, Curr. Op. Biotech. , 7, 442; Santoro et al. , 1997, Proc. Natl. Acad. Sci. , 94, 4262; Tang et
al. , 1997, RNA 3,914; Nakamaye & Eckstein, 1994, (supra); Long & Uhlenbeck, 1994, (supra); Ishizaka et al. 1995, supra; Vaish et al. , 1997, Bio
chemistry 36,6495 (all of which are hereby incorporated by reference). Each can catalyze a series of reactions involving physiological hydrolysis of phosphodiester bonds in trans (and thus cleave other RNA molecules) under physiological conditions.

  The enzymatic nature of enzymatic nucleic acid molecules has important advantages, such as the low concentration of enzymatic nucleic acid molecules required to perform therapy. This advantage reflects the ability of enzymatic nucleic acid molecules to act enzymatically. That is, one enzymatic nucleic acid molecule can cleave many target nucleic acids. Furthermore, enzymatic nucleic acid molecules are highly specific inhibitors, and the specificity of the inhibition depends not only on the base pairing mechanism of binding to the target nucleic acid but also on the mechanism of target nucleic acid cleavage. One mismatch or base substitution near the site of cleavage can be chosen to completely eliminate the catalytic activity of the enzymatic nucleic acid molecule.

A nucleic acid molecule having endonuclease enzyme activity can repeatedly cleave other nucleic acid molecules in a nucleotide base sequence specific manner. If designed properly, such enzymatic nucleic acid molecules can target RNA transcripts and achieve efficient cleavage in vitro (Zaug et al., 324, Nature 429 1986; Uhlenbeck). 1987 Nature 328, 596; Kim et al., 84 Proc. Natl. Acad. Sci. 585, 1988; Cech, 260 JAMA 3030, 1988; Jefferies et
al. , 17 Nucleic Acids Research 1371, 1989; Santoro et al. 1997 (supra)).

  Due to its sequence specificity, trans-cleaving enzymatic nucleic acid molecules can be used as therapeutic agents for human diseases (Usman & McSwigen, 1995 Ann. Rep. Med. Chem. 30, 285-294; Christoffersen and Marr, 1995 J Med.Chem.38, 2023-2037). Enzymatic nucleic acid molecules can be designed to cleave specific nucleic acid targets in the background of cellular nucleic acids. Such a cleavage event renders the nucleic acid non-functional and eliminates protein expression from the nucleic acid. In this way, the synthesis of proteins associated with disease states can be selectively inhibited (Warashina et al., 1999, Chenastyty and Biology, 6, 237-250).

Enzymatic nucleic acid molecules (“allozymes”) of the invention that are allosterically regulated can be used to down-regulate the expression of VEGF and / or VEGFr, eg, VEGFR1 and / or VEGFR2. These allosteric enzymatic nucleic acids or allozymes (eg, Usman et al., US patent application 09 / 877,526, George et al., US Pat. Nos. 5,834,186 and 5,741,679, Shih et al., US Patent 5,589,332, Nathan et al., US Pat. No. 5,871,914, Nathan and Ellington, International Publication WO 00/24931, Breaker et al., International Publication WO 00/26226 and 98/27104, and Sullenger et al. ., See US patent application 09 / 205,520) signaling agents, such as mutant VEGFR1 and / or VEGFR2 protein, wild type VEGFR1 and / or VEGFR2 protein, mutant VE. For FR1 and / or VEGFR2 RNA, wild type VEGFR1 and / or VEGFR2 RNA, other proteins and / or RNAs involved in VEGF signaling, compounds, metals, polymers, molecules and / or drugs that target VEGFR1 and / or VEGFR2 expression Designed to respond, which in turn modulates the activity of enzymatic nucleic acid molecules. The activity of allosteric enzymatic nucleic acids is activated or inhibited in response to interaction with a given signaling agent such that expression of a particular target is selectively downregulated. Targets include certain components of the wild type VEGFR1 and / or VEGFR2, mutant VEGFR1 and / or VEGFR2, and / or VEGF signaling pathway. In a particular example, allosteric enzymatic nucleic acid molecules that are activated by interaction with RNA encoding the VEGF protein are used as therapeutic agents in vivo. The presence of RNA encoding the VEGF protein activates the allosteric enzymatic nucleic acid molecule, which then cleaves the RNA encoding the VEGFR1 and / or VEGFR2 protein, resulting in inhibition of expression of the VEGFR1 and / or VEGFR2 protein Is done.

  In another non-limiting example, the allozyme is, for example, cleaved by the RNA encoded by the VEGF, VEGFR1 and / or VEGFR2 gene, so that the allozyme is of the VEGF and / or VEGFr, eg, VEGFR1 and / or VEGFR2 gene. It can be activated by VEGF and / or VEGFr, such as VEGFR1 and / or VEGFR2 protein, peptide, or variant polypeptide, which causes expression to be inhibited. In this non-limiting example, allozyme acts as a decoy, inhibits the function of VEGF, VEGFR1 and / or VEGFR2, and is also activated once by VEGF, VEGFR1 and / or VEGFR2 protein. Alternatively, it inhibits the expression of VEGFR2.

Antisense : Antisense molecules can be modified or unmodified RNA, DNA, or mixed polymer oligonucleotides that function primarily by specifically binding to matching sequences and peptide synthesis. (Wu-Pong, Nov 1994, Bio Pharm, 20-33). Antisense oligonucleotides bind to target RNA by Watson-Crick base pairing and interfere with gene expression by preventing ribosomal translation of the bound sequence by steric hindrance or by activating RNase H enzyme. Antisense molecules can also alter protein synthesis by interfering with RNA processing or nuclear to cytoplasmic transport (Mukhopadhyay & Roth, 1996, Crit. Rev. in Oncogenesis 7, 151-190).

  Furthermore, the heteroduplex is nuclease-degraded by binding of single-stranded DNA to RNA (Wu-Pong, (supra), Crooke, (supra)). So far, the only chemically modified DNAs that act as substrates for RNase H are phosphorothioate, phosphorodithioate and boron trifluoridate. Recently, 2'-arabino and 2'-fluoroarabino-containing oligos were also reported to activate RNase H activity.

  Numerous antisense molecules have been described that utilize novel configurations, secondary structures, and / or RNase H substrate domains of chemically modified nucleotides (Woolf et al; International Publication WO 98/13526; Thompson et al , International Publication No. WO 99/54459; Hartmann et al., US Patent Application 60 / 101,174, filed September 21, 1998), which are hereby incorporated by reference in their entirety.

  In addition, antisense deoxyoligoribonucleotides can be used to target RNA by DNA-RNA interaction, thereby activating RNaseH that digests the target RNA in the duplex. Antisense DNA can be expressed using intracellular expression vectors of single-stranded DNA or equivalents and variations thereof.

Triplex forming oligonucleotides (TFO) : Single stranded oligonucleotides can be designed to bind to genomic DNA in a sequence specific manner. TFO is composed of pyrimidine-rich oligonucleotides that bind to a DNA helix via Hoogsteen base pairing (Wu-Pong, supra). The resulting triple helix consisting of DNA sense, DNA antisense, and TFO disrupts RNA synthesis by RNA polymerase. Because binding is irreversible, gene expression or cell death can occur through the TFO mechanism (Mukhopadhyay & Roth, supra).

The 2′-5 ′ antisense chimera : 2-5A system is an interferon-mediated mechanism of RNA degradation found in higher vertebrates (Mitra et al; 1996, Proc Nat Acad Sci USA 93, 6780-6785). Two types of enzymes are required for RNA cleavage, namely 2-5A synthetase and RNaseL. 2-5A synthetase requires double stranded RNA to form 2'-5 'oligoadenylate (2-5A). Next, 2-5A acts as an allosteric effector for utilizing RNase L, which has the ability to cleave single-stranded RNA. This system is particularly useful for inhibiting viral replication due to the ability to form 2-5A structures with double stranded RNA.

  The (2'-5 ') oligoadenylate structure can be covalently bound to an antisense molecule to form a chimeric oligonucleotide capable of RNA cleavage (Torrance, supra). These molecules probably bind to and activate the 2-5A-dependent RNase, and then the oligonucleotide / enzyme complex binds to the target RNA molecule, which is then cleaved by the RNase enzyme. it can.

RNAi: Double-stranded RNA can suppress the expression of homologous genes through an evolutionarily conserved process called RNA interference (RNAi) or post-transcriptional gene silencing (PTGS). One mechanism underlying silencing is the degradation of target mRNA by the RNP complex, which includes short interfering RNA (siRNA) as a guide for substrate selection. Short interfering RNA is typically 21-23 nucleotides in length. It has been suggested that a bidental nuclease called Dicer is a protein responsible for siRNA production. For example, double-stranded RNA (dsRNA) having a matched gene sequence is synthesized in vitro and introduced into a cell. dsRNA is sent into the biological pathway and degraded into short fragments of short interfering (si) RNA. With the help of cellular enzymes such as Dicer, siRNA induces the degradation of messenger RNA that matches its sequence (eg Tuschl
et al. , International Publication WO 01 / 75164-, Bass, 2001, Nature, 411, 428-429; Elbashir et al. , 2001, Nature, 411, 494-498; and Kreutzer et al. , See International Publication WO 00/44895).

Target Sites Useful nucleic acid molecules of the invention, such as enzymatic nucleic acid molecules, dsRNA, and antisense nucleic acid targets are described in Draper et al. , WO 93/23569; Sullivan et al. , WO 93/23057; Thompson et al. , WO 94/02595; Draper et al. , WO 95/04818; McSwigen et al. U.S. Pat. No. 5,525,468, which is hereby incorporated by reference in its entirety. Other examples include the following PCT applications relating to inactivation of the expression of disease-related genes: WO95 / 23225, WO95 / 13380, WO94 / 02595 (herein incorporated by reference). In this specification, without repeating the guidance provided in these documents, specific examples of such methods are set forth below, but are not limited thereto. Enzymatic nucleic acid molecules and antisense against such targets are designed, synthesized as described in these applications, and tested in vitro and in vivo as also described. A computer folding algorithm is used to screen the sequence of human VEGF, VEGFR1 and / or VEGFR2 RNA for optimal nucleic acid target sites. Identify potential nucleic acid binding / cleavage sites. Stinchcomb et al. As discussed in WO 95/23225, human sequences can be screened and nucleic acid molecules can be subsequently designed, but enzymatic nucleic acid molecules that target mice must be tested for nucleic acid molecules prior to testing in humans. It may be useful to test the effectiveness of the action.

  Nucleic acid molecules, eg, enzymatic nucleic acids, antisense binding / cleavage sites are identified and dsRNA mediated binding sites are selected. For the enzymatic nucleic acid molecules of the present invention, the nucleic acid molecules are analyzed separately by computer folding (Jaeger et al., 1989 Proc. Natl. Acad. Sci. USA, 86, 7706) and the sequence is converted to an appropriate secondary structure. Evaluate whether it is folded or not. For example, nucleic acid molecules having undesirable intramolecular interactions between the binding arm and the catalyst core can be excluded from consideration. Various binding arm lengths can be selected to optimize activity.

  The binding / cleavage of nucleic acids, eg, antisense, RNAi, and / or enzymatic nucleic acid molecules is identified and designed to anneal to various sites in the nucleic acid target. The binding arm of the enzymatic nucleic acid molecule of the present invention is complementary to the target site sequence described above. Antisense and RNAi sequences are designed to have complete or partial complementarity to the nucleic acid target. Nucleic acid molecules can be synthesized chemically. The synthetic methods used are described below and are described in Usman et al. , 1987 J.H. Am. Chem. Soc. 109, 7845; Scaring et al. 1990 Nucleic Acids Res. , 18, 5433; and Wincott et al. 1995 Nucleic Acids Res. 23, 2677-2684; Caruthers et al. , 1992, Methods in Enzymology 211, 3-19.

Synthesis of nucleic acid molecules The synthesis of nucleic acids longer than 100 nucleotides is difficult using automated methods, and the therapeutic costs of such molecules are very high. In the present invention, preferably a small nucleic acid motif (“small” means a nucleic acid motif having a length of 100 nucleotides or less, preferably 80 nucleotides or less, most preferably 50 nucleotides or less, Sense oligonucleotides, enzymatic nucleic acids, aptamers, allozymes, decoys, siRNA, etc.) are used for external delivery. Since these molecules are simple in structure, the ability of nucleic acids to enter the target region of the RNA structure is high. The exemplary molecules of the present invention are chemically synthesized, but other molecules can be synthesized as well.

DNA oligonucleotides are described in Caruthers et al. , 1992, Methods in Enzymology 211, 3-19, Thompson et al. , WO 99/54459, Wincott et al. , 1995, Nucleic Acids Res. 23, 2677-2684, Wincott et al. , 1997, Methods Mol. Bio. 74, 59, Brennan et al. , 1998, Biotechnol Bioeng. , 61, 33-45, and Brennan, US Pat. No. 6,001,311, which are synthesized using protocols known in the art (all of which are hereby incorporated by reference). To quote). For the synthesis of oligonucleotides, general nucleic acid protecting groups and coupling groups such as dimethoxytrityl at the 5 ′ end and phosphoramidites at the 3 ′ end are used. In a non-limiting example, 394 Applied Biosystems, In
c. Small scale synthesis on a synthesizer with a 0.2 μmol scale protocol with a 2.5 minute coupling step for 2′-O methylated nucleotides and a 45 second coupling step for 2′-deoxynucleotides I do. Table II outlines the amount of reagents used in the synthesis cycle and the contact time. Alternatively, synthesis at the 0.2 μmol scale can be performed on a 96-well plate synthesizer, such as an apparatus manufactured by Protogene (Palo Alto, Calif.) With minimal modification to the cycle. In each coupling cycle of 2′-O-methyl residues, a 33-fold excess (60 μL of 0.11 M = 6.6 μmol) of 2′-O-methyl phosphoramidite and 105 A fold excess of S-ethyltetrazole (60 μL of 0.25M = 15 μmol) can be used. In each coupling cycle of deoxy residues, a 22-fold excess (40 μL of 0.11 M = 4.4 μmol) deoxyphosphoramidite and a 70-fold excess of S-ethyltetrazole (40 μL of polymer-bound 5′-hydroxyl) 0.25M = 10 μmol) can be used. 394 Applied Biosystems, Inc. The average coupling yield in the synthesizer is typically 97.5-99% as determined by colorimetric determination of the trityl fraction. 394 Applied
Biosystems, Inc. Other oligonucleotide synthesis reagents used in the synthesizer are: Detritylated solution is 3% TCA (ABI) in methylene chloride; capping is 16% N-methylimidazole (ABI) and THF in THF Performed in 10% acetic anhydride / 10% 2,6-lutidine (ABI); the oxidizing solution is 16.9 mM I ₂ , 49 mM pyridine, 9% water in THF (PERSEPTIVE®).
Burdick & Jackson synthetic grade acetonitrile is used directly from the reagent bottle. S-ethyltetrazole solution (0.25 M in acetonitrile) was obtained from American International Chemical, Inc. Create from solids obtained from. Alternatively, a borage reagent (3H1,2-benzodithiol-3-one 1,1-dioxide, 0.05M in acetonitrile) is used for the introduction of phosphorothioate linkages.

Deprotection of the DNA polynucleotide is performed as follows: Transfer the polymer-bound tritylone oligoribonucleotide to a 4 mL glass screw cap vial and suspend in a solution of 40% aqueous methylamine (1 mL) at 65 ° C. for 10 minutes. . After cooling to −20 ° C., the supernatant is removed from the polymer support. The support is washed 3 times with 1.0 mL EtOH: MeCN: H ₂ O / 3: 1: 1, vortexed and then the supernatant is added to the first supernatant. The combined supernatant containing oligoribonucleotides is dried to obtain a white powder.

Synthetic methods used for RNA oligonucleotides containing certain nucleic acid molecules of the present invention are described in Usman et al. , 1987 J.H. Am. Chem. Soc. 109, 7845; Scaringe et al. 1990 Nucleic Acids Res. , 18, 5433; Wincott et al. 1995 Nucleic Acids Res. 23, 2677-2684; Wincott et al. , 1997, Methods Mol. Bio. 74, 59; using conventional nucleic acid protecting and coupling groups such as dimethoxytrityl at the 5 'end and phosphoramidite at the 3' end. In a non-limiting example, small scale synthesis is performed at 394 Applied Biosystems, Inc. Using a modified 0.2 μmol scale protocol on the synthesizer, a 7.5 minute coupling step for alkylsilyl protected nucleotides and a 2.5 minute coupling step for 2′-O-methylated nucleotides I do. Table II outlines the amount of reagents used in the synthesis cycle and the contact time. Alternatively, synthesis at the 0.2 μmol scale can be performed on a 96-well plate synthesizer, such as an apparatus manufactured by Protogene (Palo Alto, Calif.) With minimal modification to the cycle. A 33-fold excess (60 μL of 0.11 M = 6.6 μmol) phosphoramidite and 75-fold excess of S-ethyl over the polymer-bound 5′-hydroxyl in each coupling cycle of 2′-O-methyl residues. Tetrazole (60 μL of 0.25M = 15 μmol) can be used. In each coupling cycle of ribo-residues, a 66-fold excess (120 μL 0.11 M = 13.2 μumol) of alkylsilyl (ribo) protected phosphoramidite and 150-fold excess of S-to the polymer-bound 5′-hydroxyl. Ethyltetrazole (120 μL of 0.25M = 30 μmol) can be used. 394 Applied Biosystems, Inc. The average coupling yield in the synthesizer is typically 97.5-99% as determined by colorimetric determination of the trityl fraction. 394 Applied Biosystems, Inc. Other oligonucleotide synthesis reagents used in the synthesizer are as follows: the detritylated solution is 3% TCA (ABI) in methylene chloride;
Performed in 16% N-methylimidazole (ABI) in F and 10% acetic anhydride / 10% 2,6-lutidine (ABI) in THF; oxidizing solution was 16.9 mM I ₂ , 49 mM pyridine, 9% in THF Water (PERSEPTIVE®). Burdick & Jackson synthetic grade acetonitrile is used directly from the reagent bottle. S-ethyltetrazole solution (0.25 M in acetonitrile) was obtained from American International Chemical, Inc. Create from solids obtained from. Alternatively, a borage reagent (3H-1,2-benzodithiol-3-one 1,1-dioxide, 0.05M in acetonitrile) is used for the introduction of phosphorothioate linkages.

RNA deprotection is performed using either a two-pot protocol or a one-pot protocol. For the two-pot protocol, the polymer-bound trityl-on-oligoribonucleotide is transferred to a 4 mL glass screw cap vial and suspended in a solution of 40% aqueous methylamine (1 mL) at 65 ° C. for 10 minutes. After cooling to −20 ° C., the supernatant is removed from the polymer support. The support is washed 3 times with 1.0 mL EtOH: MeCN: H ₂ O / 3: 1: 1, vortexed and then the supernatant is added to the first supernatant. The combined supernatant containing oligoribonucleotides is dried to obtain a white powder. Base deprotected oligoribonucleotides were resuspended in anhydrous TEA / HF / NMP solution (1.5 mL N-methylpyrrolidinone, 750 μL TEA and 1.0 mL TEA · 3HF solution 300 μL, HF concentration 1.4 M) and brought to 65 ° C. Heat. After 1.5 hours, the oligomer is quenched with 1.5M NH ₄ HCO ₃ .

Alternatively, for the one-pot protocol, polymer-bound trityl-on-oligoribonucleotides are transferred to a 4 mL glass screw cap vial and 65% in a solution of 33% ethanolic methylamine / DMSO: 1/1 (0.8 mL). Suspend at 15 ° C. for 15 minutes. Bring the vial to room temperature. TEA · 3HF (0.1 mL) is added and the vial is heated at 65 ° C. for 15 minutes. The sample is cooled to −20 ° C. and then quenched with 1.5M NH ₄ HCO ₃ .

For purification of the trityl-on oligomers, the quenched NH ₄ HCO ₃ solution, acetonitrile, followed by loading the C-18 containing cartridge that had been prewashed with 50 mM TEAA. After washing the loaded cartridge with water, the RNA is detritylated with 0.5% TFA for 13 minutes. The cartridge is then washed again with water, salt exchanged with 1M NaCl and washed again with water. The oligonucleotide is then eluted with 30% acetonitrile.

  Inactive hammerhead ribozymes or attenuated control (BAC) oligonucleotides) are synthesized by replacing G5 with U and A14 with U (numbering is described by Hertel, KJ, et al., 1992, Nucleic Acids Res., 20, 3252). Similarly, one or more nucleotide substitutions can be introduced into other enzymatic nucleic acid molecules to inactivate the molecule, and such molecules can serve as negative controls.

The average step coupling yield is typically> 98% (Wincott et al., 1995 Nucleic Acids Res. 23, 2677-2684). Those skilled in the art will appreciate that the scale of synthesis can be larger or smaller than the examples described above, for example, but not limited to, can be adapted to a 96-well format, and importantly only the proportion of chemicals used in the reaction. You will recognize that.

  Alternatively, the nucleic acid molecules of the invention may be synthesized separately and joined together after synthesis, for example by ligation (Moore et al., 1992, Science 256, 9923; Draper et al. International Publication WO 93/23569; Shabarova) et al., 1991, Nucleic Acids Research 19, 4247; Bellon et al., 1997, Nucleosides & Nucleotides, 16, 951; Belon et al., 1997, Nucleosides & Nucleo et al.

  Preferably, the nucleic acid molecules of the invention are extensively modified to give nuclease resistant groups such as 2'-amino, 2'-C-allyl, 2'-fluoro, 2'-O-methyl, 2'-H. (See Usman and Cedergren, 1992, TIBS 17, 34; Usman et al., 1994, Nucleic Acids Symp. Ser. 31, 163). Ribozymes can be purified by gel electrophoresis using common methods, or see high performance liquid chromatography (HPLC; Wincott et al., Supra), the entirety of which is hereby incorporated herein by reference. C) and resuspend in water.

Optimization of the activity of the nucleic acid molecules of the invention Chemically synthesized nucleic acid molecules having modifications (bases, sugars and / or phosphates) that prevent degradation by serum ribonucleases will increase their drag (eg, , Eckstein et al., International Publication WO 92/07065; Perrart et al., 1990 Nature 344,565; et
al. , International Publication WO 93/15187; Rossi et al. , International Publication No. WO 91/03162; Sproat, US Pat. No. 5,334,711; Gold et al. , US 6,300,074 and Burgin et al. , (Supra), all of which are hereby incorporated by reference. It is desirable to remove bases from nucleic acid molecules in order to shorten their synthesis time and reduce the need for chemicals, as well as modifications that enhance their drag in the cell. All of the above references describe various chemical modifications that can be made to the base, phosphate and / or sugar components of the nucleic acid molecules described herein.

There are several examples in the art describing sugar, base and phosphate modifications that can be introduced into nucleic acid molecules that can significantly enhance its nuclease stability and efficacy. For example, oligonucleotides are nuclease resistant groups such as 2'-amino, 2'-C-allyl, 2'-fluoro, 2'-O-methyl, 2'-O-allyl, 2'-H, nucleotide bases Modified to improve stability and / or enhance biological activity (for review see Usman and Cedergren, 1992 TITBS 17, 34; Usman et al., 1994 Nucleic Acids Symp. Ser. 31, 163; see Burgin et al., 1996 Biochemistry 35, 14090). Sugar modifications of nucleic acid molecules are widely described in the art (Eckstein et al., International Publication WO 92/07005; Perrart et al. Nature 1990, 344, 565-568; Pieken et al. Science 1991, 253, 314-317; Usman and Cedergren, Trends in B
iochem. Sci. 1992, 17, 334-339; Usman et al. International Publication WO 93/15187; Sproat, US Pat. No. 5,334,711, Beigelman et al. 1995 J. MoI. Biol. Chem. 270, 25702; Beigelman et al. , International Publication WO 97/26270; Beigelman
et al. U.S. Pat. No. 5,716,824; Usman et al. U.S. Pat. No. 5,627,053; Woolf et al. , International Publication WO 98/13526; Thompson et al. U.S. Patent Application 60 / 082,404, filed April 20, 1998; Karpeskyy et al. 1998, Tetrahedron Lett. 39, 1131; Earnshaw and Gait, 1998, Biopolymers (Nucleic Acid Sciences), 48, 39-55; Verma and Eckstein, 1998, Annu. Rev. Biochem. 67, 99-134; and Burlina et al. , 1997, Bioorg. Med. Chem. 5, 1999-2010, all of which are hereby incorporated by reference in their entirety). These publications describe general methods and strategies for determining the position to incorporate sugars, bases and / or phosphate modifications, etc. into ribozymes without inhibiting catalytic activity, and are part of this specification. As quoted here. In view of such teachings, the nucleic acid molecules of the present invention can be modified using modifications similar to those described herein.

  While chemical modification of oligonucleotide internucleotide linkages with phosphorothioate, phosphorothioate, and / or 5'-methylphosphonate linkages improves stability, excessive modification can cause some toxicity. Therefore, when designing nucleic acid molecules, the amount of these internucleotide linkages should be minimized. Decreasing the concentration of these bindings should reduce toxicity, increase the potency of these molecules and increase their specificity.

  Nucleic acid molecules having chemical modifications that maintain or enhance activity are provided. Such nucleic acids are also generally more resistant to nucleases than unmodified nucleic acids. Thus, the activity will not be significantly reduced intracellularly and / or in vivo. The exogenously delivered therapeutic nucleic acid molecule should optimally be stable in the cell until the translation of the target RNA is regulated for a time long enough to reduce the level of unwanted protein. This time will vary from hours to days depending on the disease state. Clearly, a nucleic acid molecule must be resistant to nucleases in order to function as an effective intracellular therapeutic agent. Improvements in RNA and DNA chemical synthesis (Wincott et al., 1995 Nucleic Acids Res. 23, 2677; Caruthers et al., 1992, Methods in Enzymology 211, 3-19 (herein incorporated by reference) )), As described above, the possibility of modifying nucleic acid molecules was expanded by introducing nucleotide modifications to enhance their nuclease stability.

  In one embodiment, the nucleic acid molecule of the invention comprises one or more G clamp nucleotides. G-clamp nucleotides are modified cytosine analogs, where the modification confers hydrogen bonding capabilities on both Watson-Crick and Hoogsteen faces of complementary guanines in the duplex. For example, Lin and Matteucci, 1998, J. MoI. Am. Chem. Soc. , 120, 8531-8532. A single G-clamp analog substitution in the oligonucleotide can substantially enhance helical thermal stability and mismatch discrimination when hybridized to a complementary oligonucleotide. By incorporating such nucleotides into the nucleic acid molecules of the present invention, both affinity and specificity for the nucleic acid target are enhanced. In another embodiment, the nucleic acid molecules of the invention comprise one or more LNA “locked nucleic acid” nucleotides, eg, 2 ′, 4′-C methylene bicyclonucleotides (eg, Wengel et al., International Publication). See WO 00/66604 and WO 99/14226).

  In another aspect, the invention features conjugates and / or complexes of nucleic acid molecules that target VEGF receptors, such as VEGFR1 and / or VEGFR2. Such conjugates and / or complexes can be used to facilitate delivery of the molecule to a biological system, such as a cell. Conjugates and complexes provided by the present invention treat therapeutic compounds by transporting therapeutic compounds across cell membranes, altering pharmacokinetics, and / or modulating localization of the nucleic acid molecules of the present invention. Activity can be imparted. The present invention includes molecules such as, but not limited to, small molecules, lipids, phospholipids, nucleosides, nucleotides, nucleic acids, antibodies, toxins, negatively charged polymers and other polymers such as proteins, peptides, hormones, carbohydrates, Includes the design and synthesis of new conjugates and complexes for transporting polyethylene glycol, or polyamines, across cell membranes. In general, the transporters described are designed to be used with or without degradable linkers, either individually or as part of a multicomponent system. These compounds are expected to improve the delivery and / or localization of the nucleic acid molecules of the invention to a number of cell types from different tissues in the presence or absence of serum (Sullanger and Cech). , US 5,854,038). The conjugates of the molecules described herein can be attached to a biologically active molecule via a biodegradable linker, such as a biodegradable nucleic acid linker molecule.

  As used herein, the term “biodegradable nucleic acid linker molecule” is designed as a biodegradable linker to connect one molecule to another molecule, eg, a biologically active molecule. Represents a nucleic acid molecule. The stability of a biodegradable nucleic acid linker molecule is determined by ribonucleotides, deoxyribonucleotides, and chemically modified nucleotides such as 2'-O-methyl, 2'-fluoro, 2'-amino, 2'-O-. Modifications can be made by using various combinations of amino, 2′-C-allyl, 2′-O-allyl, and other 2′-modified or base-modified nucleotides. Biodegradable nucleic acid linker molecules can be dimers, trimers, tetramers, or longer nucleic acid molecules, such as about 2,3,4,5,6,7,8,9,10,11,12,13,14,15. , 16, 17, 18, 19, or 20 nucleotides in length, or include a single nucleotide having a phosphate-based linkage, eg, a phosphoramidate or phosphodiester linkage Can do. Biodegradable nucleic acid linker molecules can also include nucleic acid backbones, nucleic acid sugars, or nucleobase modifications.

  As used herein, the term “biodegradable” refers to degradation in biological systems, such as enzymatic degradation or chemical degradation.

  As used herein, the term “biologically active molecule” refers to a compound or molecule capable of eliciting or modulating a biological response in a system. Non-limiting examples of biologically active molecules contemplated by the present invention include therapeutically active molecules such as antibodies, hormones, antiviruses, peptides, proteins, chemotherapeutic agents, small molecules, vitamins, complements. Factors, nucleosides, nucleotides, oligonucleotides, enzymatic nucleic acids, antisense nucleic acids, triplex-forming oligonucleotides, 2,5-A chimeras, siRNA, dsRNA, allozymes, aptamers, decoys and analogs thereof are included. Biologically active molecules of the present invention include molecules that can modulate the pharmacokinetics and / or pharmacodynamics of other biologically active molecules, such as lipids and polymers, such as polyamines, polyamides Polyethylene glycol and other polyethers are also included.

As used herein, the term “phospholipid” refers to a hydrophobic molecule that contains at least one phosphate group. For example, the phospholipid can include a phosphate-containing group and a saturated or unsaturated alkyl group that can be optionally substituted with OH, COOH, oxo, amine, or a substituted or unsubstituted aryl group.

  Externally delivered therapeutic nucleic acid molecules (eg, enzymatic nucleic acid molecules and antisense nucleic acid molecules) optimally modulate the translation of the target RNA for a long enough time to reduce the level of unwanted proteins Should be stable in the cell. This time will vary from hours to days depending on the disease state. These nucleic acid molecules should be resistant to nucleases in order to function as effective intracellular therapeutic agents. Improvements in the chemical synthesis of nucleic acid molecules described herein and in the art have the potential to modify nucleic acid molecules by introducing nucleotide modifications to enhance their nuclease stability, as described above. Enlarged.

  In another embodiment, nucleic acid catalysts having chemical modifications that maintain or enhance enzymatic activity are provided. Such nucleic acids are also generally more resistant to nucleases than unmodified nucleic acids. Thus, the activity of the nucleic acid will not be significantly reduced in the cell and / or in vivo. As exemplified herein, such enzymatic nucleic acids are useful in cells and / or in vivo even if the overall activity is reduced by a factor of 10 (Burgin et al., 1996, Biochemistry, 35, 14090). Such enzymatic nucleic acids are referred to herein as “maintaining” the enzymatic activity of a total RNA ribozyme or a total DNA DNAzyme.

  In another aspect, the nucleic acid molecule comprises a 5 'and / or 3'-cap structure.

  “Cap structure” means a chemical modification incorporated at either end of an oligonucleotide (eg, Wincott et al., WO 97/26270 (herein incorporated by reference)). reference). These terminal modifications will protect the nucleic acid molecule from exonucleolytic degradation and help delivery and / or localization in the cell. The cap may be present at the 5'-end (5'-cap), may be present at the 3'-end (3'-cap), or may be present at both ends. In a non-limiting example, the 5'-cap includes an inverted abasic residue (component); 4 ', 5'-methylene nucleotide; 1- (beta-D-erythrofuranosyl) nucleotide, 4'-thionucleotide Carbocyclic nucleotides; 1,5-anhydrohexitol nucleotides; L-nucleotides; alpha-nucleotides; modified base nucleotides; phosphorodithioate linkages; threo-pentofuranosyl nucleotides; Aconucleotide; acyclic 3,4-dihydroxybutyl nucleotide; acyclic 3,5-dihydroxypentyl nucleotide, 3'-3'-inverted nucleotide component; 3'-3'-inverted abasic component; 3'-2'- Inverted nucleotide component; 3'-2'-inverted abasic component; 1,4-butanediol phosphate; 3'-phospho Amidate; hexyl phosphate; aminohexyl phosphate; 3′-phosphate; 3′-phosphorothioate; phosphorodithioate; or a bridged or non-bridged methylphosphonate moiety (for details see Wincott et al., International Publication WO 97 / 26270 (herein incorporated by reference).

In another preferred embodiment, the 3'-cap includes, for example, 4 ', 5'-methylene nucleotides; 1- (beta-D-erythrofuranosyl) nucleotides; 4'-thionucleotides, carbocyclic nucleotides; 5 1, -diamino-2-propyl phosphoric acid; 3-aminopropyl phosphoric acid; 6-aminohexyl phosphoric acid; 1,2-aminododecyl phosphoric acid; hydroxypropyl phosphoric acid; 5-anhydrohexitol nucleotides; L-nucleotides; alpha-nucleotides; modified base nucleotides; phosphorodithioates; threopentofuranosyl nucleotides; acyclic 3 ', 4'seconucleotides; 3,4-dihydroxybutyl nucleotides 3,5-dihydroxypentyl nucleotide, 5'-5'-inverted nucleotide component; 5'-5'-inverted abasic component; 5'-phosphoramidate;5'-phosphorothioate; 1,4-butanediol phosphate; 5'-amino; bridged and / or unbridged 5'-phosphoramid Dating, phosphorothioates and / or phosphorodithioates, bridged or non-bridged methylphosphonates and 5′-mercapto moieties (more particularly, Beaucage and Iyer, 1993, Tetrahedron 49, 1925 (as part of this specification). See quoted here)).

  As used herein, the term “non-nucleotide” can be introduced into a nucleic acid strand in place of one or more nucleotide units and includes either sugar and / or phosphate substitutions, and the rest Means any group or compound that allows the base of to exert its enzymatic activity. A group or compound is abasic if it does not contain a commonly recognized nucleotide base, such as adenosine, guanine, cytosine, uracil or thymine.

An “alkyl” group refers to a saturated aliphatic hydrocarbon and includes straight chain, branched chain, and cyclic alkyl groups. Preferably, the alkyl group has 1-12 carbons. More preferably it is a lower alkyl having 1-7 carbons, more preferably 1-4 carbons. Alkyl may or may not be substituted. When substituted, the substituent is preferably hydroxyl, cyano, alkoxy, ═O, ═S, NO ₂ or N (CH ₃ ) ₂ , amino, or SH. The term also includes alkenyl groups that are unsaturated hydrocarbon groups containing at least one carbon-carbon double bond, including straight-chain, branched-chain, and cyclic groups. Preferably, the alkenyl group has 1-12 carbons. More preferably it is a lower alkenyl of 1-7 carbon atoms, more preferably 1-4 carbon atoms. Alkenyl may or may not be substituted. When substituted, the substituent is preferably selected from hydroxyl, cyano, alkoxy, ═O, ═S, NO ₂ , halogen, N (CH ₃ ) ₂ , amino, or SH. The term “alkyl” also includes alkynyl groups having an unsaturated hydrocarbon group containing at least one carbon-carbon triple bond, and includes straight-chain, branched-chain, and cyclic groups. Preferably, the alkynyl group has 1-12 carbons. More preferably it is a lower alkynyl having 1-7 carbons, more preferably 1-4 carbons. Alkynyl groups may or may not be substituted. When substituted, the substituent is preferably hydroxyl, cyano, alkoxy, ═O, ═S, NO ₂ or N (CH ₃ ) ₂ , amino or SH.

Such alkyl groups can also include aryl, alkylaryl, carbocyclic aryl, heterocyclic aryl, amide and ester groups. An “aryl” group refers to an aromatic group having at least one ring with a conjugated pi-electron system, and includes carbocyclic aryl, heterocyclic aryl, and diaryl groups. All of these may be optionally substituted. Preferred substituents for the aryl group are halogen, trihalomethyl, hydroxyl, SH, OH, cyano, alkoxy, alkyl, alkenyl, alkynyl, and amino groups. An “alkylaryl” group refers to an alkyl group (described above) covalently bonded to an aryl group (described above). A carbocyclic aryl group is a group in which all of the ring atoms of the aromatic ring are carbon atoms. The carbon atom may be optionally substituted. A heterocyclic aryl group is a group having 1-3 heteroatoms as ring atoms in an aromatic ring, with the remainder of the ring atoms being carbon atoms. Suitable heteroatoms include oxygen, sulfur, and nitrogen, and include, for example, furanyl, thienyl, pyridyl, pyrrolyl, N-lower alkyl pyrrolo, pyrimidyl, pyrazinyl, imidazolyl, and the like. All of these may be optionally substituted. “Amide” refers to —C (O) —NH—R, wherein R is any of alkyl, aryl, alkylaryl or hydrogen. "
"Ester" represents -C (O) -OR '(wherein R is any of alkyl, aryl, alkylaryl or hydrogen).

  "Nucleotide" means a heterocyclic nitrogen base that is N-glycosylated with a phosphorylated saccharide. Nucleotides are recognized in the art to include natural bases (standard) and modified bases well known in the art. Such bases are generally located at the 1 'position of the nucleotide sugar component. Nucleotides generally contain bases, sugars and phosphate groups. Nucleotides may or may not be modified in the sugar, phosphate and / or base components (also referred to interchangeably as nucleotide analogs, modified nucleotides, non-natural nucleotides, non-standard nucleotides, etc.). For example, Usman and McSwigen (supra); Eckstein et al., International Publication WO92 / 07005; Usman et al., International Publication WO93 / 15187; Uhlman & Peyman, (supra) (all cited herein as part of this specification). To see)). There are several examples of modified nucleobases known in the art, see Limbach et al. , 1994, Nucleic Acids Res. 22, 2183. Some non-limiting examples of chemically modified and other natural nucleobases that can be introduced into nucleic acids include, for example, inosine, purine, pyridin-4-one, pyridin-2-one, phenyl , Pseudouracil, 2,4,6-trimethoxybenzene, 3-methyluracil, dihydrouridine, naphthyl, aminophenyl, 5-alkylcytidine (eg 5-methylcytidine), 5-alkyluridine (eg ribothymidine), 5- Halo lysine (eg 5-bromouridine) or 6-azapyrimidine or 6-alkylpyrimidine (eg 6-methyluridine), propyne, kesocin, 2-thiouridine, 4-thiouridine, wybutosine, wybutoxosin, 4-acetylcytidine, 5- ( Carboxyhydroxymethyl) uridine, '-Carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluridine, beta-D-galactosylkaeosin, 1-methyladenosine, 1-methylinosine, 2,2-dimethylguanosine, 3-methylcytidine, 2 Methyladenosine, 2-methylguanosine, N6-methyladenosine, 7-methylguanosine, 5-methoxyaminomethyl-2-thiouridine, 5-methylaminomethyluridine, 5-methylcarbonylmethyluridine, 5-methyloxyuridine, 5- Methyl-2-thiouridine, 2-methylthio-N6-isopentenyladenosine, beta-D-mannosylkeosin, uridine-5-oxyacetic acid, 2-thiocytidine, threonine derivatives and others (Burgin et . L, 1996, Biochemistry, 35,14090; Uhlman & Peyman, supra). In this respect, “modified base” means a nucleotide base other than adenine, guanine, cytosine and uracil at the 1 ′ position or equivalent thereof. Such bases can be used at any position, for example, in the catalytic core of an enzymatic nucleic acid molecule and / or in the substrate binding region of the nucleic acid molecule.

"Nucleoside" means a heterocyclic nitrogenous base that is N-glycosylated with a sugar. In the art, nucleosides are recognized to include natural bases (standards) and modified bases well known in the art. Such bases are generally located at the 1 ′ position of the nucleoside sugar component. Nucleosides generally contain a base and a sugar. Nucleosides may be modified or unmodified in sugar and / or base components (referred interchangeably as nucleoside analogs, modified nucleosides, non-natural nucleosides, non-standard nucleosides and others) See, for example, Usman and McSwigen, supra; Eckstein et al., WO 92/07065; Usman et al., WO 93/15187, supra, Uhlman & Peyman, supra, all part of this specification. As quoted here). Several examples of modified nucleobases are known in the art and are described in Limbach et al. , 1994, Nucleic Acids Res. 22, 2183. Non-limiting examples of chemically modified bases and other natural nucleobases that can be introduced into nucleic acids include inosine, purine, pyridin-4-one, pyridin-2-one, phenyl, pseudouracil. 2,4,6-trimethoxybenzene, 3-methyluracil, dihydrouridine,
Naphthyl, aminophenyl, 5-alkylcytidine (eg, 5-methylcytidine), 5-alkyluridine (eg, ribothymidine), 5-halouridine (eg, 5-bromouridine) or 6-azapyrimidine or 6-alkylpyrimidine ( For example, 6-methyluridine), propyne, quesosine, 2-thiouridine, 4-thiouridine, wybutosine, wybutoxocin, 4-acetylcytidine, 5- (carboxyhydroxymethyl) uridine, 5′-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluridine, beta-D-galactosyl quesosine, 1-methyladenosine, 1-methylinosine, 2,2-dimethylguanosine, 3-methylcytidine, 2-methyladenosine, 2-methylguanosine , N6-methyladenosine, 7-methylguanosine, 5-methoxyaminomethyl-2-thiouridine, 5-methylaminomethyluridine, 5-methylcarbonylmethyluridine, 5-methyloxyuridine, 5-methyl-2-thiouridine, 2-methylthio-N6-isopentenyladenosine, beta-D-mannosyl quesosine, uridine-5-oxyacetic acid, 2-thiocytidine, threonine derivatives and others (Burgin et al., 1996, Biochemistry, 35, 14090; Uhlman & Peyman, (supra). In this respect, “modified base” means a nucleoside base other than adenine, guanine, cytosine and uracil at the 1 ′ position or an equivalent thereof. Such bases can be used at any position, for example in the catalytic core of an enzymatic nucleic acid molecule and / or in the substrate binding region of the nucleic acid molecule.

In one embodiment, the invention features a modified enzymatic nucleic acid molecule having a phosphate backbone modification, which includes one or more phosphorothioates, phosphorodithioates, methylphosphonates, morpholinos, amidates, carbamates, carboxys Includes methyl, acetamidate, polyamide, sulfonate, sulfonamide, sulfamate, formacetal, thioformacetal, and / or alkylsilyl substitution. For a review of oligonucleotide backbone modifications, see Hunziker and Leumann, 1995, Nucleic Acid Analogues: Synthesis and Properties, Modern Synthetic Methods, VCH, 331-417, and Mesmaer et al. , 1994, Novell Backbone Replacements for Oligonucleotides, Carbohydrate Modifications in Antisense.
See Research, ACS, 24-39. These references are cited herein as part of this specification.

  As used herein, the term “abasic” refers to sugar moieties that lack a base at the 1 ′ position or have other chemical groups in place of the base, eg, 3 ′, 3′— Means a bound or 5 ', 5'-linked deoxyabasic ribose derivative (for details see Wincott et al., WO 97/26270).

  “Unmodified nucleoside” means one of the bases, adenine, cytosine, guanine, thymine, uracil bonded to the 1 ′ carbon of β-D-ribo-furanose.

  By “modified nucleoside” is meant any nucleotide base that contains a modification in the chemical structure of an unmodified nucleotide base, sugar and / or phosphate.

With respect to the 2′-modified nucleotides described in the present invention, “amino” means 2′-NH ₂ or 2′-O—NH ₂ , which may or may not be modified. Such modifying groups are described, for example, in Eckstein et al. U.S. Pat. No. 5,672,695 and Matric-Adamic et al. , WO 98/28317, all of which are hereby incorporated by reference in their entirety.

  Various modifications to the nucleic acid (eg, antisense and ribozyme) structure can be made to increase the usefulness of these molecules. For example, such modifications increase product life, in vitro half-life, stability, and ease of introducing such oligonucleotides into the target site, eg, increase cell membrane permeability and target cells. Will grant the ability to recognize and combine.

  The use of nucleic acid-based molecules of the present invention will lead to better treatment of disease progression by providing the possibility of combination therapy (eg, a large number of enzymatic nucleic acid molecules targeting different genes, Enzymatic nucleic acid molecules coupled with known small molecule inhibitors, or intermittent treatment with enzymatic nucleic acid molecules (including different enzymatic nucleic acid molecule motifs) and / or other chemical or biological molecule combinations) . Treatment of patients with nucleic acid molecules also includes combinations of different types of nucleic acid molecules. Inventing a therapy comprising a mixture of enzymatic nucleic acid molecules (including different enzymatic nucleic acid molecule motifs), allozymes, antisense, dsRNA, aptamers and / or 2-5A chimeric molecules against one or more targets to treat disease Symptoms can be reduced.

Administration of nucleic acid molecules Methods for delivery of nucleic acid molecules are described in Akhtar et al. (1992, Trends Cell Bio., 2, 139) and Delivery Strategies for Antisense Oligonucleotide Therapeutics, ed. Akhtar, 1995 (all of which are hereby incorporated by reference). Sullivan et al. , PCT WO 94/02595 further describes a general method for delivering enzymatic RNA molecules. With these protocols, virtually any nucleic acid molecule can be delivered. Nucleic acid molecules can be administered to cells by various methods known to those skilled in the art, including encapsulation in liposomes, iontophoresis, or other vehicles such as hydrogels, cyclodextrins, biodegradable nanocapsules, Including but not limited to incorporation into bioadhesive spherules. Alternatively, the nucleic acid / vehicle combination is locally delivered by direct infusion or by using an infusion pump. Other delivery routes include, but are not limited to, oral (tablet or pill form) and / or intrathecal delivery (Gold, 1997, Neuroscience, 76, 1153-1158). Other methods include using various transport and carrier systems, such as using conjugates and biodegradable polymers. For a comprehensive review of drug delivery strategies including CNS delivery, see Ho et al. 1999, Curr. Opin. Mol. Ther. , 1,336-343 and Jain, Drug
Delivery Systems: Technologies and Commercial Opportunities, Decision Resources, 1998, and Groothis et al. , 1997, J. Am. Neuro Virol. 3,387-400. A more detailed description of nucleic acid delivery and administration can be found in Sullivan et al. (Supra), Draper et al. , PCTW093 / 23569, Beigelman et al. , PCTW099 / 05094, and Klimuk et al. , PCTW 099/04819, all of which are hereby incorporated by reference.

  The molecules of the present invention can be used as pharmaceuticals. The medicinal product prevents the disease state, inhibits or treats the disease in the patient (alleviates symptoms to some extent, preferably alleviates all symptoms).

The polynucleotide of the present invention can be administered (eg, RNA, DNA or protein) by any standard means by forming a pharmaceutical composition with or without stabilizers, buffers, etc. Can be introduced. If it is desirable to utilize a liposome delivery mechanism, standard protocols for forming liposomes can be followed. The compositions of the present invention are also known as tablets, capsules or elixirs for oral administration; as suppositories for rectal administration; as sterile solutions; as suspensions for infusion administration, and as known in the art. It can be formulated and used as another composition that can be used.

  The present invention also includes pharmaceutically acceptable formulations of the compounds described. These formulations include salts of the aforementioned compounds, for example, acid addition salts (eg, salts of hydrochloric acid, oxalic acid, acetic acid, and benzenesulfonic acid).

  A pharmaceutical composition or formulation refers to a composition or formulation in a form suitable for administration to a cell or patient (eg, systemic administration), preferably to a human. The appropriate form depends in part on the route of administration used (eg, oral, transdermal, or injection). Such a form should not prevent the composition or formulation from reaching the target cell (ie, the cell to which the negatively charged polymer is desired to be delivered). For example, a pharmaceutical composition that is injected into the bloodstream must be soluble. Other factors are known in the art and include, for example, consideration of toxicity and forms that prevent the composition or formulation from exerting its effect.

  “Systemic administration” means distributed systemically after in vivo systemic absorption or accumulation of the drug in the bloodstream. Routes of administration that provide systemic absorption include, but are not limited to, intravenous, subcutaneous, intraperitoneal, inhalation, oral, intrapulmonary, and intramuscular. Each of these routes of administration exposes the desired negatively charged polymer (eg, nucleic acid) to accessible diseased tissue. The rate at which drugs enter the circulation has been shown to be a function of molecular weight or size. By using liposomes or other drug carriers containing the compounds of the invention, it is possible to localize the drug, for example, to a certain type of tissue (eg, tissue of the reticuloendothelial system (RES)). Liposome formulations that can facilitate the association of the drug with the surface of cells (eg, leukocytes and macrophages) are also useful. This method involves abnormal cells due to macrophages and leukocytes (eg, endometriosis, fertility regulation, endometrial tumors, gynecological bleeding disorders, irregular menstrual cycles, ovulation, premenstrual syndrome (PMS), menopausal dysfunction , And cells that have been implicated in endometrial carcinoma) will enhance the transport of drugs to target cells.

“Pharmaceutically acceptable formulation” means a composition or formulation capable of effectively distributing a nucleic acid molecule of the present invention to the physical location most suitable for its desired activity. Non-limiting examples of agents suitable for formulation with the nucleic acid molecules of the present invention include: PEG conjugated nucleic acids, phospholipid conjugated nucleic acids, nucleic acids containing lipophilic components, phosphorothioates, various P-glycoprotein inhibitors (Pluronic P85, etc.) that can promote the invasion of drugs into other tissues, such as the CNS (Jolliet-Rient and Tillement, 1999, Fundam. Clin. Pharmacol., 13, 16-26) Biodegradable polymers for sustained release transport after intracerebral transplantation, such as poly (DL-lactide-coglycolide) microspheres (Emerich, DF et al, 1999, Cell Transplant, 8, 47-58) Alkermes, Inc .; Cambridge, MA; and packed nanoparticles made from polybutylcyanoacrylate, for example, that can transport drugs across the cerebrovascular barrier and alter neural uptake mechanisms (Prog Neuropsychopharmacol Biol Psychiatry, 23, 941-949, 1999). Other non-limiting examples of delivery strategies, such as CNS delivery of the nucleic acid molecules of the present invention, include Boado et al. 1998, J. MoI. Pharm. Sci. 87, 1308-1315; Tyler et al. 1999, FEBS Lett. , 421, 280-284; Pardridge e
t al. , 1995, PNAS USA. , 92, 5592-5596; Boado, 1995, Adv. Drug Delivery Rev. , 15, 73-107; Aldrian-Herrada et al. 1998, Nucleic Acids Res. , 26, 4910-4916; and Tyler et al. 1999, PNAS USA. 96, 7053-7058. All of these documents are hereby incorporated by reference.

  The invention also features the use of compositions comprising surface-modified liposomes, including poly (ethylene glycol) lipids (PEG-modified, or long-circulating liposomes or stealth liposomes). The nucleic acid molecules of the present invention also include PEG molecules of various molecular weights linked by covalent bonds. These formulations provide a way to increase drug accumulation in the target tissue. This type of drug carrier is resistant to opsonization and elimination by the mononuclear phagocyte system (MPS or RES), thus increasing the circulation time of the encapsulated drug and enhancing tissue exposure (Lasic et al. Chem. Rev. 1995, 95, 2601-2627; Ishiwata et al., Chem. Pharm. Bull. 1995, 43, 1005-1101). Such liposomes have been shown to accumulate selectively in tumors, possibly due to extravasation and capture in angiogenic target tissues (Lasic et al., Science 1995, 267, 1275-1276; Oku et al. al., 1995, Biochim. Biophys. Acta, 1238, 86-90). Long-circulating liposomes enhance the pharmacokinetics and pharmacodynamics of DNA and RNA, especially compared to conventional cationic liposomes known to accumulate in MPS tissues (Liu et al., J. Biol. Biol.Chem. 1995, 42, 24864-24870; Quoted here as part of the book). Long-circulating liposomes also appear to protect drugs more strongly from nuclease degradation compared to cationic liposomes based on their ability to avoid accumulation in metabolically aggressive MPS tissues such as the liver and spleen . All of these references are hereby incorporated by reference herein.

The present invention also includes compositions prepared for storage or administration that include a pharmaceutically effective amount of the desired compounds in a pharmaceutically acceptable carrier or diluent. Acceptable carriers or diluents for use in therapeutic applications are well known in the pharmaceutical art, for example, Remington's Pharmaceutical Sciences, Mack Public.
ishing Co. (AR Gennaro edit. 1985), which is hereby incorporated by reference. For example, preservatives, stabilizers, dyes, and flavoring agents can be used. These include sodium benzoate, sorbic acid, and esters of p-hydroxybenzoic acid. In addition, antioxidants and suspending agents may be used.

  A pharmaceutically effective dose is that dose necessary to prevent, inhibit, or treat a disease state (alleviate symptoms to some extent, preferably alleviate all symptoms). The pharmaceutically effective dose depends on the type of disease, the composition used, the route of administration, the type of mammal being treated, the physical properties of the particular mammal under consideration, the drugs being co-administered, and the pharmaceutical field It depends on other factors that those skilled in the art will recognize. Generally, 0.1 mg / kg-100 mg / kg body weight / day of active ingredient is administered depending on the potency of the negatively charged polymer.

The nucleic acid molecules of the invention can be administered orally, topically, parenterally, by inhalation or spray, in dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants and vehicles. Or it can be administered rectally. As used herein, the term parenteral includes percutaneous, subcutaneous, intravascular (eg, intravenous), intramuscular, or intracapsular injection or infusion techniques. Further provided is a pharmaceutical formulation comprising a nucleic acid molecule of the present invention and a pharmaceutically acceptable carrier. One or more nucleic acid molecules of the present invention are present together with one or more non-toxic pharmaceutically acceptable carriers and / or diluents and / or adjuvants and other active ingredients as desired. can do. The pharmaceutical composition containing the nucleic acid molecule of the present invention is in a form suitable for oral use, such as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard capsules or It can be in the form of a soft capsule or syrup or elixir.

  Compositions intended for oral use may be prepared according to any method known in the art for the manufacture of pharmaceutical compositions, and such compositions may be prepared using a pharmaceutically sophisticated mouthpiece. One or more of such sweetening, flavoring, coloring or preserving agents may be included to provide a product that suits. Tablets contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients that are suitable for the manufacture of tablets. These excipients include, for example, inert diluents such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents such as corn starch, or alginic acid; binders such as starch , Gelatin or gum arabic, and lubricants such as magnesium stearate, stearic acid or talc. Tablets may be uncoated or may be coated by known techniques. In some cases, such coatings can be prepared by known techniques to delay disintegration and absorption in the gastrointestinal tract, thereby providing a longer duration of action. For example, a time delay material such as glyceryl monostearate or glyceryl distearate can be employed.

  Formulations for oral use include hard gelatin capsules in which the active ingredient is mixed with an inert solid diluent such as calcium carbonate, calcium phosphate or kaolin, or the active ingredient is water or an oily medium such as peanut oil, liquid paraffin Or it may be a soft gelatin capsule mixed with olive oil.

  Aqueous suspensions contain the active materials in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients are suspending agents, for example sodium carboxymethylcellulose, methylcellulose, hydropropyl-methylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum arabic. Dispersants or wetting agents are naturally occurring phosphatides, such as lecithin, or condensation products of alkylene oxide and fatty acids, such as polyoxyethylene stearate, or condensation products of ethylene oxide and long chain aliphatic alcohols, For example, heptadecaethyleneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol, such as polyoxyethylene sorbitol monooleate, or moieties derived from ethylene oxide and fatty acids and anhydrous hexitol It may be a condensation product with an ester, for example, polyethylene sorbitan monooleate. Aqueous suspensions also contain one or more preservatives, such as ethyl- or n-propyl-p-hydroxybenzoate, one or more colorants, one or more fragrances, and one or more. It may contain the above sweeteners such as sucrose or saccharin.

  Oily suspensions may be formulated by suspending the active ingredient in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in an inorganic oil such as liquid paraffin. Oily suspensions may contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol. Sweetening and flavoring agents can be added to provide a palatable oral product. These compositions can be preserved by the addition of an anti-oxidant such as ascorbic acid.

  Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water include the active ingredient in a mixture with a dispersing or wetting agent, suspending agent and one or more preservatives. Give in. Suitable dispersing or wetting agents or suspending agents are as exemplified above. Still other excipients may be present, for example sweetening, flavoring and coloring agents.

  The pharmaceutical composition of the present invention may also be in the form of an oil-in-water emulsion. The oily phase may be a vegetable oil or a mineral oil or mixtures of these. Suitable emulsifiers include naturally occurring gums such as gum arabic or tragacanth, naturally occurring phosphatides such as soy, lectin, and esters or partial esters derived from fatty acids and hexitols, anhydrides such as sorbitan mono Examples include oleate and condensation products of the partial ester and ethylene oxide, such as polyoxyethylene sorbitan monooleate. The emulsion may contain sweeteners and fragrances.

  Syrups and elixirs can be formulated with sweetening agents, for example glycerol, propylene glycol, sorbitol, glucose or sucrose. Such formulations may also contain a demulcent, a preservative and sweetening and coloring agents. The pharmaceutical compositions may be in the form of a sterile injectable aqueous or oleagenous suspension. This suspension may be formulated as known in the art using suitable dispersing or wetting agents and suspending agents described above. A sterile injectable product may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example, a solution in 1,3-butanediol. Also good. Examples of acceptable vehicles and solvents that can be used are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterilized and coagulated oil can be conveniently used as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid can be used in the manufacture of injectable medicaments.

  The nucleic acid molecules of the invention can also be administered in the form of suppositories, eg, for rectal administration of the drug. These compositions are made by mixing the drug with a suitable non-irritating excipient that is solid at normal temperature but liquid at rectal temperature and therefore melts in the rectum to release the drug be able to. Such materials include cocoa butter and polyethylene glycol.

  The nucleic acid molecules of the invention can be administered parenterally in a sterile medium. Depending on the vehicle and concentration used, the drug may be suspended or dissolved in the vehicle. It may also be advantageous to dissolve adjuvants such as local anesthetics, preservatives and buffering agents in the vehicle.

  For the treatment of the above conditions, dosage levels on the order of about 0.1 mg to about 140 mg per day per kilogram of body weight are useful (about 0.5 mg to about 7 g per day per patient). The amount of active ingredient that can be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. Dosage unit forms will generally contain about 1 mg to about 500 mg of an active ingredient.

  The particular dose level for a particular patient can vary depending on various factors such as the activity of the particular compound used, age, weight, general health, sex, diet, time of administration, route of administration, and elimination rate, It will be understood that this depends on the combination and the severity of the particular disease being treated.

  For administration to non-human animals, the composition may be added to animal feed or drinking water. It may be convenient to formulate animal feed and drinking water compositions so that the animal can be inoculated with the feed in a therapeutically appropriate amount of the composition. It may also be convenient to produce the composition as a premix for addition to feed or drinking water.

  The nucleic acid molecules of the invention can also be administered to a patient in combination with other therapeutic compounds to enhance the overall therapeutic effect. The use of multiple compounds in the treatment of an indication can increase the beneficial effects while reducing the presence of side effects.

Alternatively, certain nucleic acid molecules of the invention can be expressed in cells from eukaryotic promoters (see, eg, Izant and Weintraub, 1985 Science 229, 345; McGarry and Lindquist, 1986 Proc. Natl. Acad. Sci.USA 83,399;
et al. 1991, Proc. Natl. Acad. Sci. USA, 88, 10591-5; Kasani-Sabet et al. , 1992 Antisense Res. Dev. 2, 3-15; Dropulic et al. , 1992 J. et al. Virol 66, 1432-41; Weerasinghe et al. , 1991 J. et al. Virol, 65, 5531-4; Ojwang et al. , 1992 Proc. Natl. Acad. Sci. USA 89, 10802-6; Chen et al. , 1992 Nucleic Acids Res. , 20, 4581-9; Sarver et al. , 1990 Science 247, 1222-1225; Thompson et al. 1995 Nucleic Acids Res. 23, 2259; Good et al. 1997, Gene Therapy, 4, 45; all references are hereby incorporated by reference in their entirety)). One skilled in the art will recognize that any nucleic acid can be expressed from a suitable DNA / RNA vector in a eukaryotic cell. The activity of such nucleic acids can be increased by releasing them from primary transcripts with enzymatic nucleic acids (Draper et al., PCT WO 93/23569, Sullivan et al., PCT WO 94/02595; Ohkawa et al. al., 1992 Nucleic Acids Symp. Ser., 27, 15-6; Taira et al., 1991, Nucleic Acids Res., 19, 5125-30; Ventura
et al. , 1993 Nucleic Acids Res. , 21, 3249-55; Chowira et al. , 1994 J. et al. Biol. Chem. 269, 25856; all references are hereby incorporated by reference in their entirety). Gene therapy methods specific for CNS are described in Blesch et al. , 2000, Drug News Perspect. , 13, 269-280; Peterson et al. , 2000, Cent. Nerv. Syst. Dis. 485-508; Peel and Klein, 2000, J. MoI. Neurosci. Methods, 98, 95-104; Hagihara et al. 2000, Gene Ther. , 7, 759-763; and Herringer et al. , 2000, Methods Mol. Med. , 35, 287-312. AAV-mediated delivery of nucleic acids to neural cells is further described in Kaplitt et al. , US 6,180,613.

In another aspect of the invention, the RNA molecule of the invention is preferably expressed from a transcription unit inserted into a DNA or RNA vector (see, e.g., Couture et al., 1996, TIG., 12, 510). ). The recombinant vector is preferably a DNA plasmid or a viral vector. Viral vectors that express ribozymes can be constructed based on, but not limited to, adeno-associated virus, retrovirus, adenovirus, or alphavirus. Preferably, the recombinant vector capable of expressing the nucleic acid molecule is delivered as described above and remains in the target cell. Alternatively, viral vectors that give transient expression of nucleic acid molecules can be used. Such vectors can be repeatedly administered as necessary. Once expressed, the nucleic acid molecule binds to the target mRNA. Delivery of vectors expressing nucleic acid molecules can be systemic (eg, by intravenous or intramuscular administration), administered to target cells explanted from the patient, then reintroduced into the patient, or desired target cells. It can be done by any other means that allow introduction into it (for review see Couture et al., 1996, TIG., 12, 510).

  In one aspect, the invention features an expression vector that includes a nucleic acid sequence encoding at least one of the nucleic acid molecules of the invention. Nucleic acid sequences encoding the nucleic acid molecules of the invention are operably linked in a manner that allows expression of the nucleic acid molecule.

In another aspect, the invention features an expression vector comprising: a) a transcription initiation region (eg, the initiation region of a eukaryotic pol I, II or III); b) a transcription termination region (eg, a eukaryotic region). A termination region of the organism pol I, II or III); c) comprising a nucleic acid sequence encoding at least one of the nucleic acid catalysts of the invention, said sequence being in a manner allowing the expression and / or delivery of said nucleic acid molecule , Operatively connected to the start area and the end area. The vector optionally comprises an open reading frame (ORF) of the protein operably linked 5 ′ or 3 ′ to the sequence encoding the nucleic acid catalyst of the present invention; and / or an intron (intervening sequence). May be included.

Transcription of nucleic acid molecule sequences is driven by eukaryotic RNA polymerase I (pol I), RNA polymerase II (pol II), or RNA polymerase III (pol III) promoters. Transcripts from the pol II or pol III promoter will be expressed at high levels in all cells. The level of a given pol II promoter in a given cell type will depend on the nature of nearby gene regulatory sequences (enhancers, silencers, etc.). A prokaryotic RNA polymerase promoter is also used as long as the prokaryotic RNA polymerase enzyme is expressed in a suitable cell (Elroy-Stein and Moss, 1990 Proc. Natl. Acad. Sci. USA, 87, 6743-7; Gao and Huang 1993 Nucleic Acids Res., 21, 2867-72; Lieberet. al., 1993 Methods Enzymol., 217, 47-66; Are hereby incorporated by reference as part of this specification). Several researchers have shown that nucleic acid molecules expressed from such promoters, such as ribozymes, can function in mammalian cells (eg, Kasani-Sabet et al., 1992 Antisense Res. Dev., Ojwang et al., 1992 Proc.Natl.Acad.Sci.USA, 89, 10802-6; Chen et al., 1992 Nucleic Acids Res., 20, 4581-9; Yu et al., 1993 Proc. Natl. Acad. Sci. USA, 90, 6340-4;
, 1992 EMBO J. et al. 11, 4411-8; Lisziewicz et al. 1993 Proc. Natl. Acad. Sci. U. S. A. , 90, 8000-4; Thompson et al. 1995 Nucleic Acids Res. 23, 2259; Sullanger & Cech, 1993, Science, 262, 1566). More particularly, transcription units such as those derived from genes encoding U6 micronuclei (snRNA), transfer RNA (tRNA) and adenovirus VA RNA are highly concentrated in the cell of the desired RNA molecule (eg ribozyme). (Thompson et al., Supra); Couture and Stin
chcomb, 1996, (supra); Nonberg et al. , 1994, Nucleic Acid Res. , 22, 2830; Nononberg et al. U.S. Pat. No. 5,1624,803; Good et al. , 1997, Gene Ther. 4, 45; Beicrelman et al. , International Publication WO 96/18736; (all these publications are hereby incorporated by reference). The ribozyme transcription unit described above can be incorporated into various vectors for introduction into mammalian cells. Vectors include, but are not limited to, plasmid DNA vectors, viral DNA vectors (eg, adenovirus or adeno-associated virus vectors), or viral RNA vectors (eg, retrovirus or alphavirus vectors) (for review, see: and Stinchcomb, 1996, supra).

  In another aspect, the invention features an expression vector that includes a nucleic acid sequence encoding at least one of the nucleic acid molecules of the invention in a manner that allows expression of the nucleic acid molecule. In one embodiment, the expression vector comprises a) a transcription initiation region; b) a transcription termination region; c) a nucleic acid sequence encoding at least one of said nucleic acid molecules; said sequence comprising expression of said nucleic acid molecule and / or Alternatively, it is operably connected to the start region and the stop region in a manner that allows delivery.

In another embodiment, the expression vector comprises a) a transcription initiation region; b) a transcription termination region; c) an open reading frame; d) a nucleic acid sequence encoding at least one of said nucleic acid molecules, said sequence comprising: Operably linked to the 3'-end of an open reading frame, wherein the sequence is responsive to the start region, the open reading frame and the end in a manner that allows expression and / or delivery of the nucleic acid molecule. Operatively linked to the area. In yet another embodiment, the expression vector comprises a) a transcription initiation region; b) a transcription termination region; c) an intron; d) a nucleic acid sequence encoding at least one of said nucleic acid molecules; Operatively linked to the initiation region, the intron and the termination region in a manner that allows for expression and / or delivery of molecules.

In another embodiment, the expression vector comprises a) a transcription initiation region; b) a transcription termination region; c) an intron; d) an open reading frame; e) a nucleic acid sequence encoding at least one of said nucleic acid molecules, A sequence is operably linked to the 3'-end of the open reading frame, and the sequence is operatively linked to the initiation region, the intron, in a manner that allows expression and / or delivery of the nucleic acid molecule. Operatively connected to the open reading frame and the termination region.

  Flt-1 (VEGFR1), KDR (VEGFR2) and / or flk-1 are attractive nucleic acid-based therapeutic targets by several criteria. The interaction between VEGF and VEGF-R is well established. Efficacy can be tested in well-defined and predictable animal models. Finally, the disease state is severe and current therapies are inadequate. Although protein-based therapies that affect VEGF activity have been designed, nucleic acid-based therapies based on the molecules and methods described herein directly modulate flt-1, KDR and / or flk-1 expression. Provide a direct and sophisticated way to do.

Because VEGFR1 and VEGFR2 mRNA are highly homologous in certain regions, some nucleic acid target sites are also homologous. In this case, one nucleic acid molecule of the present invention can target both VEGFR1 mRNA and VEGFR2 mRNA. For sites that are partially homologous, one nucleic acid molecule can be designed to accommodate sites on both mRNAs, possibly by including G / U base pairs. For example, if G is present in the enzymatic nucleic acid target site in VEGFR1 mRNA and the same position is A in the VEGFR2 enzymatic nucleic acid target site, the enzymatic nucleic acid is synthesized to have U at the complementary position. Which will bind to both positions. The advantage of one enzymatic nucleic acid that targets both VEGFR1 and VEGFR2 mRNA is particularly apparent when both VEGF receptors may contribute to the progression of angiogenesis in disease states.

  The following are non-limiting examples showing the selection, isolation, synthesis, and activity of representative nucleic acids of the invention.

  The following examples illustrate the selection and design of antisense, aptamer, dsRNA, allozyme, hammerhead, DNAzyme, NCH, amberzyme, tinzyme, or G-cleaving ribozyme molecules, and within VEGF, VEGFR1, and / or VEGFR2 RNA The binding / cleavage sites are shown.

Example 1: Enzymatic nucleic acid-mediated inhibition of angiogenesis in vivo A hammerhead targeting the Flt-1 4229 site (SEQ ID NO: 5977) in a rat corneal model of VEGF-induced angiogenesis by performing the studies described below. The anti-angiogenic activity of ribozymes was evaluated. These ribozymes have a catalytic core, either active or inactive, and bind and cleave or simply bind to the Flt-1 subtype VEGF-R mRNA. Active ribozymes capable of binding to and cleaving target RNA have been shown to inhibit VEGF binding ( ^125I- labeled) in cultured endothelial cells and reduce VEGF-induced endothelial cell proliferation in those cells in a dose-dependent manner. ing. The catalytically inactive forms of these ribozymes, which can bind RNA but cannot catalyze RNA cleavage, did not inhibit VEGF binding and did not reduce VEGF-induced endothelial cell proliferation . Ribozyme and VEGF were co-delivered using the filter disk method: as described by Pandey et al. (Supra), a 0.057 diameter nitrocellulose filter disk (Millipore®) was immersed in a suitable solution and applied to the rat cornea. Surgically implanted. This delivery method has been shown to deliver rhodamine-labeled free ribozymes to scleral cells and possibly cells of the vascular network surrounding the cornea. It is very important to evaluate these ribozymes for in vivo anti-angiogenic activity because active ribozymes show cell culture efficacy and can be delivered to target sites using the disc method.

Stimulation of angiogenesis in this study was performed by treating a filter disk implanted in the corneal matrix (stroma) with 30 μM VEGF. This dose results in reproducible angiogenesis caused by the growth of the pericorneal vascular network to the disc in a 5 day post-implant dose-response study. Filter disks treated with VEGF vehicle alone do not show an angiogenic response. Ribozyme was co-administered with VEGF on the disc at two different ribozyme concentrations. One concern with co-administration is that ribozymes cannot inhibit angiogenesis because VEGF receptors can be stimulated. However, at low doses of VEGF, an angiogenic response has been observed to reverse to normal, suggesting that VEGF stimulation is essential to maintain the angiogenic response. Inhibition of VEGF receptor production using co-administration of anti-VEGF-R mRNA ribozymes can reduce normal angiogenesis induced by VEGF-treated filter discs.

Sample and method:
1． Hammerhead ribozyme stock solution:
a. Flt-1 4229 (786 μM) -activity b. Flt-1 4229 (736μM)-Inactive

2． Test solution / group:
Group 1 Solution 1 Control VEGF solution: 30 μM in 82 mM Tris base
Group 2 Solution 2 Flt-1 4229 (1 μg / μL) in 30 μM VEGF / 82 mM Tris base
Group 3 Solution 3 Flt-1 4229 (10 μg / μL) in 30 μM VEGF / 82 mM Tris base
Group 4 Solution 4 VEGF-free, 82 mM Tris base, Flt-1 4229 (10 μg / μL)
Group 5 Solution 5 VEGF-free, ribozyme-free 82 mM Tris 10 eyes per group, 5 animals (molar concentrations should be essentially similar due to similar molecular weights).
Each solution (VEGF and ribozyme) was prepared as a 2X solution so that the final concentration was obtained by mixing 1: 1. However, in solution 1, VEGF was 2X and diluted with ribozyme diluent (sterile water).

3． VEGF solution
2X VEGF solution (60 μM) was prepared from a 0.82 μg / μL stock solution in 50 mM Tris base. A 200 μL VEGF stock solution was concentrated by speedback to a final volume of 60.8 μL, with a final concentration of 2.7 μg / μL or 60 μM. Divided into 6 10 μL for daily mixing. A 2X solution of VEGF and ribozyme was stored at 4 ° C until the day of surgery. The solution was mixed on each surgery day. The original 2X solution was prepared the day before the first day of surgery.

Four. Surgical solution:
anesthesia:
Stock ketamine hydrochloride 100mg / mL
Stock xylazine hydrochloride 20mg / mL
Stock acepromazine 10mg / mL
Final anesthetic solution: 50 mg / mL ketamine, 10 mg / mL xylazine, and 0.5 mg / mL acepromazine
5% povidone iodine (for surgical eyewash)
2% lidocaine (sterile) (instillation (2 drops per eye))
Aseptic 0.9% NaCl (for eye wash)

Five. Surgery:
Standard surgical procedure as described in Pandey et al. (Supra). The filter disk was incubated in 1 μL of each solution for about 30 minutes before being implanted.

6． Experimental protocol:
Animal corneas were treated with treatment groups as described above. Animals were allowed to recover for 5 days after treatment with daily observation (score 0-3). On day 5, animals were euthanized and digital images of each eye were obtained and quantified using Image Pro Plus. After analyzing the quantified angiogenic surface area by ANOVA, two post-hoc tests including Dunnets and Tukey-Kramer tests were performed to test significance at 95% confidence level. Dunnets provides information on the significance of the treatment group means versus the differences in the controls, and Tukey-Kramer provides information on the significance of the differences in the means of each group.

  Flt-1 4229 (SEQ ID NO: 5977) active hammerhead ribozyme was effective at inhibiting angiogenesis at either concentration, whereas inactive ribozyme did not show a significant reduction in angiogenesis. A statistically significant reduction in angiogenic surface area was only observed with active ribozymes. This result clearly shows that ribozymes have the ability to significantly inhibit angiogenesis in vivo. In particular, according to the mechanism of action of a given ribozyme, the observed inhibition is due to the binding and cleavage of the target RNA by the ribozyme.

Example 2: Biological activity of anti-angiogenic ribozymes targeting Flt-1 and kdrRNA Samples and methods Ribozymes: Hammerhead ribozymes and controls designed to have attenuated activity (attenuated controls) Synthesized and purified as described above. The attenuated ribozyme control retains the binding arm sequence of the parent ribozyme and thus retains the ability to bind to the mRNA target. However, two nucleotides in the core sequence have changed, which substantially reduces the ability to perform the cleavage reaction. Ribozymes were designed to target conserved Flt-1 or KDR mRNA sites in humans, mice, and rats. In general, ribozymes with 7 nucleotide binding arms were designed and tested. However, if only the six nucleotides surrounding the cleavage site are conserved in all three species, a 6 nucleotide binding arm was used. Presented herein are data for 2′-NH ₂ uridine modified ribozymes in cell proliferation studies, and RNase protection, in vitro cleavage, and 2′-C-allyl uridine in corneal studies.

In vitro ribozyme cleavage assay: RNA cleavage rates for 15 nucleotide synthetic RNA substrates in vitro were measured as described above.

Cell culture: human skin microvascular endothelial cells (HMVEC-d, Clonetics), 1.5% porcine skin gelatin (300 clones) in growth medium (Clonetics) supplemented with 10-20% fetal calf serum (FBS, Hyclone)
bloom, Sigma) solution and kept at 37 ° C. in a flask or plate coated with the solution.
Cells were grown to confluence and used up to passage 7. The stimulation medium consisted of 50% Sigma 99 medium and 50% RPMI 1640 (with L-glutamine), which was further supplemented with 10 μg / mL insulin-transferrin-selenium (Gibco BRL) and 10% FBS. Incubation in stimulation medium supplemented with either 20 ng / mL VEGF ₁₆₅ or bFGF stimulated cell proliferation. VEGF ₁₆₅ (165 amino acids) was selected for cell culture and animal experiments because it is the main of the four wild-types of VEGF generated by alternative mRNA splicing. Cell culture assays were performed in triplicate measurements.

Preparation of ribozymes and ribozymes / LipofectAMINE ^™ :
Cell culture: Ribozymes or attenuated controls (50-200 nM) were prepared for cell culture studies and used immediately. The preparation was performed by using LipofectAMINE ^™ (Gibco BRL) with a lipid / phosphate charge ratio of 3: 1 in a serum-free medium (OPTI-MEM ^™ , Gibco BRL) and mixing at room temperature for 20 minutes. For example, a lipid / phosphate charge ratio of 3: 1 is established by complexing 200 nM ribozyme with 10.8 μg / μL LipofectAMINE ^™ (13.5 μM DOSPA).

  In vivo: For corneal studies, lyophilized ribozymes or attenuated controls were resuspended in sterile water at a final stock concentration of 170 μg / μL (maximum dose). Lower doses (1.7-50 μg / μL) were prepared by serial dilution with sterile water.

Proliferation assay: HMVEC-d was seeded in 48-well plates (Costar) (5 × 10 ³ cells / well) and incubated in growth medium at 37 ° C. for 24-30 hours. After removal of the growth medium, cells were treated with 50-200 nM ribozyme or attenuated control LipofectAMINE ^™ complex in OPTI-MEM ^™ for 2 hours. Ribozyme / control containing medium was removed and cells were washed thoroughly in 1X PBS. The medium was then replaced with stimulation medium or stimulation medium supplemented with 20 ng / mL VEGF ₁₆₅ or bFGF. After 48 hours, cell numbers were measured using a Coulter ^™ cell counter. Data are expressed as the number of cells per well 48 hours after VEGF stimulation.

RNase protection assay: Seed HMVEC-d in 6-well plates (Costar) (2x10 ⁵ cells /
Wells), grown in growth medium at 37 ° C. for 32-36 hours. Cells are treated with LipofectAMINE ^™ complex containing 200 nM ribozyme or attenuation control as described in the “Proliferation Assay” section for 2 hours and then incubated in growth medium containing 20 ng / mL VEGF ₁₆₅ for 24 hours. did. Cells were harvested and RNase protection assays were performed using the Ambion Direct Protect kit and protocol. However, 50 mM EDTA was added to the lysis buffer to eliminate the possibility of ribozyme cleavage during sample preparation. Antisense RNA probes targeting Flt-1 and KDR were prepared by transcription in the presence of [ ³² P] -UTP. Samples were analyzed on polyacrylamide gels and the level of protected RNA fragments was quantified using a Molecular Dynamics PhosphorImager. In each sample, the levels of Flt-1 and KDR were normalized to the level of cyclophilin (human cyclophilin probe template, Ambion). The variation count of cyclophilin levels was 11% under all conditions tested here (ie in the presence of either active ribozyme or attenuated control) [265940 cpm ± 29386 (SD)]. Cyclophilin is therefore useful as an internal standard for these studies.

Rat corneal pocket assay for VEGF-induced angiogenesis:
Animal guidelines and anesthesia. Animal housing and experimentation was carried out in accordance with the criteria outlined in the 1996 Guide for the Care and Use of Laboratory Animals (National Researsh Council). Male Sprague Dawley rats (250-300 g) were anesthetized with intramuscular (im) administration of ketamine (50 mg / kg), xylazine (10 mg / kg), and acepromazine (0.5 mg / kg). The level of anesthesia was monitored every 2-3 minutes by squeezing the hind limb paw and examining limb withdrawal.
Atropine (0.4 mg / kg, im) was also administered to eliminate the possibility of bradycardia due to corneal reflex.

Preparation of VEGF immersion disc. For corneal implants, a 0.57 mm diameter nitrocellulose disc prepared from a 0.45 μm pore size nitrocellulose filter membrane (Millipore) was placed on ice, in a Petri dish with lid, 30 μM in 82 mM Tris-HCl (pH 6.9). It was immersed in 1 μL of VEGF ₁₆₅ for 30 minutes.

Corneal surgery. The rat cornea model used in this study is a modification of Koch et al. (Above) and Pandey et al. (Above). Briefly, the cornea was washed with 0.5% povidone iodine solution followed by normal saline and 2 drops of 2% lidocaine. A substrate pocket was generated under a dissecting microscope (Leica MZ-6), and a pre-soaked filter disc (above) was inserted into the pocket so that its edge was 1 mm from the corneal edge.

Intraconjunctival injection of test solution. Immediately after the insertion of the disc, the tip of a syringe with an outer diameter of 40-50 μm (prepared by Applicant's laboratory) was inserted into the conjunctival tissue just beside the VEGF immersion filter disc, 1 mm away from the edge of the cornea. A 600 nanoliter test solution (ribozyme, attenuated control, or sterile aqueous medium) was injected at a rate of 1.2 μL / min using a syringe pump (Kd Scientific). The syringe was then removed, washed sequentially with 70% ethanol and sterile water, and immersed in sterile water between each injection. After injection of the test solution, the vagina was kept closed using a microaneurysm clip until the animal's general activity was restored. After treatment, the animals were warmed on a 37 ° C heating pad.

Animal treatment group / experiment protocol. Ribozymes targeting Flt-1 site 4229 (SEQ ID NO: 5977) and KDR mRNA site 726 (SEQ ID NO: 5978) were tested in a corneal model along with their attenuated controls. Five treatment groups were selected, and the effects on VEGF-stimulated angiogenesis when five doses of each test substance over the 1-100 μg dose range were tested. Negative (30 μM VEGF soaked filter disk, 600 nL sterile water intraconjunctival injection) and unstimulated (Tris soaked filter disk, sterile water intraconjunctival injection) control groups were also included. Each group consisted of 5 animals (10 eyes) and received the same treatment.

Quantification of angiogenic response. Five days after the disc implant, the animals were euthanized with 0.4 mg / kg atropine and digitally imaged the cornea. Using computerized morphometry (Image Pro Plus, Media Cybernetics, v2.0), postvascular measurements of neovascular surface area (NSA, expressed in pixels) were made from corneal blood vessels filled with blood. Individual average NSA was measured in triplicate from three regions of the same size in the largest angiogenic segment between the filter disc and corneal border. The number of pixels corresponding to blood-filled corneal vessels in these areas was summed to calculate the NSA index. The group average NSA was then calculated. Data from each treatment group was normalized to VEGF / ribozyme vehicle treated control NSA and finally expressed as percent inhibition of VEGF-induced angiogenesis.

statistics. After confirming the normality of the mean value of the treatment group, a one-way analysis of variance was performed for the group mean VEGF-induced angiogenesis inhibition percentage. This was followed by two post-hoc tests (alpha = 0.05) for significance including Dunnett's (compared to VEGF control) and Tukey-Kramer (compared to the mean of all other groups). Statistical analysis was performed using JMP v.3.1.6 (SAS Laboratories).

Results Reduced VEGF-induced cell proliferation mediated by ribozymes: Ribozyme cleavage of Flt-1 or KDR mRNA should result in a decrease in cell surface VEGF receptor density. This reduction limits VEGF binding and consequently interferes with VEGF-induced mitogenic signaling. To confirm whether cell growth is affected by anti-Flt-1 and / or anti-KDR ribozyme treatment, a proliferation assay was performed using cultured human microvascular cells. First, ribozymes used in proliferation assays were selected for their ability to reduce VEGF binding levels on treated cells. In these initial tests, ribozymes targeting 20 sites in the coding region of each mRNA were screened. The most effective ribozymes for two sites in each target (Flt-1 sites 1358 and 4229, and KDR sites 726 and 3950) were used in the proliferation assay reported here. In addition, attenuated analogs of each ribozyme were used as controls. These attenuated controls retain the binding arm sequence and thus have the ability to bind to the mRNA target. However, these controls have two nucleotide changes in the core sequence, which substantially reduces the ability to perform cleavage reactions.

The active ribozymes tested decreased the relative proliferation of HMVEC-d after VEGF stimulation, and this effect increased with ribozyme concentration. This concentration dependence was not observed after treatment with the attenuation control designed for these sites. In fact, little or no change in cell proliferation was observed after attenuated control treatments, despite their ability to bind to specific target sequences. At 200 nM, there was a clear “window” between the antiproliferative effects of each ribozyme and its attenuated control, and this trend was observed even at low doses. This window of growth inhibition (56-77% based on total cells / well) reflects the contribution of ribozyme-mediated activity. In contrast, anti-Flt-1 or anti-KDR ribozyme had no effect on bFGF-stimulated cell proliferation. Furthermore, an irrelevant but active ribozyme whose binding sequence was not found in either Flt-1 or KDR mRNA had no effect in this assay. These data are consistent with the basic ribozyme mechanism where binding and cleavage are necessary elements. Although the relative surface distribution of Flt-1 and KDR receptors in this cell type is unknown, the antiproliferative effects of these ribozymes are functionally linked to proliferation, at least in cell culture medium. It shows that.

Specific reduction of Flt-1 or KDR mRNA by ribozyme treatment: To confirm that anti-Flt-1 and anti-KDR ribozymes reduce their respective mRNA targets, an RNase protection assay is performed with specific Flt-1 or KDR Used with the probe to quantify intracellular levels of Flt-1 or KDR. For each target, one ribozyme / attenuated control pair was selected for subsequent testing. F
Exposure of HMVEC-d to an active ribozyme targeting lt-1 site 4229 decreased Flt-1 mRNA but not KDR mRNA. Similarly, treatment with an active ribozyme targeting KDR site 726 reduced KDR mRNA but not Flt-1. Both ribozymes reduced their respective target RNA levels by more than 50%. The degree of decline for the corresponding attenuation control was less than 13%.

Activity of anti-Flt-1 and anti-KDR ribozymes in vitro.
To further confirm the need for an active ribozyme core, in vitro cleavage activity was measured for the Flt-1 site 4229 ribozyme and the KDR site 726 ribozyme and their associated attenuation control. The first order rate constants calculated from the time course of cleavage of the short substrate for the anti-Flt-1 ribozyme and its attenuation control were 0.081 ± 0.0007 / min and 0.001 ± 6 × 10 ⁻⁵ / min, respectively. For the anti-KDR ribozyme and the control paired with it, the first-order rate constants were 0.434 ± 0.024 / min and 0.002 ± 1 × 10 ⁻⁴ / min, respectively. The attenuated control retains only a negligible level of cleavage activity under these optimized conditions, but the reduction in in vitro cleavage activity between each active ribozyme and its counterpart attenuation control is approximately two orders of magnitude. It is a size. Therefore, the active core is essential for in vitro cleavage activity and also for ribozyme activity in cell culture.

  Reduction of VEGF-induced angiogenesis mediated by ribozymes in vivo. To assess whether ribozymes targeting VEGF receptor mRNA affect complex angiogenesis processes, prototype anti-Flt-1 and KDR ribozymes identified in cell culture studies were tested for VEGF-induced angiogenesis. Screened in rat corneal pocket assay. In this assay, corneas implanted with VEGF-containing filter discs showed a strong angiogenic response in the corneal region between the disc and the limbus (where new blood vessels emerge). The disc containing the media solution did not induce an angiogenic reaction. In another study, intraconjunctival injection of sterile aqueous media did not affect the magnitude of the VEGF-induced angiogenic response. Furthermore, angiogenesis was not induced by ribozyme injection alone.

The dose-related effects of anti-Flt-1 or KDR ribozyme on VEGF-induced angiogenic response were then tested. The anti-angiogenic effects of anti-Flt-1 (site 4229) and KDR (site 726) ribozymes and their attenuated controls were measured over a dose range of 1 to 100 μg, respectively. For both ribozymes, the maximum anti-angiogenic response (48 and 36% for anti-Flt-1 and KDR ribozymes, respectively) was observed at a dose of 10 μg.

Anti-Flt-1 ribozyme induced a significantly higher anti-angiogenic response at 3 and 10 μg than its attenuated control (p <0.05). The attenuated control showed a slight but significant anti-angiogenic response compared to the vehicle-treated VEGF control at doses> 10 μg (p <0.05). This response was at most not significantly higher than that observed with the lowest dose of active anti-Flt-1 ribozyme. Anti-KDR ribozyme significantly inhibited angiogenesis at 3 to 30 μg (p <0.05). Anti-KDR attenuated controls had no significant effect at any dose tested.

Example 3: Inhibition of tumor growth and metastasis by VEGF-R ribozyme in vitro Lewis lung cancer mouse model: Ribozymes were chemically synthesized as described above. The sequence of ANGIOZYME ^™ bound to the target RNA is shown in FIG.

The tumors in this study are from a cell line (LLC-HM) that, when grown in vivo, causes a reproducible number of spontaneous lung metastases. The LLC-HM system was obtained from Dr. Michele O'Reilly (Harvard University). Tumor angiogenesis in Lewis lung cancer has been shown to be VEGF-dependent. Tumors from mice bearing LLC-HM (selected for advanced metastatic phenotype by subculture) were harvested 20 days after inoculation. Tumor brei suspensions were prepared from these tumors according to standard protocols. On day 0 of the study, 0.5 × 10 ⁶ viable LLC-HM tumor cells were injected subcutaneously (sc) into the dorsal or flank of untreated mice (100 μL injectate). Tumor growth for 3 days followed by saline or 30 mg / kg / day ANGIOZYME ^TM
Were injected intravenously continuously with an Alzet mini pump. One set of animals was dosed from day 3 to day 17. Tumor length and width dimensions and volume were calculated according to the following formula: Volume = 0.5 (length) (width) ² . On day 25 after inoculation, animals were euthanized and lungs were collected. The number of lung metastases that were visible to the naked eye was counted. Note that metastases were quantified 8 days after discontinuation. Prepare ribozyme solution and transfer to another set of animals at 100, 10, 3, or 1 mg / kg /
Sun ANGIOZYME ^™ was delivered by an Alzette mini pump. An osmotic minipump pre-filled with each test solution was surgically implanted in the left flank of a total of 10 animals for each administration group or saline control group. A pump was connected to the indwelling jugular vein catheter.

FIG. 2 shows the antitumor effect of ANGIOZYME ^™ . LLC-HM primary tumors in tumors that grew in the flank region showed statistically significant inhibition compared to saline controls (p <0.05). ANGIOZYME ^™ significantly reduced the number of lung metastases in animals inoculated in any of the flank areas (p <0.05). It shows the dose-dependent anti-metastatic effects of ANGIOZYME ^TM as compared to saline controls in FIG.

B. Mouse colorectal cancer model. KM12L4a-16 is a human colorectal cancer cell line. On day 0 of the study, 0.5 × 10 ⁶ KM12L4a-16 cells were implanted into the spleen of nude mice. Tumor inoculation 3
One day later, an Alzette minipump was implanted and continuous subcutaneous delivery of either saline or 12, 36, or 100 mg / kg / day of ANGIOZYME ^™ was initiated. On day 5, the spleen containing the primary tumor was removed. On the 18th day, the Alzette mini pump was replaced with a new pump so that saline or ANGIOZYME ^TM could be delivered continuously for 28 days from the 3rd to the 32nd day. On day 41, animals were euthanized and liver tumor burden was assessed.

Incidence and median of liver metastases after 100mg / kg / day ANGIOZYME ^TM treatment
Decreased significantly (Figure 4). In saline-treated animals, the median metastasis was 10. However, at high doses of ANGIOZYME ^™ (100 mg / kg / day), the median metastasis was zero.

Example 4: ANGIOZYME ^™ alone or in combination with chemotherapy in a mouse Lewis lung cancer model
Method of effect Tumor inoculation. Male C57 / BL6 mice (6 to 8 weeks old) were inoculated subcutaneously in the flank with 5 × 10 ⁵ LLC-HM cells derived from Bry preparations generated from tumors grown in mice.

Ribozymes and controls. RPI.4610 (also known as ANGIOZYME ^™ (SEQ ID NO: 5977)) is an anti-Flt-1 ribozyme that targets site 4229 of the human Flt-1 receptor mRNA (EMBL approval number X51602). Controls tested include RPI.13141, which is an attenuated version of RPI.4610 that changes four nucleotides in the catalytic core, which dramatically reduces cleavage activity. However, RPI.13141 retains the base composition and binding arm of RPI.4610 and therefore possesses the ability to bind to the target site. In the second control (RPI.13030), the catalytic core is changed to inhibit the cleavage activity (3), but the binding arm sequence is scrambled and can no longer bind to the target sequence. One nucleotide in the arm of RPI.13030 also changes while maintaining the same base composition as RPI.4610.

Ribozyme administration. Ribozymes and controls were resuspended in normal saline. Administration began 7 days after tumor inoculation. Animals were injected daily subcutaneously from day 7 to day 20 (30 mg / kg test substance) or fitted with an Alzette osmotic minipump containing ribozyme or control solution (flow rate 12 μL / day). Test substance (30 mg / kg / day) by subcutaneous infusion pump up to 7-20 days (14 days of infusion, total 420 mg / kg of test substance) or 7-34 days (28 days of infusion, total 840 mg / kg) Test substance). When indicated, chemotherapy drugs were administered with ribozyme treatment. On days 7, 9, and 11, cyclophosphamide was administered intraperitoneally (125 mg / kg). On days 8, 11, and 14, gemcitabine was administered intraperitoneally (125 mg / kg). Untreated and unworn animals were used as a comparison. Five animals were included in each group.

Results The antiangiogenic ribozyme, ANGIOZYME ^™ , was tested in the Lewis lung cancer model alone and in combination with two chemotherapeutic agents. Already (see above), 30 mg / kg / day of ANGIOZYME ^™ alone has been shown to inhibit both primary tumor growth and lung metastasis in a highly metastatic variant of Lewis (14 consecutive days with an Alzette minipump) iv delivery).

In this study, 30 mg / kg / day of ANGIOZYME ^™ was delivered daily by subcutaneous bolus injection or by continuous infusion from an Alzette minipump, resulting in delayed tumor growth. On average, animals treated with ANGIOZYME ^TM delayed tumor growth to 500 mm ³ compared to the untreated group by 7 days. Tumor growth in animals treated with either of the two attenuated controls was delayed only by 2 days.

ANGIOZYME ^™ delivered by subcutaneous bolus injection was also tested in combination with either gemcitabine or cyclophosphamide. In the presence of ANGIOZYME ^TM and gemcitabine combination therapy, tumor growth delay was prolonged to about 3 days beyond either treatment alone. The combination of ANGIOZYME ^™ and cyclophosphamide did not delay tumor growth more than cyclophosphamide alone, but this study did not investigate suboptimal doses of cyclophosphamide. None of the attenuated controls enhanced the effects of chemotherapeutic drugs.

The effect of ANGIOZYME ^{™ on} lung metastases was also measured in the presence and absence of further chemotherapy treatment. Lung metastases visible to the naked eye were counted on day 20 in 2 animals in each treatment group. In the presence of ANGIOZYME ^™ , lung metastases were reduced to zero with or without chemotherapeutic drugs. Single treatment with either gemcitabine or cyclophosphamide (mean metastasis numbers 4.5 and 4 respectively) was not as effective as ANGIOZYME ^™ alone or in combination with ANGIOZYME ^™ . None of the attenuated controls enhanced the effects of chemotherapeutic drugs.

The effect on lung metastases was also measured after continuous ANGIOZYME ^TM treatment. On day 20, an average of 8 in the treatment group fitted with an Alzette minipump (either 14 or 28 day pump)
Metastases up to were confirmed with the naked eye. This is a reduction in metastasis by 50% over the untreated group. Delivery of ANGIOZYME ^™ by daily subcutaneous bolus injection resulted in zero metastasis in the two animals counted (Figure 4), so further loading with the minipump contributed to a slight decrease in response to ANGIOZYME ^™ . There is a possibility.

Example 5: Identification of Potential Target Sites in Human VEGFR1 and / or VEGFR2 RNA Using computer folding algorithms, the sequence of the human VEGFR1 and / or VEGFR2 gene was screened for available sites. We identified a region of RNA that has no folding secondary structure and that contains potential enzymatic nucleic acid molecules and / or antisense binding / cleavage sites. Representative sequences of enzymatic nucleic acid molecules of the invention are shown in Formula I and / or Formula II (SEQ ID NOs: 5977 and 5978, respectively). Other nucleic acid molecules and targets contemplated by the present invention are described in Pavco et al. (US Patent No. 09 / 870,161, which is hereby incorporated by reference in its entirety). Similarly, other nucleic acid molecules of the present invention (antisense, aptamer, dsDNA
, SiRNA, and / or 2,5-A chimeras), Pavco et al. (US 09 / 870,161)
Can be designed to modulate the expression of the nucleic acid target described in.

Example 6: Selection of enzymatic nucleic acid cleavage sites in human VEGFR1 and / or VEGF2 RNA To select target sites for enzymatic nucleic acid molecules, human VEGFR1 receptor (eg Genbank accession number NM_002019) and VEGFR2 receptor (eg Genbank accession number) NM_002253) Gene sequences were analyzed and sites were prioritized based on folding. Enzymatic nucleic acid molecules are designed to bind to their respective targets and individually analyzed by computer folding (Christoffersen et al., 1994 J. Mol. Struc. Theochem., 311, 273; Jaeger
1989, Proc. Natl. Acad. Sci. USA, 86, 7706), and evaluated whether the enzymatic nucleic acid molecule sequence was folded into a suitable secondary structure. Enzymatic nucleic acid molecules that have adverse intermolecular interactions between the binding arm and the catalytic core can be excluded from consideration. As discussed herein, different binding arm lengths can be selected for optimal activity. In general, at least 4 bases on each arm can bind to or interact with the target RNA.

Example 7: Efficient cleavage and / or blocking of VEGFR1 and / or VEGFR2 RNA
Chemical synthesis and purification of ribozymes and antisense for RNA Enzymatic nucleic acid molecules and antisense constructs are designed to anneal to various sites in an RNA message. The binding arm of the enzymatic nucleic acid molecule is complementary to the target site sequence, and the antisense construct is completely complementary to the target site sequence. RNAi molecules (dsRNA) also have a single strand of RNA or a portion of RNA that is complementary to a target site sequence or part of a target site sequence. For example, complementarity within a double-stranded RNAi structure is generated from two different individual RNA strands or from topologically closed, self-complementary regions of individual RNA strands (which may be circular if desired) . Nucleic acid molecules were chemically synthesized. The synthesis methods used are those described above and Usman et al. (1987 J. Am. Chem. Soc., 109, 7845), Scaringe et al. (1990).
Nucleic Acids Res., 18, 5433), and the standard RNA described by Wincott et al. (Supra)
According to the method of synthesis, common nucleic acid protecting groups and coupling groups such as dimethoxytrityl at the 5 ′ end and phosphoramidite at the 3 ′ end were used. The average coupling yield per stage was generally> 98%.

Nucleic acid molecules were also synthesized from DNA templates using bacteriophage T7 RNA polymerase (Milligan and Uhlenbeck, 1989, Methods Enzymol. 180, 51). The nucleic acid molecules of the invention can be purified by gel electrophoresis using common methods, or by high performance liquid chromatography (HPLC; see Wincott et al. (Supra); incorporated herein by reference in its entirety). Purified and resuspended in water. Examples of sequences of chemically synthesized enzymatic nucleic acid molecules can be found in Formula I (SEQ ID NO: 5977), Formula II (SEQ ID NO: 5978), and US Patent No. 09 / 870,161 to Pavco et al.

Example 8: Enzymatic nucleic acid molecule cleavage of VEGFR1 and / or VEGFR2 RNA targets in vitro
Sectional human VEGFR1 and / or VEGFR2 RNA enzymatic nucleic acid molecules targeting were designed and synthesized as described above. These enzymatic nucleic acid molecules can be tested for in vitro cleavage activity using, for example, the following method. Target sequences and nucleotide positions within VEGFR1 and / or VEGFR2 RNA are described in US Pat. No. 09 / 870,161 to Pavco et al.

Cleavage reaction: In vitro transcription in the presence of [a- ³² P] CTP and spin to prepare full-length or partially full-length internally labeled target RNA for enzymatic nucleic acid molecule cleavage assays It was passed through a G50 Sephadex column by chromatography and used as substrate RNA without further purification. Or substrate was ^5'32 P-end labeled using T4 polynucleotide kinase enzyme. To perform the assay, an enzymatic nucleic acid molecule cleavage buffer (
2X concentrate of purified enzymatic nucleic acid molecules mixed in 50 mM Tris-HCl, pH 7.5, 37 ° C, 10 mM MgCl ₂ ) is pre-warmed, and 2X enzymatic nucleic acid molecule mixture is pre-warmed in cleavage buffer The same amount of substrate RNA (up to 1-5 nM) was added to initiate the cleavage reaction. For initial screening, a final concentration of 40 nM or 1 mM enzymatic nucleic acid molecules (ie, excess enzymatic nucleic acid molecules)
The assay was performed for 1 hour at 37 ° C. Equivalent amounts of 95% formamide, 20 mM EDTA, 0.05% bromophenol blue, and 0.05% xylene cyanol are added to quench the reaction, after which the sample is heated to 95 ° C for 2 minutes, rapidly cooled, and denatured Loaded onto polyacrylamide gel. Specific RNA cleavage products generated by substrate RNA and enzymatic nucleic acid molecule cleavage are visualized with an autoradiograph on the gel. The percentage of cleavage is determined by quantification with the Phosphor Imager® of bands corresponding to intact substrate and cleavage products.

Example 9: Phase I / II study of repeated administration of ANGIOZYME ^™ targeting the VEGFR1 (FLT-1) receptor of VEGF About 31 patients with refractory solid tumors in a daily I / II clinical trial The ribozyme therapeutic ANGIOZYME ^™ (SEQ ID NO: 5977) was evaluated by subcutaneous administration. Demographic information about patients enrolled in the study is shown in Table III. The end point of the initial test was to confirm the safety and maximum allowable amount of ANGIOZYME ^™ . The second endpoint evaluated the pharmacokinetics and clinical response of ANGIOZYME ^™ . Patients were treated with the following doses: 3 patients received 10 mg / m ² / day, 4 patients received 30 mg / m ² / day, 20 patients received 100 mg / m ² / day, and 4 patients received 300 mg / m ² / day. All but one patient were administered for a minimum of 29 consecutive days, and a 24-hour pharmacokinetic analysis was performed on days 1 and 29. Clinical response was assessed once a month.

result
Data from 20 patients showed that ANGIOZYME ^TM was well tolerated and had no systemic adverse reactions. FIG. 5 shows a plasma concentration profile of ANGIOZYME ^™ after one subcutaneous administration of 10, 30, 100, or 300 mg / m ² . A summary of the pharmacokinetic parameters of ANGIOZYME ^™ after subcutaneous bolus administration is shown in Table IV. MTD (maximum tolerance) could not be established. One patient in the 300 mg / m ² / day group experienced 3 injection site reactions. The other group of patients experienced 1 to 2 injection site reactions intermittently with erythema and sclerosis. No systemic or laboratory toxicity was observed. Pharmacokinetic analysis demonstrated dose-dependent plasma concentrations, good bioavailability (70-90%), t1 / 2 = 209-384 minutes, and no accumulation after repeated doses . To date, 17/28 (61%) of evaluable patients have had stable disease for 1 to 6 months, and 2 patients (nasopharyngeal squamous cell carcinoma and melanoma) have had a slight clinical response. MRI revealed necrosis of the central tumor in a patient with nasopharyngeal carcinoma. So far, the longest treatment period is 8 months at 100 mg / m ² / day for 2 patients (breast cancer, peritoneal mesothelioma).

Example 10: Suppressive modulation of VEGFR1 gene expression to treat symptoms dependent on gynecological angiogenesis One patient enrolled in ANGIOZYME ^™ monotherapy in the phase I / II clinical trial described in Example 19 There was menstruation before. 1-2 months after the trial, the patient's menstrual cycle stopped. The patient continued the study for about 11 months, but did not have menstruation. Later, the patient's study was discontinued for about 4 months, and menstruation resumed. Upon reenrollment in the ANGIOZYME ^TM trial, the patient's menstrual cycle stopped again. This clinical finding shows that ANGIOZYME ^TM is probably due to inhibition of neovascularization of uterine tissue
This suggests interfering with the patient's menstrual cycle. This data also suggests that ANGIOZYME ^TM has a direct effect on endometrial tissue or an effect on LH / FSH stimulation. These results indicate that various clinical targets and / or processes related to female reproductive and gynecological angiogenesis (eg, endometrium) using ANGIOZYME ^™ (SEQ ID NO: 5977) and / or other nucleic acid molecules of the invention. Syndrome, fertility regulation, gynecological bleeding disorders, menstrual irregularities, ovulation, premenstrual syndrome (PMS), menopausal dysfunction, endometrial cancer, or other symptoms related to VEGFR1 and / or VEGFR2 VEGF receptor expression) Suggests treatment or control.

Example 11: Inhibitory modulation of VEGFR1 in the clinical environment Serum samples from day 1 (baseline) and day 43 (6 weeks) of 27 patients enrolled in the phase I / II study described in Example 19 Harvested and assayed for VEGFR1 biomarker. VEGFR1 levels changed statistically after 6 weeks of ANGIOZYME treatment (Figure 9). Statistical analysis, including all 27 patients, confirmed the effect statistically, but not all patients showed elevated VEGFR1 levels. Since the effect of ANGIOZYME on VEGF-R1 can only be revealed when sufficient levels exist at baseline, a 100 pg / mL cut-off was chosen and this change in VEGF-R1 was reanalyzed. Ten of 27 patients showed baseline VEGFR1 levels above 100 pg / mL. In this subgroup, VEGF-R1 levels were 3 times lower (p <.001). After 6 weeks of treatment, the mean (geometric mean) of VEGF-R1 decreased from 419 pg / ml to 132 pg / ml in this subgroup (p <.001). These results indicate that VEGFR1 expression was statistically significantly reduced by treatment with ANGIOZYME.

Example 22: Inhibition of angiogenesis by VEGF-R ribozyme in an animal model of the eye in vivo Mouse model summary: A mouse model of proliferative retinopathy (Aiello et al., 1995, Proc. Natl. Acad. Sci. USA 92: Robinson et al., 1996, Proc. Natl. Acad. Sci. USA 93: 4851-4856; Pierce et al., 1996, Archives of Ophthalmology 114: 1219-1228). This involves exposing 7-day-old mice to 75% oxygen to induce neovascularization of the mouse retina and then returning to normal room air. In the early stages of high oxygen, obstruction of the developing blood vessels in the retina occurs. If exposed to room air after 5 days, the retina with poor blood flow is perceived as hypoxic. The result is an immediate upregulation of VEGF mRNA and VEGF protein followed by a high degree of retinal neovascularization, which peaks by day 5. This model is more typical of retinopathy of prematurity than diabetic retinopathy, but is an acceptable small animal model for studying the angiogenic pathophysiology of the retina. In fact, intravitreal injection of certain antisense DNA constructs targeting VEGF mRNA has been shown to have anti-angiogenic activity in this model, which binds to VEGF in the vitreous humor (Aiello et al., 1995, Proc. Natl. Acad. Sci. USA 92: 10457-10461; Robinson et al., 1996, Proc. Natl. Acad. Sci. USA 93: 4851-4856; Pierce et al., 1996, Archives of Ophthalmology 114: 1219-1228).

Experimental Summary: The effect of anti-KDR / Flk-1 ribozyme on peak levels of angiogenesis was tested in the mouse model described above. As shown in FIG. 10, P7 mice were removed from the hyperoxia and 10 g of RPI.4731 anti-KDR / Flk-1 ribozyme was injected intraocularly into the right eye twice (P12 and P13). The left eye of each mouse was treated as a control and saline was injected intraocularly. Five days after exposure to room air, neovascular nuclei in the retinas of both eyes were counted. The data is shown in FIG. A significant reduction in retinal neovascularization was observed compared to control eyes injected with saline (up to 40%).

Sequence and chemical composition of RPI.4731: 5'-us _s a _s c _s a _s au ucU GAu Gag gcg aaa gcc Gaa Aag aca aB-3 '(SEQ ID NO: 5978)
(Where uppercase G, A = ribonucleotide, lowercase = 2′-OMe, U = 2′-C-allyluridine, B = reverse abasic nucleotide, S = phosphothioate internucleotide linkage)

index
1) Tumor angiogenesis: Angiogenesis has been shown to be necessary for tumors to grow and become pathological size (Folkman, 1971, PNAS 76, 5217-5221; Wellstein and Czubayko, 1996, Breast Cancer Res and Treatment 38, 109-119). In addition, angiogenesis allows tumor cells to travel throughout the circulatory system during metastasis. Increased levels of gene expression of many angiogenic factors (eg, vascular endothelial growth factor (VEGF)) have been reported in angiogenesis and edema-related brain tumors (Berkman et al., 1993 J. Clini. Invest. 91, 153). A more direct demonstration of the role of VEGF in tumor angiogenesis was made by Jim Kim et al. (1993 Nature 362,841), where rhabdomyosarcoma and pleomorphic glia were utilized in nude mice using monoclonal antibodies to VEGF. Succeeded in inhibiting proliferation of blastoma cells. Similarly, expression of a dominant negative mutant of the Flt-1 VEGF receptor inhibits angiogenesis induced by human edema cells in nude mice (Millauer et al., 1994, Nature 367, 576).
Specific tumor / cancer types that can be targeted using the nucleic acid molecules of the present invention include, but are not limited to, the tumor / cancer types listed in “Diagnosis” in Table III.

2) Eye disease: Angiogenesis causes or exacerbates eye disease (including but not limited to macular degeneration, neovascular glaucoma, diabetic retinopathy, myopic degeneration, and trachoma) Has been clarified (Norrby, 1997, APMIS 105, 417-437). Aiello et al. (1994 New Engl. J. Med. 331, 1480) found that most ophthalmic fluids in patients with diabetic retinopathy and other retinal disorders contained high levels of VEGF. Miller et al. (1994 Am. J. Pathol. 145, 574) reported increased levels of VEGF mRNA in patients with retinal ischemia. These findings support a direct role for VEGF in ocular diseases. Other factors, including those that stimulate VEGF synthesis, can also contribute to these indications.

3) Skin disorders: Many indications that may be angiogenesis-dependent have been identified, including but not limited to: psoriasis, common warts, vascular fibers of tuberous sclerosis Tumor, flaming nevus, Sturge-Weber syndrome, Klipel-Tornonnay-Weber syndrome, and Osler-Weber Lange syndrome (Norrby, supra). It was revealed that angiogenesis occurs in nude mice by intradermal injection of the angiogenic factor b-FGF (Weckbecker et al., 1992, Angiogenesis: Key principles-Science-Technology-Medicine, edited by R. steiner). Detmar et al. (1994, J. Exp. Med. 180, 1141) reported that VEGF and its receptor are overexpressed in psoriatic skin and psoriatic cutaneous microvessels, indicating that VEGF is present in psoriasis. Suggests it plays an important role.

4) Rheumatoid arthritis: Immunohistologic and in situ hybridization studies on tissues from the joints of patients with rheumatoid arthritis have shown elevated levels of VEGF and its receptors (Fava et al., 1994 J. Exp. Med. 180, 341). Furthermore, Koch et al. (1994 J. Immunol. 152, 4149) report that VEGF-specific antibodies significantly reduce mitogenic activity in synovial membrane tissue from patients with rheumatoid arthritis. These findings support a direct role for VEGF in rheumatoid arthritis. Other angiogenic factors, including those of the present invention, can also be associated with arthritis.

5) Endometriosis: Various studies have shown that VEGF is directly related to endometriosis.
One study found that VEGF concentrations in peritoneal fluid as measured by ELISA were significantly higher in women with endometriosis than in women without endometriosis (24.1 ± 15 ng) / ml
In contrast, 13.3 ± 7.2 ng / ml is normal. In patients with endometriosis, VEGF concentrations (33 ± 13 ng / ml) detected during the proliferative phase of the menstrual cycle were higher than those in the secretory phase (10.7 ± 5 ng / ml). Cycles were not seen in body fluids from normal patients (McLaren et al., 1996, Human Reprod. 11, 220-223). In another study, women with moderate to severe endometriosis had significantly higher levels of VEGF in the peritoneal fluid than women without endometriosis. There was a clear correlation between the severity of endometriosis and the concentration of VEGF in peritoneal fluid. In human endometrial biopsies, VEGF expression was increased approximately 1.6-fold, 2-fold, and 3.6-fold in metaphase, late phase, and secretory endometrium compared to early phase (Shifren et al., 1996, J. Clin. Endocrinol. Metab. 81, 3112-3118).

In a third study, VEGF positive staining of human ectopic endometrium revealed localization in macrophages (two-way immunofluorescence staining with CD14 marker). Peritoneal macrophages were found to stain VEGF in patients with and without endometriosis. However, body fluids from women with endometriosis increased macrophage activity (acid phosphatase activity) compared to controls. In peritoneal fluid macrophage preparation medium from patients with endometriosis, cell proliferation in HUVEC cells ([ ³ H
] Thymidine incorporation) was significantly higher than control. The percentage of peritoneal macrophages with VEGFR2 mRNA is high in the secretory phase and is significantly higher (80 ± 15%) in body fluids from women with endometriosis compared to controls (32 ± 20%). Flt-mRNA was detected in peritoneal macrophages from women with and without endometriosis, but there was no difference between groups or cycle-dependent evidence (McLaren et al., 1996 , J. Clin. Invest. 98, 482-489).

In female mice, it has been shown that VEGF is expressed in the secretory columnar epithelium (estrogen-responsive) that covers both the fallopian tube and uterus in the early stages of the menstrual cycle. During the secretory phase, VEGF expression was found to have shifted to the underlying stroma that forms the functional endometrium. In addition to endometrial studies, follicular and luteal angiogenesis and angiogenesis at the embryo implant site were also analyzed. In these processes, VEGF was expressed in spatial and temporal proximity to the forming blood vessels (Shweiki et al., 1993, J. Clin. Invest. 91, 2235-2243).

Most of the current findings in VEGFR1 and / or VEGFR2 research are methods for assaying VEGFR1 and / or VEGFR2 activity and compounds that can modulate VEGFR1 and / or VEGFR2 expression for use in research, diagnosis, and therapy Indicates the need for As described herein, the nucleic acid molecules of the invention can be used in assays to diagnose pathologies related to VEGF, VEGFR1, and / or VEGFR2 levels. In addition, the nucleic acid molecules can be used to treat conditions associated with VEGF and / or VEGFr (eg, VEGFR1 and / or VEGFR2 levels).

Specific processes, diseases, or symptoms that may be related to VEGFR1 and / or VEGFR2 levels include, but are not limited to, gynecological-related angiogenesis such as endometriosis, endometrial cancer, gynecological bleeding Disorders, irregular menstruation, ovulation, premenstrual syndrome (PMS), menopausal dysfunction, other diseases and disorders discussed here, and levels of VEGF and / or VEGFr (eg, VEGFR1 and / or VEGFR2) in cells or tissues There are other diseases or symptoms that are related to or respond to alone or in combination with other therapeutic agents.

GnRH (gonadotropin-releasing hormone) agonist, Lupron Depot (leuprolide acetate), Synarel (naferaline acetate), Zolodex (goserelin acetate), Suprefact (buserelin acetate), or oral contraceptives (but not limited to Depo-Provera) Or the use of Provera (with medroxyprogesterone acetate), or other estrogen / progesterone) is all non-limiting examples of compounds and methods that can be combined or combined with the nucleic acid molecules of the invention . Various chemotherapies can be readily combined with the nucleic acid molecules of the invention for the treatment of endometrial cancer. Common chemotherapies that can be combined with the nucleic acid molecules of the present invention include various combinations of cytotoxic substances to kill cancer cells. These drugs include, but are not limited to, paclitaxel (taxol), docetaxel, cisplatin, methotrexate, cyclophosphamide, doxorbin, fluorouracil carboplatin, edatrexate, gemcitabine, vinorelbine. As will be appreciated by those skilled in the art, other drug compounds and therapies can be readily combined with the nucleic acid molecules of the present invention and are therefore within the scope of the present invention.

Animal model
There are several animal models that can test the anti-angiogenic effect of nucleic acids of the invention (eg ribozymes) that target VEGF-R mRNA. The corneal model has generally been used to study angiogenesis in rats and rabbits because vascular recruitment readily occurs in this normal avascular tissue (Pandey et al., 1995 Science 268: 567-569). These models inserted small Teflon or Hydron discs pretreated with angiogenic factors (eg bFGF or VEGF) into a surgically generated pocket in the cornea. Angiogenesis was monitored from 3 to 5 days later. Ribozymes targeting VEGF-R mRNA were similarly delivered in disc or instilled over the duration of the experiment. In another eye model, hypoxia has been shown to cause both increased expression of VEGF and angiogenesis in the retina (Pierce et al., 1995 Proc. Natl. Acad. Sci. USA 92: 905-909; Shweiki et al., 1992 J.
Clin. Invest. 91: 2235-2243).

In human glioblastoma, VEGF has been shown to be at least partially responsible for tumor angiogenesis (Plate et al., 1992 Nature 359, 845). Animal models have been developed to study the progression of tumor growth and angiogenic status by implanting glioblastoma cells subcutaneously in nude mice (Kim et al., 1993 supra; Milleruer et al., 1994 supra).

Another animal model for angiogenesis involves Matrigel, which is an extract of the basement membrane and becomes a solid gel when injected subcutaneously (Passaniti et al., 1992 Lab. Invest. 67: 519-528). When Matrigel is supplemented with angiogenic factors such as VEGF, blood vessels grow in Matrigel over 3 to 5 days and angiogenesis can be assessed. A ribozyme targeting VEGF-R mRNA can be delivered into Matrigel to evaluate its anti-angiogenic effect.

There are several animal models for screening for anti-angiogenic substances. These include: corneal surgery (Burger et al., 1985 Cornea 4: 35-41; Lepri et al., 1994 J. Ocular Pharmacol. 10: 273-280; Ormerod et al., 1990 Am. J. Pathol. 137: 1243- 1252) or corneal angiogenesis after intracorneal growth factor implant (Grant et al., 1993 Diabetologia 36: 282-291; Pandey et al., 1995 supra; Zieche et al., 1992 Lab. Invest. 67: 711-715), containing growth factors Vascular proliferation into Matrigel matrix (Passaniti et al., 1992 supra), female reproductive organ angiogenesis after hormonal treatment (Shweiki et al., 1993 Clin. Invest. 91: 2235-2243), in highly vascularized solid tumors Several models with inhibition of tumor growth (O'Reilly et al., 1994 Cell 79: 315-328; Senger et al., 1993 Cancer and Metas. Rev. 12: 303-324; Takahasi et al., 1994 Cancer Res. 54: 4233 -4237; Kim et al., 1993 supra), and transient hypoxia-induced angiogenesis in the mouse retina (Pierce et al., 1995 Proc. Natl. Acad. Sci. USA 92: 905-909).

The corneal model (described in Pandey et al., Supra) is the most common and is a model for screening the effectiveness of well-characterized anti-angiogenic substances. This model involves avascular tissue, and blood vessels are recruited into the tissue by stimulants (growth factors, burns or alkali burns, endotoxins). The corneal model uses a Teflon® pellet in-matrix corneal implant soaked in a VEGF-hydron solution to replenish the pellet with blood vessels using standard microscopy and image analysis techniques. Can be quantified. To assess anti-angiogenic efficacy, ribozymes are applied topically to the eye or bound to themselves in hydron on Teflon® pellets. This avascular cornea as well as Matrigel (see below) allows low background assays to be performed. The cornea model has been extensively used in rabbits, but has also been studied in rats.

The mouse model (Passaniti et al., Supra) is a non-tissue model that uses Matrigel (Kleinman et al., 1986) or Millipore® filter discs, which are extracts of the basement membrane, and are used as growth factors and anti-antibodies in liquid form. It may be injected after being immersed in an angiogenic substance. When administered subcutaneously at body temperature, Matrigel or Millipore® filter discs become solid grafts. VEGF embedded in Matrigel or Millipore® filter disc
Is used to replenish blood vessels within the matrix of Matrigel or Millipore® filter discs for endothelial cell-specific vWF (factor VIII antigen) immunohistochemistry, trichrome masson staining, or hemoglobin content Can be processed histologically. Like the cornea, Matrigel or Millipore® filter discs are avascular; however, they are not tissues. In the Matrigel or Millipore® filter disc model, the ribozyme is administered into a matrix of Matrigel or Millipore® filter disc to test its anti-angiogenic effect. Therefore, the delivery problem in this model is due to the homogeneous presence of ribozymes within each matrix, as in the case of ribozyme delivery with hydrone-coated Teflon® pellets in the rat cornea model. Minimal.

These models offer another advantage over several other angiogenesis models listed previously. The ability to use VEGF as a pro-angiogenic stimulator in both models is highly desirable because the ribozyme targets only VEGFr mRNA. In other words, the involvement of other non-specific types of stimulants in the cornea and Matrigel models is not beneficial in terms of understanding the pharmacological mechanism by which anti-VEGFr mRNA ribozymes produce their effects. In addition, this model allows the specificity of anti-VEGFr mRNA ribozymes to be tested using either aFGF or bFGF as pro-angiogenic factors. Vascular recruitment using FGF should not be affected by anti-VEGFr mRNA ribozymes in any model. Other angiogenesis models (angiogenesis in the female reproductive system using hormone treatment (Shweiki et al., 1993 supra); various vascular solid tumors with an indirect correlation with angiogenesis (O'Reilly et al., 1994 supra); Senger et al., 1993 above; Takahasi et al., 1994 above; Kim et al., 1993 above); and retinal neovascularization after transient hypoxia (including Pierce et al., 1995 above) are effective because of their non-specificity Although it was not selected for screening, it has proved a correlation between VEGF and angiogenesis and can be a useful model.

  Other model systems for studying tumor angiogenesis are reviewed by Folkman (1985 Adv. Cancer Res. 43, 175).

Use of mouse model A typical systemic study involving 10 mice (20 g each) in each treatment group (5 treatment groups: 1, 3, 10, 30, and 100 mg / kg / day for 14 days) Use approximately 400 mg of ribozyme prepared by mixing with saline. Similar studies in young adult rats (200g) require more than 4g. Parallel pharmacokinetic studies involve the use of similar amounts of ribozymes, further justifying the use of mouse models.

Ribozyme and Lewis Lung Cancer and B-16 Melanoma Mouse Model Identifying a common animal model for systemic efficacy testing of ribozymes is an efficient screening method for ribozymes for systemic efficacy.

The Lewis lung cancer and B-16 mouse melanoma models are well-tolerated models of primary and metastatic cancer and are used for initial screening for anticancer drugs. These mouse models do not rely on the use of immunodeficient mice, are relatively inexpensive, and have minimal housing problems. Both Lewis lung and B-16 melanoma models are approximately from metastatically active tumor cell lines (Lewis lung line 3LL or D122, LLc-LN7; B-16-BL6 melanoma) in C57BL / 6J mice. With ^{6 6} subcutaneous implants of tumor cells. Alternatively, the Lewis lung model can be generated by a surgical implant of a tumor sphere (approximately 0.8 mm in diameter). Metastasis can also be modeled by direct injection of tumor cells into a vein. In the Lewis lung model, microscopic metastases are observed approximately 14 days after implantation, resulting in quantifiable gross metastases within 21-25 days. B-16 melanoma shows similar changes over time and tumor angiogenesis begins 4 days after the implant. Since both primary and metastatic tumors are present after 21-25 days in the same animal in these models, multiple measurements can be taken as an indication of efficacy. The primary tumor volume and growth latency, as well as the number of microscopic and macroscopic lung metastases or animals showing metastasis can be quantified. The percentage of life extension can also be measured. Thus, these models provide a suitable initial efficacy assay for screening systemically administered ribozymes / ribozyme preparations.

In Lewis lung and B-16 melanoma models, systemic drug therapy with a wide range of drugs usually begins 1-7 days after tumor implant / inoculation and is given on a continuous or multiple dose regimen. At the same time, pharmacokinetic studies can be performed to determine whether the ribozyme level is sufficient for the expected pharmacodynamic effects. In addition, the primary tumor and secondary lung metastases were removed,
It can be applied to various in vitro tests (eg reduction of target RNA).

Flt-1, KDR, and / or flk-1 protein levels can be measured clinically and experimentally by FACS analysis. MRNA levels encoding Flt-1, KDR, and / or flk-1 can be assessed by Northern analysis, RNase protection, primer extension analysis, and / or quantitative RT-PCR. Ribozymes can be identified that block mRNA encoding Flt-1, KDR, and / or flk-1 proteins, thus reducing Flt-1, KDR, and / or flk-1 activity levels by 20% or more in vitro .

  These animal model experiments (as described above) can be performed by free delivery, liposome delivery, cationic lipid delivery, adeno-associated virus vector delivery, adenovirus vector delivery, retrovirus delivery, or plasmid vector delivery of the ribozymes and / or genes encoding them. ).

  Subjects can be treated by topical administration of a nucleic acid targeting VEGF-R by direct injection. Routes of administration include but are not limited to intravascular, intramuscular, subcutaneous, intraarticular, aerosol inhalation, oral (in the form of tablets, capsules or pills), local, systemic, ocular, intraperitoneal, and / or Or there is intrathecal delivery.

Surgical guided models of endometriosis have been developed in rats, mice, and rabbits. Non-human primates show spontaneous endometriosis, but surgical induction can also be used. In addition to surgical techniques, primates can be monitored for cycles by daily vaginal cytology. All surgically induced models of endometriosis use the following general procedure. An initial laparotomy is performed and tissue is implanted from the donor animal. Remove part of one uterine horn (or one whole uterine horn in the case of mice). The endometrium of this uterine segment is separated from the myometrium and cut into small sections (4-10 mm ² ). Sections (approximately 3) are sutured to various locations in the abdominal cavity (peritoneum, mesenteric blood vessels, uterus, and mesentery). Cummings and Metcalf (1996) attached the entire section of the mouse uterus without separating the endometrium from the myometrium. The implant piece is allowed to grow for 3-6 weeks. Occasionally, a second laparotomy is performed to confirm the formation of endometriosis-like foci (angiogenesis and cysts filled with clear fluid). This second laparotomy was performed in a study by Quereda et al. (1996) and Stoeckmann et al. (1995). Drug treatment begins and continues for the prescribed period 3-6 weeks after surgery and / or after visualization of endometriosis. Animals were euthanized at the end of these studies. At the end, there is a change in the surface area of the tissue mass of the implant piece and the ectopic endometrial implant, including but not limited to (eg Brogniez et al., 1995, Human Reprod. 10, 927-931; Cummings et al., 1996). , Tox. Appl. Pharm. 138, 131-139; Cummings and Metcalf, 1996, Proc. Soc. Exp. Biol. Med. 212, 332-337; D'Hooghe et al., 1996, Fertility and S
terility. 66, 809-813; see Quereda et al., 1996, Eur. J. Obstet. Gynecol. Rep. Biol. 67, 35-40; and Stoeckemann et al., 1995, Human Reprod. 10, 3264-3271) .

Combination therapy gemcitabine and cyclophosphamide are non-limiting examples of chemotherapeutic agents that can be mixed or used in combination with nucleic acid molecules of the invention (eg, ribozymes and antisense molecules). As will be appreciated by those skilled in the art, other anti-angiogenic and / or anti-cancer compounds and therapies can be readily mixed with the nucleic acid molecules of the invention (eg, ribozymes and antisense molecules) as well, so that they Within range. Those compounds and therapies are well known to those skilled in the art (eg Cancer: Principles and Practice of Oncology, Volumes 1 and 2, Devita, VT, Hellman, S., and Rosenberg) , SA, JB Lippincott, Philadelphia, USA; incorporated herein by reference), and include, but are not limited to: folic acid, antifolate, pyrimidine analogs, fluoropyrimidine, Purine analogs, adenosine analogs, topoisomerase type I inhibitors, anthrapyrazoles, retinoids, antibiotics, anthracyclines, platinum analogs, alkylating agents, nitrosoureas, plant-derived compounds (eg vinca alkaloids), epipodophyllo Toxin, tyrosine kinase inhibitor, taxol, radiation therapy, surgery, dietary supplement, gene therapy, radiation therapy, eg 3D-CRT, immunology Toxin therapy, such as lecithin, and monoclonal antibodies. Specific examples of chemotherapeutic compounds that can be mixed or combined with the nucleic acid molecules of the invention include, but are not limited to: paclitaxel; docetaxel; methotrexate; doxorbin; edatrexate; vinorelbine; tomaxifene; Uridine (5-FU); irinotecan (CAMPTOSAR® or CPT-11 or camptothecin-11 or campto); cisplatin; carboplatin; amsacrine; cytarabine; bleomycin; mitomycin C; dactinomycin;
Hexamethylmelamine; dacarbazine; L-asperginase; nitrogen mustard; melphalan, chlorambucil; busulfan; ifosfamide; 4-hydroperoxycyclophosphamide, thiotepa; tamoxifen, herceptin; IMC C225;
And combinations thereof.

Diagnostic Uses The nucleic acid molecules of the invention (eg, enzymatic nucleic acid molecules) are used as a diagnostic tool to examine genetic drift and mutations in diseased cells, or in a cell, VEGF and / or VEGFr, such as VEGFR1 and The presence of / and VEGFR2 RNA can be detected. Due to the close relationship between the enzymatic nucleic acid molecule activity and the structure of the target RNA, mutations that alter base pairing and three-dimensional structure of the target RNA can be detected in any region of the molecule. By using multiple enzymatic nucleic acid molecules described in the present invention, nucleotide changes important to RNA structure and function in vitro and in cells and tissues can be mapped. Cleavage of target RNA by enzymatic nucleic acid molecules can be used to inhibit gene expression and reveal (essentially) the role of specific gene products in disease progression. In this way, other gene targets can be identified as important mediators of the disease. These experiments will lead to better treatment of disease progression by providing the possibility of combination therapy (eg, numerous enzymatic nucleic acid molecules targeting different genes, known small molecule inhibitors and Intermittent treatment in combination with coupled enzymatic nucleic acid molecules, enzymatic nucleic acid molecules and / or other chemical or biological molecules). Other in vitro uses of the enzymatic nucleic acid molecules of the invention are well known in the art and include the detection of the presence of mRNA associated with conditions associated with VEGF, VEGFR1 and / or VEGFR2. . Such RNA is detected by determining the presence of cleavage products after treatment with enzymatic nucleic acid molecules using standard methodologies.

  In certain instances, enzymatic nucleic acid molecules that can only cleave only wild-type or mutant forms of the target RNA are used in the assay. The first enzymatic nucleic acid is used to identify the presence of wild-type RNA in the sample, and the second enzymatic nucleic acid is used to identify mutant RNA in the sample. Cleave both wild-type and mutant RNA synthetic substrates as reaction controls with both enzymatic nucleic acid molecules, revealing the relative efficiency of enzymatic nucleic acid molecules in the reaction and not cleaving "non-target" RNA species . The cleavage products from the synthetic substrate also serve to generate size markers for the analysis of wild type and mutant RNA in the sample population. Thus, each analysis requires two enzymatic nucleic acid molecules, two substrates, and one unknown sample, which are combined to perform six reactions. The presence of cleavage products is confirmed using an RNase protection assay, allowing the full length and cleavage fragments of each RNA to be analyzed in one lane of a polyacrylamide gel. The results need not necessarily be quantified in order to gain insight into the expression of the mutant RNA in the target cells and the presumed risk of the desired phenotypic change. Expression of mRNA suggested that the protein product is involved in the development of the phenotype (ie, VEGFR1 and / or VEGFR2) is sufficient to establish risk. If equivalent specific activity probes are used for both transcripts, a qualitative comparison of RNA levels is sufficient and the cost of initial diagnosis is reduced. Whether comparing RNA levels qualitatively or quantitatively, a higher mutant to wild type ratio will correlate with higher risk.

  The use of enzymatic nucleic acid molecules in diagnostic applications contemplated herein is described, for example, in Usman et al. US patent application 09 / 877,526, George et al. U.S. Pat. Nos. 5,834,186 and 5,741,679, Shih et al. U.S. Pat. No. 5,589,332, Nathan et al. U.S. Pat. No. 5,871,914, Nathan and Ellington, International Publication WO 00/24931, Breaker et al. , International Publications WO 00/26226 and 98/27104, and Sullenger et al. , U.S. patent application Ser. No. 09 / 205,520.

Additional Uses The potential utility of the sequence-specific enzymatic nucleic acid molecules of the present invention has many applications for RNA studies similar to those that DNA restriction endonucleases have for DNA studies (Nathans). et al., 1975 Ann. Rev. Biochem.44: 273). For example, a restriction fragment pattern can be used to establish a sequence relationship between two related RNAs, and large RNAs can be specifically cleaved into fragments of a more useful size for research. The ability to manipulate the sequence specificity of enzymatic nucleic acid molecules is ideal for cleaving RNAs of unknown sequence. Applicants describe the use of nucleic acid molecules to down-regulate gene expression of target genes in bacteria, microorganisms, fungi, viruses, and eukaryotic systems such as plants or mammalian cells.

  All patents and publications mentioned in this specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All references cited in this specification are cited as part of this specification to the same extent as if each reference was individually cited herein in its entirety as part of this specification. The

  Those skilled in the art will readily appreciate that the present invention is well adapted to carry out its objectives and obtain the results and advantages described, as well as those inherent herein. The methods and compositions described herein are representative of presently preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention. Those skilled in the art will make modifications and other uses that fall within the spirit of the invention as defined in the claims.

  Those skilled in the art will readily appreciate that various substitutions and modifications can be made to the invention disclosed herein without departing from the scope and spirit of the invention. That is, such additional aspects are within the scope of the present invention and claims.

The invention described herein as an example can be practiced appropriately without any elements or limitations not specifically disclosed herein. That is, for example, in each example in this specification, the terms “including”, “consisting essentially of ...” and “consisting of ...” are replaced with either of the other two. be able to. The terms and expressions used in this specification are used as terms of description and are not limiting. The use of such terms and expressions is not intended to exclude the features shown and described, or equivalents thereof, but is within the scope of the invention as set forth in the claims. It will be understood that various modifications are possible. That is, although the invention has been specifically disclosed by preferred embodiments and optional features, those skilled in the art can make changes and variations in the concepts described herein, and such changes and variants are also claimed. It should be understood that it is considered to be within the scope of the present invention as defined in

  Furthermore, if the features and aspects of the invention are described in terms of Markush groups or other alternative groups, those skilled in the art will also describe the invention in terms of individual members or subgroups of Markush groups or other groups. You will recognize that.

Other embodiments are within the scope of the claims.
Table 1

FIG. 1 shows the secondary structure model of ANGIOZYME ™ ribozyme bound to its RNA target. FIG. 2 shows the time course of inhibition of primary tumor growth after systemic administration of ANGIOZYME ™ in an LLC mouse model. FIG. 3 shows the inhibition of primary tumor growth after systemic administration of ANGIOZYME ™ according to a dosing regimen in an LLC mouse model. FIG. 4 shows dose-dependent inhibition of tumor metastasis after systemic administration of ANGIOZYME ™ in a mouse colorectal model. FIG. 5 is a graph showing the plasma concentration profile of ANGIOZYME ™ after a single subcutaneous administration (SC) at a dose of 10, 30, 100 or 300 mg / m ² . FIG. 6 shows an example of a chemically stabilized ribozyme motif. FIG. 7 shows an example of a chemically stabilized tinzyme A ribozyme motif. FIG. 8 shows an example of a DNAzyme motif. FIG. 9 shows data showing the inhibition of soluble VEGFR1 in clinical trials with ANGIOZYME (SEQ ID NO: 5977). FIG. 10 shows a generalized outline for a mouse model of proliferative retinopathy. FIG. 11 shows a graph showing the effectiveness of enzymatic nucleic acid molecules targeting the VEGF-receptor in a mouse model of proliferative retinopathy.

Claims (103)

Formula II: (SEQ ID NO: 5978)
5'-u _s a _s c _s a _s au uc U GAu Gag gcg aaa gcc Gaa Aag aca aB-3 '
[Wherein each a is a 2′-O-methyladenosine nucleotide, each g is a 2′-O-methylguanosine nucleotide, each c is a 2′-O-methylcytidine nucleotide, and each u is 2 '-O-methyluridine nucleotides, each A is adenosine, each G is guanosine, each s independently represents a phosphorothioate internucleotide linkage, U is 2'-deoxy-2'-C -Allyluridine, and B is the inverted deoxy abasic component]
A compound having A composition comprising the compound of claim 1 and a pharmaceutically acceptable carrier or diluent. A method of administering to a cell a compound of claim 1, comprising contacting said cell with a compound under conditions suitable for said administration. 4. The method of claim 3, wherein the cell is a mammalian cell. 4. The method of claim 3, wherein the cell is a human cell. 4. The method of claim 3, wherein the administration is performed in the presence of a delivery reagent. The method of claim 6, wherein the delivery reagent is a lipid. The method of claim 7, wherein the lipid is a cationic lipid. The method of claim 7, wherein the lipid is a phospholipid. The method of claim 6, wherein the delivery reagent is a liposome. 2. A method of administering a compound of claim 1 to a cell together with one or more other agents comprising contacting the cell with the compound and other agent under conditions suitable for the administration. A method of inhibiting ocular angiogenesis in a subject comprising the step of contacting said subject with a compound of claim 1 under conditions suitable for said inhibition. 13. The method of claim 12, wherein the angiogenesis is associated with diabetic retinopathy. 13. The method of claim 12, wherein the angiogenesis is associated with age related diabetic retinopathy. A method for cleaving RNA comprising a sequence of KDR RNA, comprising contacting the compound of claim 1 with said RNA under conditions suitable for cleaving said RNA. The method according to claim 15, wherein the cleavage is performed in the presence of a divalent cation. The method of claim 16, wherein the divalent cation is Mg ²⁺ . A method of administering a compound of claim 1 to a mammal comprising contacting said mammal with the compound under conditions suitable for said administration. The method of claim 18, wherein the mammal is a human. The method of claim 18, wherein the administration is performed in the presence of a delivery reagent. The method of claim 18, wherein the delivery reagent is a lipid. The method of claim 21, wherein the lipid is a cationic lipid. The method of claim 21, wherein the lipid is a phospholipid. 21. The method of claim 20, wherein the delivery reagent is a liposome. A method of treating a subject having endometriosis comprising contacting said subject with a nucleic acid molecule that modulates the expression of VEGF, VEGFR1, and / or VEGFR2 under conditions suitable for said treatment. 26. The method of claim 25, wherein the nucleic acid molecule is an enzymatic nucleic acid molecule. 26. The method of claim 25, wherein the nucleic acid molecule is an antisense nucleic acid molecule. 26. The method of claim 25, wherein the nucleic acid molecule is a dsRNA nucleic acid molecule. 26. The method of claim 25, wherein the nucleic acid molecule is a nucleic acid aptamer. 26. The method of claim 25, wherein the nucleic acid molecule comprises a sequence having SEQ ID NO: 5977. 27. The method of claim 26, wherein the enzymatic nucleic acid molecule has endonuclease activity that cleaves RNA encoded by a VEGFR1 and / or VEGFR2 gene. 27. The method of claim 26, wherein the enzymatic nucleic acid molecule is of a hammerhead configuration. 27. The method of claim 26, wherein the enzymatic nucleic acid molecule is of an inozyme configuration. 27. The method of claim 26, wherein the enzymatic nucleic acid molecule is of a tinzyme configuration. 27. The method of claim 26, wherein the enzymatic nucleic acid molecule is of a DNAzyme configuration. 27. The method of claim 26, wherein the enzymatic nucleic acid molecule is of a G-cleaving agent configuration. 27. The method of claim 26, wherein the enzymatic nucleic acid molecule is of an amberzyme configuration. 27. The method of claim 26, wherein the enzymatic nucleic acid molecule is an allozyme. 26. The method of claim 25, wherein the nucleic acid molecule is chemically synthesized. 26. The method of claim 25, wherein the nucleic acid molecule comprises at least one 2'-sugar modification. 26. The method of claim 25, wherein the nucleic acid molecule comprises at least one nucleobase modification. 26. The method of claim 25, wherein the nucleic acid molecule comprises at least one phosphate backbone modification. 26. The method of claim 25, wherein the subject is a human. A method of treating a subject having endometriosis, comprising administering to the subject a nucleic acid molecule that modulates expression of VEGF, VEGFR1, and / or VEGFR2 under conditions suitable for said treatment. 45. The method of claim 44, wherein the administration is performed in the presence of a delivery reagent. 46. The method of claim 45, wherein the delivery reagent is a lipid. 48. The method of claim 46, wherein the lipid is a cationic lipid. 48. The method of claim 46, wherein the lipid is a phospholipid. 46. The method of claim 45, wherein the delivery reagent is a liposome. 45. The method of claim 44, further comprising administering one or more other agents. The other drug is selected from GnRH (gonadotropin releasing hormone) agonist, leupron depot (leuprolide acetate), cinalel (naferaline acetate), zolodex (goserelin acetate), suprefact (buserelin acetate), donazole, and oral contraceptives 51. The method of claim 50, wherein: Formula I: (SEQ ID NO: 5977)
5'-g _s a _s g _s u _s ugc U GAuGagg ccgaaa ggccGaaAgucugB-3 '
[Wherein each a is a 2′-O-methyladenosine nucleotide, each g is a 2′-O-methylguanosine nucleotide, each c is a 2′-O-methylcytidine nucleotide, and each u is 2 '-O-methyluridine nucleotides, each A is adenosine, each G is guanosine, each s independently represents a phosphorothioate internucleotide linkage, U is 2'-deoxy-2'-C -Allyluridine, and B is the inverted deoxy abasic component]
A compound having 53. A composition comprising the compound of claim 52 in a pharmaceutically acceptable carrier or diluent. 53. A method of administering to a cell a compound of claim 52 comprising contacting said cell with a compound under conditions suitable for said administration. 55. The method of claim 54, wherein the cell is a mammalian cell. 55. The method of claim 54, wherein the cell is a human cell. 55. The method of claim 54, wherein the administration is performed in the presence of a delivery reagent. 58. The method of claim 57, wherein the delivery reagent is a lipid. 59. The method of claim 58, wherein the lipid is a cationic lipid. 59. The method of claim 58, wherein the lipid is a phospholipid. 58. The method of claim 57, wherein the delivery reagent is a liposome. 53. A method of administering a compound of claim 52 to a cell with a chemotherapeutic agent, comprising contacting the cell with the compound and the chemotherapeutic agent under conditions suitable for the administration. 64. The method of claim 62, wherein the chemotherapeutic agent is 5-fluorouridine. 64. The method of claim 62, wherein the chemotherapeutic agent is leucovorin. 63. The method of claim 62, wherein the chemotherapeutic agent is selected from irinotecan, CAMPTOSAR®; CPT-11, camptothecin-11, or campto. 64. The method of claim 62, wherein the chemotherapeutic agent is paclitaxel. 64. The method of claim 62, wherein the chemotherapeutic agent is carboplatin. 53. A mammalian cell comprising the compound of claim 52. 69. The mammalian cell of claim 68, wherein the mammalian cell is a human cell. 53. A method of inhibiting angiogenesis in a subject comprising the step of contacting said subject with a compound under conditions suitable for said inhibition. 72. The method of claim 70, wherein the angiogenesis is tumor angiogenesis. 53. A method of treating a subject having a condition associated with increased levels of VEGF receptor, comprising contacting the subject's cells with a compound of claim 52 under conditions suitable for the treatment. 73. The method of claim 72, further comprising using one or more drug therapies under conditions suitable for said treatment. 54. A method of cleaving RNA comprising the sequence of VEGFR1 (flt-1), comprising contacting the compound of claim 52 with said RNA under conditions suitable for cleaving said RNA. 75. The method of claim 74, wherein the cleavage is performed in the presence of a divalent cation. 76. The method of claim 75, wherein the divalent cation is Mg2 ⁺ . 75. The method of claim 72, wherein the condition is cancer. 78. The method of claim 77, wherein the cancer is breast cancer. 78. The method of claim 77, wherein the cancer is lung cancer. 78. The method of claim 77, wherein the cancer is colorectal cancer. 78. The method of claim 77, wherein the cancer is kidney cancer. 78. The method of claim 77, wherein the cancer is melanoma. 78. The method of claim 77, wherein the cancer is pancreatic cancer. 80. The method of claim 79, wherein the lung cancer is non-small cell lung carcinoma. 82. The method of claim 81, wherein the renal cancer is renal cell carcinoma. 74. The method of claim 73, wherein the other therapy is 5-fluorouridine. 74. The method of claim 73, wherein the other therapy is leucovorin. 75. The method of claim 73, wherein the other therapy is irinotecan, CAMPTOSAR (R), CPT-11, camptothecin-11, or campto. 74. The method of claim 73, wherein the other therapy is paclitaxel. 74. The method of claim 73, wherein the other therapy is carboplatin. 53. A method of administering a compound of claim 52 to a mammal comprising contacting said mammal with the compound under conditions suitable for said administration. 92. The method of claim 91, wherein the mammal is a human. 92. The method of claim 91, wherein the administration is performed in the presence of a delivery reagent. 94. The method of claim 93, wherein the delivery reagent is a lipid. 95. The method of claim 94, wherein the lipid is a cationic lipid. 95. The method of claim 94, wherein the lipid is a phospholipid. 94. The method of claim 93, wherein the delivery reagent is a liposome. 53. A method of administering a compound of claim 52 together with a chemotherapeutic agent to a mammal comprising contacting said mammal with the compound and chemotherapeutic agent under conditions suitable for said administration. 99. The method of claim 98, wherein the chemotherapeutic agent is 5-fluorouridine. 99. The method of claim 98, wherein the chemotherapeutic agent is leucovorin. 99. The method of claim 98, wherein the chemotherapeutic agent is irinotecan, CAMPTOSAR (R), CPT-11, camptothecin-11, or campto. 99. The method of claim 98, wherein the chemotherapeutic agent is paclitaxel. 99. The method of claim 98, wherein the chemotherapeutic agent is carboplatin.

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