JP2009526861A - GPCR as an angiogenic target - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for regulating angiogenesis.
The present invention relates to a G-protein coupled receptor (GPCR), namely, Rectomedin-3 (LEC3), endothelin-1 receptor (EDNRA) and C-X-. Methods are provided for modulating angiogenesis by enhancing or attenuating the expression or function of C chemokine receptor 4 (CXCR-4: C—X—C Chemokine Receptor 4). The present invention also provides methods for inhibiting and promoting GPCR-dependent angiogenesis in vertebrates, including certain mammals, including humans, for the treatment of angiogenesis-related diseases. The present invention also provides a method of identifying a compound that promotes or inhibits angiogenesis through interaction with all or part of LEC3, EDNRA or CXCR-4. The present invention further provides a method for regulating angiogenesis through regulation of these GPCRs and VEGF.
[Selection] FIG.

Description

(Cross reference for related applications)
This application claims the benefit of US Provisional Application No. 60 / 772,991, filed Feb. 14, 2006, which is hereby incorporated by reference in its entirety.

The present invention relates to three G protein-coupled receptor (GPCR) proteins and their relationship to angiogenesis. Specifically, this application relates to Lectinedin-3 (LEC3), endothelin-1A receptor (EDNRA) as a drug target for therapies related to anti-angiogenesis and pro-angiogenesis. A) and the role of C—X—C chemokine receptor 4 (CXCR4: C—X—C Chemokine Receptor 4).

Angiogenesis is the process by which new blood vessels are formed from existing blood vessels. This includes proliferation, differentiation and migration of endothelial cells as well as possibly other cell types found in blood vessels such as smooth muscle cells and fibroblasts. Changing this process, whether by activation or by inhibition, is for the treatment of human diseases such as cancer, macular degeneration, rheumatoid arthritis, Alzheimer's disease, wound healing, atherosclerosis and anemia. Can be beneficial.

  Inhibition of angiogenesis represents a powerful and novel approach to cancer treatment. Analytical methods that can rapidly screen for compounds for anti-angiogenic activity are essential to firmly realize the potential of this method for cancer treatment. Solid tumors require a sufficient supply of nutrients from the blood to survive, grow and metastasize (Non-Patent Document 1, Non-Patent Document 2). New blood vessels grow growing tumors by sprouting from existing blood vessels, a process known as angiogenesis. In recent years, angiogenesis has received considerable attention as a novel process that targets cancer. Already in clinical trials, many drugs have been proven to have anti-angiogenic activity, and several new drugs have been developed specifically for their ability to inhibit blood vessel growth (Non-Patent Document 3). ).

Abnormal angiogenesis in the retina is a major factor in age-related macular degeneration, diabetic retinopathy and retinopathy of prematurity, which is the root cause of vision loss. Thus, the treatment of angiogenesis-related diseases is particularly important in retinal pathology.

Anti-angiogenic drugs have been successfully mixed in clinical applications. Many new compounds will need to be tested in vivo to identify agents that can treat various tumors. Therefore, in vivo assays that are suitable for screening potential anti-angiogenic compounds are becoming increasingly important. U.S. Patent No. 6,057,077 provides an analytical method using zebrafish (Danio rerio) that provides not only the potential for high-throughput drug screening but also the relevance of the in vivo environment. This technique is, for example, a zebra that expresses a reporter protein such as a green leaf coral fluorescent protein (G-RCFP: non-patent document 4) or a red fluorescent protein (dsRed2) that is specifically expressed in blood vessels. Generating a transgenic line of fish.

Zebrafish has become a good model for research on spine formation. Unlike mice, zebrafish embryos develop outside the mother and are transparent, facilitating the observation of differentiating tissues and organs. In particular, the zebrafish vascular system has been described in detail and proved to be highly conserved from zebrafish to humans. (Non-patent document 5, Non-patent document 6). In addition, zebrafish embryos can live for days without much blood supply, thus facilitating research on embryos with vascular defects. Intersegmental blood vessels in zebrafish embryos are formed by angiogenic collapse that is very similar to tumor angiogenesis, and this process has also been demonstrated to be necessary for blood vessel growth in mammals It seems to require the same protein as For example, the anti-angiogenic compound PTK787 / ZK222258, which is an inhibitor of vascular endothelial growth factor (VEGF) receptor tyrosine kinase, has been shown to affect the formation of zebrafish blood vessels ( Non-patent document 7).

Currently, methods for visualizing zebrafish blood vessels include whole-mount in situ hybridization of vascular endothelial cell markers (Non-patent Documents 8 and 9), detection and circulation of endogenous alkaline phosphatase activity in blood vessels. Includes cardiovascular microangiography. The latter technique involves the injection of fluorescent beads into the circulation of live zebrafish larvae (Non-Patent Document 10) and is useful for visualization of blood vessels in a complete circulatory system. The formation of blood vessels between somites blocked by the application of tyrosine kinase inhibitors targeting VEGF receptors can be easily visualized in transgenic fish expressing fluorescent proteins in the vascular system found.

Zebrafish is an excellent model for angiogenesis studies. There is a need in the art to find targets that act to promote or inhibit angiogenesis (depending on the desired outcome) and the zebrafish model is for pro-angiogenic and anti-angiogenic therapies. Can be used to demonstrate the usefulness of these targets.

Toxicity is a major cause of failure during drug development. Many drugs that have been shown to be safe in cell culture have been proven toxic in animal studies. Thus, although zebrafish is a useful animal model for early toxicity screening, further research is often needed in mammalian systems that are believed to more closely mimic the genetic and biological basis of human disease . Therefore, there is a need for a mammalian model of disease as a second stage for screening for compounds that are useful in angiogenesis related abnormalities such as retinopathy.

US Patent Application Publication No. 20040144385 (Rubinstein et al.) Hanahan Dee-Jay Volkman (1996) Cell 86: 353-364 Lee Sea Y et al. (2000) Cancer Met. Rev. 19: 7-11 Rosen L (2000) Oncologist 5 (suppl.l): 20-27 Mats MV et al. (1999) Nat. Biotechnol. 17: 969-973 Isogai S et al. (2001) Dev. Biol. 230 (2): 278-301 Vogel AM BM Weinstein (2000) Trends Cardiovasc. Med. 10 (8): 352-360 Chang Jay et al. (2002) Cancer Cell 1: 257-267 Hooket Bee et al. (1997) Dev. Biol. 183: 37-48 Liao Wu Wu et al. (1997) Development 124: 381-389 Weinstein BM et al. (1996) Nat. Med. 1: 1143-1147

Therefore, an object of the present invention is to satisfy the above-mentioned necessity or necessity.

  The present invention provides methods of promoting angiogenesis using isolated nucleic acid sequences for all or part of CXCR4, LEC3 or EDNRA. In some embodiments, the nucleic acid encoding CXCR4 has the polynucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 7, or SEQ ID NO: 32. In some embodiments, the nucleic acid encoding LEC3 has the polynucleotide sequence of SEQ ID NO: 3, or SEQ ID NO: 9. In some embodiments, the nucleic acid encoding EDNRA has the polynucleotide sequence of SEQ ID NO: 5, SEQ ID NO: 11, SEQ ID NO: 19, or SEQ ID NO: 35. The invention also provides polynucleotides encoding all or part of CXCR4, LEC3 or EDNRA and their portions, homologues and fragments from other vertebrate species used in the methods of the invention. In some embodiments, the polynucleotide is derived from species such as, for example, humans, mice, rats, and cows.

The present invention also provides isolated zebrafish CXCR4, LEC3 and EDNRA polypeptides. In some embodiments, the polypeptide has the amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively. The invention also provides CXCR4, LEC3 and EDNRA polypeptides and their portions, homologues and fragments from other vertebrate species used in the methods of the invention. In some embodiments, the polypeptide is derived from species such as, for example, humans, mice, rats, and cows. In some embodiments, the CXCR4 polypeptide has an amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 8, or SEQ ID NO: 33. In some embodiments, the LEC3 polypeptide has the amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 10, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 27, or SEQ ID NO: 28. In some embodiments, the EDNRA polypeptide has an amino acid sequence of SEQ ID NO: 6, SEQ ID NO: 12, SEQ ID NO: 20, SEQ ID NO: 22, or SEQ ID NO: 23.

The invention also includes an isolated zebrafish peptide ligand, stromal cell derived factor 1 (SDF-1 to CXCL12) for CXCR4 receptor and endothelin-1 (ET-1 for EDNRA receptor). Or ETA). In some embodiments, the polypeptide has the amino acid sequence of SEQ ID NO: 37 (SDF-1: NP_954637) and SEQ ID NO: 39 (ET-1: NP_001946), respectively. The polynucleotides encoding these SDF-1 and ET-1 proteins are SEQ ID NO: 38 (NM_199168) and SEQ ID NO: 40 (NM_001955), respectively. The present invention also provides SDF-1 and ET-1 polypeptides and their portions, homologues and fragments from other vertebrate species used in the methods of the invention. In some embodiments, the polynucleotide is derived from species such as, for example, humans, mice, rats, and cows. In some embodiments, the peptide SDF-1 ligand for CXCR4 is SEQ ID NO: 42 (NP_000600 for Homo sapiens (human)), SEQ ID NO: 44 (NP_068350, Mus musculus, mouse), SEQ ID NO: 46 (Rattus norvegicus) (NP_071513 for rat), SEQ ID NO: 48_ (NP_989841 for Gallus gallus), SEQ ID NO: 50 (NP_001009580 for Sus scrofa). The polynucleotides encoding these CXCR4 proteins are SEQ ID NO: 41 (for Homo sapiens (human)), SEQ ID NO: 43 (for Mus musculus (mouse)), SEQ ID NO: 45 (for Rattus norvegicus (rat)), SEQ ID NO: 47 (For Gallus gallus (chicken)) and SEQ ID NO: 49 (for Sus scrofa (pig)). The present invention also provides a method of promoting angiogenesis in an animal. Peptide ET-1 ligands for EDNRA are SEQ ID NO: 39 (NP_001946 for Homo sapiens (human)), SEQ ID NO: 52 (NP_034234 for Mus musculus (mouse)), SEQ ID NO: 54 (NP_036680 for Rattus norvegicus (rat)), It has the amino acid sequence of SEQ ID NO: 56 (NP_851353 for Bos taurus (bovine)) and SEQ ID NO: 58 (NP_9999047 for Sus scrofa (pig)) Polynucleotides encoding these ET-1 ligands are SEQ ID NO: 40 (for Homo sapiens (human)), SEQ ID NO: 51 (NP_034234 for Mus musculus (mouse)), SEQ ID NO: 53 (for Rattus norvegicus (rat)), SEQ ID NO: 55 (for Bos taurus (bovine)) and SEQ ID NO: 57 (for Sus scrofa (pig)). In some embodiments, the method provides an animal with an effective amount of peptide ligand SDF-1 for CXCR4 and an effective amount of ET for activation of the EDNRA receptor. -1 is provided, thereby promoting angiogenesis. In addition, antibodies against the ligands SDF-1 and ET-1 can possibly be used to block the bioavailability of endogenous ligands for their respective receptors CXCR4 and EDNRA, thereby allowing anti-vascular It can be used to block activation of CXCR4 and EDNRA for neogenesis. This is a similar scenario for Avastin, an anti-VEGF antibody, to block the bioavailability of VEGF to its receptor Flk-I in tumor blood vessels for anti-angiogenic therapy to treat cancer (See: Ferrara, N., Hillan, KJ & Novotny, W. (2005) “Bevacizumab (Avastin), a humanized-antibody fortification. Biothem. Biother. : 328-335).

The present invention also provides a method for isolating endogenous artificial peptide ligands for orphan GPCR, LEC3 receptor. Since LEC3 is classified as an orphan GPCR, there is no known endogenous ligand for this receptor. However, we propose to screen a combinatorial peptide phage library to isolate high affinity peptide sequences that bind to the LEC3 receptor. Once the peptide sequence has been identified, we will generate and screen additional variants of peptide clones with higher affinity, functional blocking or enhancement for the LEC3 receptor Can do. This identified recombinant peptide can have either a functional blocking or potentiating effect on the LEC3 receptor, so that it inhibits angiogenesis (anti-angiogenesis) or induces (promoting angiogenesis), respectively. ) Can be used to. In some embodiments, the method comprises administering to the animal an effective amount of this recombinant peptide ligand for the LEC3 receptor. Which leads to receptor activation or deactivation depends on the amino acid aspect on the peptide ligand. Accordingly, the present invention also provides each of the methods of anti-angiogenic or promoting angiogenesis in an animal.

The present invention also provides a method of promoting angiogenesis in an animal. In some embodiments, the method comprises administering to the animal an effective amount of CXCR4, LEC3 and EDNRA polypeptide alone or in admixture. In order to facilitate passage through the cell membrane barrier, the polypeptide may be conjugated with various effective delivery reagents. The CXCR4, LEC3 and EDNRA polypeptide may be any spinal CXCR4, LEC3 and EDNRA polypeptide (ie, may be derived from any species). In some embodiments, the CXCR4 polypeptide has an amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 8, or SEQ ID NO: 33. In some embodiments, the LEC3 polypeptide has the amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 10, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 27, or SEQ ID NO: 28. In some embodiments, the EDNRA polypeptide has an amino acid sequence of SEQ ID NO: 6, SEQ ID NO: 12, SEQ ID NO: 20, SEQ ID NO: 22, or SEQ ID NO: 23.

In other embodiments of the invention, the method of promoting angiogenesis comprises an effective amount of a polynucleotide encoding a CXCR4, LEC3 or EDNRA polypeptide, alone or in an animal in need of angiogenesis. Including administering in combination. The polynucleotide may encode any of CXCR4, LEC3 or EDNRA polypeptide. In some embodiments, the polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 1. In other embodiments, the polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 7. In other embodiments, the polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 32. In other embodiments, the polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 3. In other embodiments, the polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 9. In other embodiments, the polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 5. In other embodiments, the polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 11. In other embodiments, the polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 19. In other embodiments, the polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 35.

The invention also provides a method of promoting angiogenesis for an animal in need thereof, wherein the animal has an effective amount of a first polynucleotide encoding a CXCR4, LEC3 or EDNRA polypeptide, and VEGF. Administering an effective amount of a second polynucleotide encoding the polypeptide. In some embodiments, the VEGF polypeptide comprises the group consisting of SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, and SEQ ID NO: 76. Comprising an amino acid sequence selected from

The present invention also provides a method for inhibiting angiogenesis in an animal. In some embodiments, the method comprises subjecting an animal to an antisense oligonucleotide that suppresses expression of all or part of a CXCR4, LEC3, EDNRA polypeptide, ie, phosphoramidite morpholino oligonucleotide (PMO). administering an effective amount of a polynucleotide such as morpholino oligonucleotide. The CXCR4, LEC3 and / or EDNRA polypeptide may be any vertebrate CXCR4, LEC3 and / or EDNRA polypeptide (human, mouse, cow, rat, zebrafish and all other vertebrates). Including.) To improve the efficient absorption of PMO into cells, peptides such as arginine rich peptide may be added to PMO (Moulton, HM et al. (2004) Cellular Uptake of Antisense. Morpholino Oligomers Conjugated to Arginine-Rich Peptides. Bioconjugate Chem. 15: 290-299). In some embodiments, the CXCR4 polypeptide has an amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 8, or SEQ ID NO: 33. In some embodiments, the LEC3 polypeptide has the amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 10, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 27, or SEQ ID NO: 28. In some embodiments, the EDNRA polypeptide has an amino acid sequence of SEQ ID NO: 6, SEQ ID NO: 12, SEQ ID NO: 20, SEQ ID NO: 22, or SEQ ID NO: 23. In some embodiments, the nucleic acid encoding CXCR4 has the polynucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 7, or SEQ ID NO: 32. In some embodiments, the nucleic acid encoding LEC3 has the polynucleotide sequence of SEQ ID NO: 35 or SEQ ID NO: 9. In some embodiments, the nucleic acid encoding EDNRA has the polynucleotide sequence of SEQ ID NO: 5, SEQ ID NO: 11, SEQ ID NO: 19, or SEQ ID NO: 35.

The present invention also provides for a patient in need of treatment for an angiogenesis-related disease in an amount sufficient to inhibit angiogenesis, ie, a polynucleotide that inhibits expression of CXCR4, LEC3 and / or EDNRA, ie Provided is a method of treating an angiogenesis-related disease comprising administering an amount that enhances the effectiveness of a regimen of any combination of a therapy such as anti-VEGF and chemotherapy. Angiogenesis-related diseases include: angiogenesis-dependent cancers; benign tumors; rheumatoid arthritis; psoriasis; visual angiogenic diseases; Ausler-Webber syndrome; myocardial neovascularization; Fibroangiopathies; fibroangioma; wound granulation; intestinal adhesions, atherosclerosis, scleroderma, hyperplastic scars, cat scratch disease, and Helicobacter pylori ulcers .

As such, the method includes treating a patient with an angiogenesis-dependent tumor by administering a polynucleotide, a modified polynucleotide. These administrations enable effective delivery of the polynucleotide into cells and tissues and, together with excipients that suppress the expression of CXCR4, LEC3 and / or EDNRA, together with the regression or stabilization of cancerous tumors Administration, in an amount sufficient to provide the effect of the regimen combined with any therapy. The polynucleotide or modified polynucleotide may be an antisense oligonucleotide, siRNA, morpholino oligonucleotide, or a ribozyme that hybridizes specifically to nucleic acids encoding CXCR4, LEC3 and / or EDNRA. The present invention also provides a method of treating a subject with a cell-penetrating peptide to inhibit the interaction of CXCR4, LEC3 and / or EDNRA with their ligands or to impair downstream effector function. I will provide a. The method according to the present invention may further comprise cell penetrating peptide inhibition of VEGF activity.

In some embodiments, the invention suppresses VEGF expression and a first reagent that suppresses CXCR4, LEC3 and / or EDNRA expression for a patient in need of treatment for angiogenesis-related disease. A method of treating an angiogenesis-related disease comprising administering a second reagent. There, the first reagent and the second reagent act synergistically together to suppress angiogenesis. The first reagent may comprise a polynucleotide directed to a polynucleotide encoding CXCR4, LEC3 and / or EDNRA (such as RNAis, siRNAs, miRNAs, modified nucleic acids, PNA, or morpholino oligonucleotides or ribozymes, etc. Such antisense molecules). In some embodiments, the first reagent has a sequence selected from the group consisting of SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, and combinations thereof.

It's not as simple as limiting only a small portion of the targeted sequence for an antisense morpholino or other siRNA. The blocking antisense morpholino oligo for translation acts anywhere 25 bp downstream from the 5 'untranslated region of the ATG start codon. Splicing-inhibiting antisense morpholino oligos also act between exon / intron boundaries. Thus, any exon / intron boundary of the CXCR4, LEC3 and / or EDNRA gene is the target sequence. Thus, any region of CXCR4, LEC3 and / or EDNRA mRNA transcription for translation block antisense and any genomic region of the CXCR4, LEC3 and / or EDNRA gene for splicing repression can be used. . The second reagent may comprise a polynucleotide directed to a polynucleotide encoding VEGF (including antisense molecules, RNAis, siRNAs, miRNAs, modified nucleic acids, PNAs, morpholino oligonucleotides, ribozymes, etc.). . In some embodiments, the first reagent has a sequence selected from the group consisting of SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, and combinations thereof. The human VEGF accession number is NMJ303376 (Unigene cluster: Hs. 73793 (Ensembl gene ID: ENSG00000123715)). In some embodiments, the angiogenesis-related disease is associated with tumor or cancer progression.

In order to design oligonucleotides that disrupt the action of CXCR4, LEC3 and / or EDNRA and / or VEGF, in the technical field to which the invention belongs to predict sequences that will have the desired effect on the suppression of RNA expression Various known computer-based algorithms may be used. An example of such an algorithm includes, but is not limited to, Sfold. Sfold is sfold. wadsworth, org. Available on the Sfold web server on the World Wide Web under the domain address. This algorithm is described in Ding, Y. et al. et al. (2004) Nucl. Acids Res. 32: W135-W141.

In some embodiments, the first reagent is a small molecule, a natural compound or a synthetic compound from combinatorial chemistry that suppresses the expression or action of CXCR4, LEC3 and / or EDNRA, and VEGF. Such compounds may be known in the art to which this invention belongs and may be identified using the screening methods described herein. Such compounds may, for example, suppress CXCR4, LEC3 and / or EDNRA expression, CXCR4, LEC3 and / or EDNRA post-translational modification, or alter the allosteric organization of CXCR4, LEC3 and / or EDNRA. Or may compete with the binding of endogenous ligands to the receptors CXCR4, LEC3 and EDNRA.

The present invention relates to a first compound that suppresses the function or expression of CXCR4, LEC3, and / or EDNRA, and to suppress the expression or function of VEGF signal for a patient in need of treatment for angiogenesis-related disease. Further provided is a method of treating an angiogenesis-related disease comprising administering two compounds (including its receptor and a downstream signaling molecule). The first compound and the second compound are provided in an amount sufficient to inhibit angiogenesis. In some embodiments, the compound that inhibits the function of CXCR4 is an antibody that specifically binds to CXCR4.

In a specific embodiment according to the present invention, the present invention relates to a first compound that suppresses the expression or action of CXCR4, LEC3 and / or EDNRA, and the expression or action of A method of treating a patient having an angiogenesis-dependent tumor comprising administering two compounds (including natural compounds, synthetic compounds or small molecules from combinatorial chemistry). The first compound and the second compound are provided in an amount sufficient to cause tumor regression. In some embodiments, the compound that inhibits the expression or action of CXCR4, LEC3 and / or EDNRA is an antisense, morpholino that specifically hybridizes to a nucleic acid comprising a sequence encoding a CXCR4, LEC3 and / or EDNRA polypeptide. -An oligonucleotide or a polynucleotide such as a ribozyme. In other embodiments, the compound that inhibits the expression or action of CXCR4, LEC3 and / or EDNRA is an antibody that specifically binds to CXCR4, LEC3 and / or EDNRA polypeptide. In some embodiments, the compound that inhibits VEGF expression or action is a polynucleotide such as an antisense, morpholino oligonucleotide, or ribozyme that specifically hybridizes to a nucleic acid comprising a sequence encoding a VEGF polypeptide. is there. In other embodiments, the compound that inhibits the action of VEGF is an antibody that specifically binds to a VEGF polypeptide. In some embodiments, the compound that inhibits CXCR4, LEC3 and / or EDNRA and / or VEGF expression and / or action is a combination of a polynucleotide and an antibody.

In another specific method according to the present invention, a polynucleotide that suppresses the expression of angiogenesis-related proteins is administered to the eye of a patient in need of treatment in an amount sufficient to suppress angiogenesis. This provides a method for preventing or treating retinopathy. The protein is, for example, LEC3, EDNRA and CXCR4. The method may comprise administration of a polynucleotide such as an antisense oligonucleotide or a phosphoramidite morpholino oligomer. Such an antisense oligonucleotide or PMO may be conjugated to a peptide to promote cellular absorption of the polynucleotide. In other embodiments, the method of inhibiting CXCR4, LEC3 and / or EDNRA expression or action is an antibody that specifically binds to a CXCR4, LEC3 and / or EDNRA polypeptide. These methods are useful for preventing or treating retinopathy such as age-related macular degeneration, diabetic retinopathy or retinopathy of prematurity. Oligonucleotide and PMO sequences may be those described elsewhere to suppress angiogenesis elsewhere in this specification.

Three drug-contaminable targets have been identified that are essential for the formation of new blood vessels (ie, angiogenesis), a critical step during tumor pathogenesis. Suppression of any one of these genes prevents angiogenesis in zebrafish, a vertebrate model organism. Small, transparent, and fast-growing zebrafish offer the hope of becoming the leading vertebrate model for studying human diseases associated with angiogenesis. The results from this in vivo study on angiogenesis using the zebrafish model reveals three G protein-coupled receptors (GPCRs) that have an important role in angiogenesis and provide a method for anticancer therapy .

GPCRs accounted for approximately 50% of all drug targets on the market over the past 20 years. Over 30% of the top 50 best-selling drug-targeted GPCRs include well-known drugs for allergies, schizophrenia and bipolar disorder, heartburn and hypertension. Thus, the discovery of GPCRs essential for angiogenesis enables us to take advantage of drug-containable targets to regulate pathological angiogenesis and promote drug discovery processes to fight cancer . We identified three important molecules whose function is essential for angiogenesis. These molecules are CX-C chemokine receptor type 4 (CXCR4), Lectomedin-3 (LEC3) and endothelin-1 receptor (EDNRA). First, we have demonstrated that these genes are expressed in human vascular endothelial cells and zebrafish vasculature using zebrafish as an in vivo vertebrate model. Next, using reverse genetic tools, targeted knockdown of each of these genes suppressed angiogenesis in animal models.

The present invention provides polynucleotides and polypeptides for zebrafish (and other vertebrate species, particularly human) CXCR4, LEC3 and EDNRA, as well as CXCR4, LEC3 and EDNRA sense and antisense polynucleotides. Also provided are methods for promoting and inhibiting angiogenesis in animals.

The referenced work, patents, patent applications, and scientific literature, including the accession numbers to the GenBank database sequences referenced herein, confirm the knowledge of those skilled in the art and are each incorporated by reference. To the same extent as specifically and individually shown, it is incorporated herein by reference in its entirety. In case of any conflict between the references cited herein and the specific teachings herein, the latter shall be resolved to the benefit of the latter.

Various definitions are made throughout this document. Most words have the meaning attributed to them by those skilled in the art. The terms specifically defined below or elsewhere in this document are given in the context of the present invention as a whole and have the meanings typically understood by those skilled in the art. If there is a conflict between the technically understood definition of a word or phrase and the definition specifically taught herein for that word or phrase, it is resolved to the benefit of the latter. It will be done. The headings used herein are for convenience only and should not be construed as limiting.

Standard reference work describing general principles of recombinant DNA technology known to those skilled in the art include Ausbel et al. , CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, 1998; Sambrook et al. , MOLECULAR CLONING: A LABORATORY MANUAL, 2D ED. , Cold Spring Harbor Laboratory Press, Plainview, New York, 1989; Kaufman et al. Eds. McHerson, Ed., HANDBOOK OF MOLECULAR AND CELLULAR METHODS IN BIOLOGY AND MEDICINE, CRC Press, Boca Raton, 1995; , DIRECTED MUTAGENESIS: A PRACTICAL APPROACH, IRL Press, Oxford, 1991. Standard work describing general principles and protocols for studies using zebrafish that are known to those skilled in the art include: THE ZEBRAFISH BOOK: M.M. , 4th ed. , Univ. of Oregon Press, Eugene, 2000, but is not limited thereto.

As used herein, “isolated” means that the material is removed from its original environment (eg, the natural environment if it is naturally present). For example, a naturally occurring polynucleotide or polypeptide that appears in a living animal is not isolated, but the same polynucleotide or isolated from all or part of the coexisting material in a natural system The polypeptide has been isolated. Such a polynucleotide may be part of a vector and / or such a polynucleotide or polypeptide may be part of a composition, and such a vector or composition may be part of its natural composition. It may still be isolated if it is not part of the environment.

A “purified” or “fully purified” polynucleotide or polypeptide refers to other cellular components that naturally accompany the native (or wild-type) nucleic acid or polypeptide and / or other impurities It means being sufficiently separated from (eg, agarose gel). A purified polypeptide or protein comprises about 60% to 99% w / w or more of the sample and may be about 90%, about 95%, or about 98% pure.

As used herein, “about” refers to +/− 10% of the reference value.

As used herein, “variant” nucleic acid or amino acid sequence refers to homologues, including, for example, isoforms, species variants, allelic variants, and fragments of the sequence of interest. A “homologous nucleotide sequence” or “homologous amino acid sequence” or variant thereof refers to at least about 60%, at least about 70% with respect to a reference sequence or portion or fragment thereof encoding or having a functional domain. At least about 75%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, preferably at least about 90%, at least about 95%, at least about 98%, or at least about 99%, more preferably 100% refers to a sequence characterized by identity (when associated with a polynucleotide) or homology (when associated with a polypeptide).

The term “therapeutically effective amount” of CXCR4, LEC3 and EDNRA antagonists, such as CXCR4, LEC3 or EDNRA antisense oligonucleotides, is used in disease states (eg, tumors when applied to tumors) or systemically. When administered, an amount calculated to achieve and maintain a therapeutically effective level in plasma to inhibit angiogenesis (when applied to cancer, inhibit the growth of cancer cells) Means. For example, a therapeutic amount sufficient to inhibit the growth of about 50% or more of cancer cells, eg, KS cells, in vitro. Of course, the therapeutic dose depends on the efficacy of each CXCR4, LEC3 and EDNRA antagonist in inhibiting cancer cell growth in vitro and the rate of elimination or metabolism of the CXCR4, LEC3 and EDNRA antagonist by the body in tumor tissue and / or plasma. It will change. The therapeutic dose is also compatible with the amount of CXCR4, LEC3 and EDNRA antagonist that will enhance the efficacy of any combination therapy of treatment, such as anti-VEGF and chemotherapy.

1. Polynucleotide A. Vertebrate CXCR4, LEC3 and EDNRA

The present invention provides polynucleotides encoding zebrafish CXCR4, LEC3 and EDNRA, and also provides polynucleotides encoding CXCR4, LEC3 and EDNRA from other vertebrates for use in the methods of the present invention. . As used herein, “polynucleotide” refers to a nucleic acid molecule and includes genomic DNA, cDNA, RNA, mRNA, mixed polymers, recombinant nucleic acids, fragments and variants thereof, and the like. A fragment of a polynucleotide useful in the present invention is at least 10, preferably at least 12, 14, 16, 18, 20, 25, 30, 35, 40, 45, 50, 75, or 100 contiguous sequences of a reference polynucleotide. Nucleotides. The polynucleotide of the present invention includes a sense strand and an antisense strand. The polynucleotide of the present invention may be a naturally occurring polynucleotide or a non-naturally occurring polynucleotide. As used herein, “synthesized polynucleotide” refers to a polynucleotide made purely by chemical methods, apart from enzymatic methods. Thus, a “fully” synthesized DNA sequence is created entirely by chemical means, and a “partially” synthesized DNA is a DNA fragment in which only a portion of the resulting DNA is produced by chemical means. Include. The polynucleotide of the present invention may be single-stranded or double-stranded. The polynucleotides of the present invention may be chemically modified and may comprise non-natural nucleotide bases or derived nucleotide bases, as will be readily appreciated by those skilled in the art. Such modifications include, for example, labeling, methylation, substitution of one or more nucleotides with analogs, internucleotide modifications, such as uncharged bonds (eg, methylphosphonates, phosphotriesters, phosphoramidates, Carbamates, etc.), charged bonds (eg, phosphorothioate, phosphorodithioate, etc.), pendant moieties (eg, polypeptides, etc.), intercalators (eg, acridine, psoralen, etc.), chelators, alkylators, and modified bonds (eg, α -Anomeric nucleic acid etc.). Also included are synthetic molecules that mimic polynucleotides in their ability to bind to a specified sequence through hydrogen bonding and other chemical interactions. Such molecules are known in the art and include, for example, molecules in which a peptide bond replaces a phosphate bond in the molecular backbone.

The polynucleotides of the invention include those encoding the polypeptide sequences of SEQ ID NO: 2 (human CXCR4), SEQ ID NO: 4 (human LEC3) and SEQ ID NO: 6 (human EDNRA). In some embodiments, the polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 1 (human CXCR4), SEQ ID NO: 4 (human LEC3), and SEQ ID NO: 6 (human EDNRA). A polynucleotide may contain mutations that result in amino acid substitutions, whether conservative or non-conservative. Mutations can be introduced into the nucleic acid sequences of the present invention by standard techniques such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions may be made at one or more predicted non-essential amino acid residues. A “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been defined in the art. These families include basic side chains (eg, lysine, arginine, and histidine), acidic side chains (eg, aspartic acid, glutamic acid), uncharged polar side chains (eg, glycine, asparagine, glutamine, serine, threonine) , Tyrosine, cysteine), nonpolar side chains (eg, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (eg, threonine, valine, isoleucine), and aromatic side chains ( For example, amino acids having tyrosine, phenylalanine, tryptophan, histidine). Thus, a predicted nonessential amino acid residue is replaced by another amino acid residue from the same side chain family. Alternatively, mutations can be introduced randomly along all or part of the coding sequence, for example by saturation mutation, and the resulting mutant is biological to identify variants that retain activity. Can be screened for activity. Following mutation, the encoded protein may be expressed by any recombinant technique known in the art, and the activity of the protein can be determined.

Nucleotide sequences are presented by single strands only in the 5'-3 'direction from left to right. Nucleotides are represented in the manner recommended by the IUPAC-IUB Biochemical Nomenclature Commission.

In some embodiments of the invention, the mutation will not significantly alter the post-translational modification or biological activity of the CXCR4, LEC3 and EDNRA proteins.

B. Expression vector

Another aspect of the invention relates to vectors, preferably expression vectors, containing nucleic acids encoding zebrafish CXCR4, LEC3 and EDNRA polypeptides, or derivatives, fragments, analogs or homologues thereof. As used herein, the term “vector” refers to a nucleic acid molecule capable of transporting a nucleic acid to which it is linked. One type of vector is a “plasmid”, which refers to a circular double stranded DNA loop into which additional DNA segments can be joined. Another type of vector is a viral vector, in which the added DNA segment can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (eg, bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (eg, non-episomal mammalian vectors) are integrated into the host cell genome upon introduction into the host cell, and thereby are replicated along with the host genome. In addition, certain vectors can direct the expression of genes to which they are operably linked. Such vectors are referred to herein as “expression vectors”. In general, expression vectors useful in recombinant DNA technology often exist in the form of plasmids. In the present specification, “plasmid” and “vector” can be used interchangeably as the plasmid is the most commonly used form of vector. However, the present invention is intended to include such other forms of expression vectors, such as viral vectors (eg, replication defective retroviruses and adeno-associated viruses), which serve an equivalent function.

The recombinant expression vector of the present invention comprises the nucleic acid of the present invention, which is in a form suitable for expression of the nucleic acid in a host cell. This means that the recombinant expression vector contains one or more regulatory sequences that are chosen based on the host cell used for expression and are operably linked to the nucleic acid sequence to be expressed. In a recombinant expression vector, “operably linked” means in a manner that allows the nucleotide sequence of interest to express the nucleotide sequence (eg, in an in vitro transcription / translation system or when the vector is introduced into a host cell). It is intended to mean to be bound to regulatory sequences (in the host cell when done). The term “regulatory sequence” is intended to include promoters, enhancers and other expression control elements (eg, polyadenylation signals). Such regulatory sequences are described, for example, by Goeddel; GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Regulatory sequences include sequences that direct constitutive expression of nucleotide sequences in many host cells, and sequences that direct expression of nucleotide sequences only in certain host cells (eg, tissue specific regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression vector may depend on factors such as the choice of the host cell to be transformed and the level of expression of the desired protein. An expression vector of the invention is introduced into a host cell and thereby a protein or peptide (eg, CXCR4, LEC3) including a fusion protein or peptide encoded by a nucleic acid as described herein from any vertebrate species. And EDNRA polypeptides, CXCR4, LEC3 and EDNRA mutant forms, fusion proteins, etc.). In some embodiments, CXCR4, LEC3 or EDNRA is obtained from a mammal. In some other embodiments, CXCR4, LEC3 or EDNRA is obtained from a mouse or rat. In some other embodiments, CXCR4, LEC3 or EDNRA is obtained from a human. In some other embodiments, CXCR4, LEC3 or EDNRA is obtained from zebrafish. A comparison of the amino acid sequences of human and zebrafish CXCR4, LEC3 and EDNRA is shown in FIG. 2, FIG. 4 and FIG.

The recombinant expression vectors of the present invention can be designed for the expression of vertebrate CXCR4, LEC3 and EDNRA in prokaryotic or eukaryotic cells. For example, zebrafish CXCR4, LEC3 and EDNRA can be expressed in bacterial cells such as E. coli, insect cells (using baculovirus expression vectors), yeast cells or mammalian cells. Suitable host cells include: Goeddel; GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990) for further discussion. The recombinant expression vector may also be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.

Protein expression in prokaryotes is often carried out in E. coli with vectors containing constitutive or inducible promoters that direct the expression of fusion or non-fusion proteins. A fusion vector adds a number of amino acids to the amino terminus of the protein encoded therein, usually a recombinant protein. Such fusion vectors typically have three purposes: (1) enhance the expression of the recombinant protein; (2) increase the solubility of the recombinant protein; and (3) a ligand in affinity purification. Helps to purify the recombinant protein by acting as Often, in fusion expression vectors, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to allow separation of the recombinant protein from the fusion moiety and subsequent purification of the fusion protein. Such enzymes and their cognate recognition sequences include Factor Xa, thrombin and enterokinase. A typical fusion expression vector is pGEX (Pharmacia Biotech Inc .; Smith and Johnson (1988) Gene 67, which fuses glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein. : 31-40), pMAL (New England Biolabs, Beverly, MA.) And pRIT5 (Pharmacia, Piscataway, NJ).

Suitable inducible non-fused E. coli expression vectors include pTrc (Amann et al., (1988) Gene 69: 301-315) and pET11d (Studer et al., GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic S Calif. (1990) pp. 60-89).

One strategy to maximize recombinant protein expression in E. coli is to express the protein in a host bacterium that has been impaired in its ability to proteolytically cleave the recombinant protein. See, Gottesman, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990) 119-128. Another strategy is to change the nucleic acid sequence of the nucleic acid inserted into the expression vector so that individual codons for each amino acid are preferentially utilized in E. coli (Wada et al., (1992) Nucleic acids Res. 20: 2111-2118). Such alteration of the nucleic acid sequences of the present invention can be performed by standard DNA synthesis techniques.

In another embodiment, the vertebrate CXCR4, LEC3 and EDNRA expression vectors are yeast expression vectors. Examples of vectors for expression in Saccharomyces cerevisiae include pYepSec1 (Baldari, et al., (1987) EMBO J. 6: 229-234), pMFa (Kurjan and Herskowitz, (1982) Cell 30: 933). -943), pJRY88 (Sechutz et al., (1987) Gene 54: 113-123), pYES2 (Invitrogen Corporation, San Diego, CA), and picZ (Invitrogen Corp. San Diego, CA).

Vertebrate CXCR4, LEC3 and EDNRA can also be expressed in insect cells using baculovirus expression vectors. A baculovirus vector that can be used for expression of proteins in cultured insect cells (eg, Spodoptera frugiperda SF9 cells) is the pAc series (Smith et al., (1983) Mol. Cell. Biol. 3: 2156. -2165) and the pVL series (Lucklow and Summers (1989) Virology 170: 31-39).

In yet other embodiments, the nucleic acids of the invention are expressed in mammalian cells using mammalian expression vectors. Examples of mammalian expression vectors include pCDM8 (Seed (1987) Nature 329: 840) and pMT2PC (Kaufman et al., (1987) EMBO J. 6: 187-195). When used in mammals, the expression vector's control functions are often provided by viral regulatory elements. For example, the promoters typically used are derived from polyoma, adenovirus 2, cytomegalovirus and simian virus 40. Representative examples of promoters include, but are not limited to, controlling LTR or SV40 promoter, E. coli, lac or trp, the λ phage P _L promoter and a prokaryotic or eukaryotic cells or their viruses gene expression Other promoters are known. Other suitable expression systems for both prokaryotic and eukaryotic cells are described, for example, in Sambrook et al. , MOLECULAR CLONING: A LABORATORY MANUAL 2nd ed. , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N .; Y. , 1989, Chapters 16 and 17. Many suitable vectors and promoters are known to those skilled in the art and are commercially available. The following vectors are provided as examples; Bacteria: pQE70, pQE60, pQE-9 (Qiagen), pBS, pD10, pagesscript, psiX174, pBluescript SK, pbsks, pNH8A, pNH16a, pNH18A, pNH46A (Strak99; -3, pDR540, pRITS (Pharmacia); eukaryotes: pWLNEO, pSV2CAT, pOG44, pXT1, pSG (Stratagene); pSVK3, pBPV, pMSG, pSVL (Pharmacia). However, other plasmids and vectors can be used as long as they are replicable and viable in the host. In addition, the complete mammalian transcription unit and selectable marker may be inserted into a prokaryotic plasmid. The resulting vector is then amplified in bacteria and then transfected into cultured mammalian cells. Examples of this type of vector include pTK2, pHyg and pRSVneo.

In other embodiments, the recombinant mammalian expression vector can preferentially direct nucleic acid expression in specific cell types (eg, tissue-specific regulatory elements are used to express the nucleic acid). ). Tissue specific regulatory elements are known in the art. Non-limiting examples of suitable tissue specific regulatory elements include albumin promoters (liver specific; Pinkert et al., (1987) Gene Dev. 1: 268-277), lymphoid specific promoters (Callame and Eaton). (1988) Adv. Immunol. 43: 235-275), in particular T cell receptors (Winoto and Baltomore (1989) EMBO J. 8: 729-733) and immune globulin (Banerji et al. (1983) Cell 33: 729- 740; Queen and Baltimore (1983) Cell 33: 741-748), neuron specific promoters (eg, neurofilament promoters; Byme and Ruddle (1989) P roc.Natl.Acad.Sci.USA86: 5473-5477), pancreas specific promoter (Edrund et al., (1985) Science 230: 912-916) and mammary gland specific promoter (eg, whey promoter; , 873,316 and European Patent Application Publication No. 264,166). Further, developmentally regulated promoters include, for example, mouse hox promoter (Kessel and Gross (1990) Science 249: 374-379) and α-fetoprotein promoter (Campes and Tilghman (1989) Gene Dev. 3: 537-546). Is included. Selection of appropriate vectors and promoters is well known within the level of ordinary skill in the art.

The expression vector may also contain a ribosome binding site for initiation of translation and a transcription terminator. The vector may also include appropriate sequences for amplifying expression. Further, in some embodiments, the expression vector is a phenotypic characteristic for selection of transformed host cells, such as dihydrofolate reductase (DHFR) or neomycin (NEO) resistance for eukaryotic cell culture, or It contains one or more selectable marker genes to provide tetracycline (TET) or ampicillin (AMP) resistance in E. coli.

Furthermore, the present invention provides a recombinant expression vector comprising a DNA molecule of the present invention cloned in an expression vector in the antisense orientation. That is, the DNA molecule is operably linked to regulatory sequences in a manner that allows expression of RNA molecules that are antisense to vertebrate CXCR4, LEC3 and EDNRA mRNA (by transcription of the DNA molecule). Regulatory sequences that are operably linked to nucleic acids cloned in the antisense orientation, such as viral promoters and / or enhancers, may be chosen that direct the continuous expression of antisense RNA molecules in various cell types, or Regulatory sequences that direct constitutive, tissue specific or cell type specific expression of the antisense RNA may be selected. Antisense expression vectors may exist in the form of recombinant plasmids, phagemids or attenuated viruses in which antisense nucleic acids are produced under the control of highly efficient regulatory regions, and this activity is determined by the cell type into which the vector is introduced. can do. In the discussion of the regulation of gene expression using antisense genes, see Weintraub et al. (1986) "Antisense RNA as a molecular tool for genetic analysis," Reviews-Trends in Genetics, Vol. 1 (1): 22-25.
C. Antisense oligonucleotide

Antisense oligonucleotides may be used as therapeutic moieties in the treatment of disease states in animals and humans. Antisense oligonucleotide drugs, including ribozymes, have been safely and effectively administered to humans, and many clinical trials are currently underway. Thus, it is established that oligonucleotides can be useful therapeutic modalities that can be configured to be useful in treatment methods for the treatment of cells, tissues and animals, particularly humans.

In the context of the present invention, the term “oligonucleotide” refers to an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mimetics thereof. The term includes oligonucleotides consisting of naturally occurring nucleobases, sugars and internucleoside (backbone) covalent bonds, as well as oligonucleotides having non-naturally occurring moieties that function similarly. Such modified or substituted oligonucleotides are often preferred over the original form because of the desired properties, such as enhanced cellular uptake, enhanced affinity for nucleic acid targets and increased stability in the presence of nucleases.

Antisense oligonucleotides are contemplated by the present invention, but other oligomeric antisense compounds are also included, including but not limited to oligonucleotide mimetics as described below. Antisense compounds according to the present invention preferably comprise about 8 to about 50 nucleobases (ie, about 8 to about 50 linked nucleosides). In some embodiments, the antisense oligonucleotide comprises about 8 to about 15 nucleobases. In other embodiments, the antisense oligonucleotide comprises about 16 to about 25 nucleobases. In other embodiments, the antisense oligonucleotide comprises about 20 to about 30 nucleobases. In other embodiments, the antisense oligonucleotide comprises about 25 to about 35 nucleobases. In other embodiments, the antisense oligonucleotide comprises about 30 to about 45 nucleobases. In other embodiments, the antisense oligonucleotide comprises about 40 to about 50 nucleobases. Antisense compounds include ribozymes, external guide sequence (EGS) oligonucleotides (oligozymes), and other short catalytic RNAs or catalytic oligonucleotides that hybridize to and modulate the expression of target nucleic acids. .

As is known in the art, a nucleoside is a base-sugar combination. The base portion of the nucleoside is normally a heterocyclic base. The two most common types of such heterocyclic bases are purines and pyrimidines. A nucleotide is a nucleoside that further comprises a phosphate group covalently linked to the sugar portion of the nucleoside. For those nucleosides that include a pentofuranosyl sugar, the phosphate group can be linked to either the 2 ', 3' or 5 'hydroxyl moiety of the sugar. In the formation of oligonucleotides, phosphate groups covalently link adjacent nucleosides to other nucleosides to form linear polymer compounds. In turn, each end of the linear polymer structure may be further joined to form a cyclic structure, although open linear structures are generally preferred. In oligonucleotide structures, phosphate groups are usually directed to the formation of the internucleoside backbone of the oligonucleotide. The normal linkage or backbone of RNA and DNA is a 3'-5 'phosphodiester linkage.

Particular examples of suitable antisense compounds useful in the present invention include oligonucleotides containing modified backbones or non-natural internucleoside linkages. As defined herein, oligonucleotides having modified backbones include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone. For purposes of this specification, and as sometimes cited in the art, modified oligonucleotides that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleotides.

In some embodiments, the modified oligonucleotide backbone comprises, for example, phosphorothioate, chiral phosphorothioate, phosphorodithioate, phosphotriester, aminoalkyl phosphotriester, 3′-alkylene phosphonate, 5′-alkylene phosphonate and chiral phosphonate. Containing methyl and other alkyl phosphonates, phosphinates, phosphoramidates including 3'-aminophosphoramidates and aminoalkyl phosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters Selenophosphates and boranophosphates having normal 3'-5 'linkages, their 2'-5' linkage analogues, and one or more internucleotide linkages of 3'-3 ', 5'-5' or 2 Including those of them having an inverting polarity is to 2 'linkage. Suitable oligonucleotides with inversion polarity are single 3 ′ to 3 ′ linkages in 3′-major internucleotide linkages, ie single inverted nucleoside residues that may be basic (losing nucleobases). Or have a hydroxyl group instead).

In other suitable oligonucleotide mimetics, the linkage between both sugars and nucleosides, ie the backbone of the nucleotide unit, is replaced by a new group. Base units are maintained for hybridization with the appropriate nucleic acid target compound. One such oligomeric compound, an oligonucleotide mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar skeleton of the oligonucleotide is replaced by an amide containing a skeleton, in particular an aminoethylglycine skeleton. The nucleobase is retained and attached directly or indirectly to the aza nitrogen atom of the backbone amide moiety. Representative US patents that teach the preparation of PNA compounds include, but are not limited to, US Pat. Nos. 5,539,082; 5,714,331; and 5,719,262. Each of which is incorporated herein by reference. Further teachings of PNA compounds can be found in Nielsen et al. (1991) Science 254: 1497-1500.

Modified oligonucleotides may also contain one or more substituted sugar moieties.

Further modifications include Locked Nucleic Acids (LNA) in which the 2'-hydroxyl group is attached to the 3 'or 4' carbon atom of the sugar ring to form a bicyclic sugar moiety. The bond is preferably a methylene (-CH2-) n group bridging a 2 'oxygen atom and a 4' carbon atom, where n is 1 or 2, and its preparation is described in WO 98 / 39352 and WO 99/14226.

Other modifications include 2'-methoxy _(2'-OCH 3), 2'- aminopropoxy _{(2'-OCH 2 CH 2 CH} 2 NH 2), 2'- allyl _(2'-CH 2 -CH = _CH 2), including 2'-O- allyl _{(2'-O-CH 2 -CH} = CH 2) and 2'-fluoro and (2'-F). The 2'-modification may be in the arabino (up) position or ribo (down) position. A preferred 2'-arabino modification is 2'-F. Similar modifications may also be made at other positions in the oligonucleotide, specifically the 3 ′ position of the sugar at the 3 ′ terminal nucleotide or the 5 ′ position of the 2′-5 ′ linked oligonucleotide and the 5 ′ terminal nucleotide. Good. Oligonucleotides may also have sugar mimetics such as cyclobutyl moieties instead of pentofuranosyl sugars.

Oligonucleotides also include nucleobases (often referred to in the art simply as “bases”) modifications or substitutions. As used herein, “unmodified” or “natural” nucleobases include purine bases adenine (A) and guanine (G), and pyrimidine bases thymine (T), cytosine (C) and uracil (U). including. Modified nucleobases are other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, adenine and guanine 6-methyl. And other alkyl derivatives, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (—C═C—CH ₃ ) uracil And other alkyl derivatives of cytosine and pyrimidine bases, 6-azouracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-hydroxyl and 8-substituted Adenine and guanine, 5-halo, especially 5-bromo, 5- Rifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-aminoadenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and Contains 3-deazaguanine and 3-deazaadenine. Further modified nucleobases include tricyclic pyrimidines such as phenoxazine cytidine (1H-pyrimido [5,4-b] [1,4] benzoxazin-2 (3H) -one), phenothiazine cytidine (1H-pyrimido [5, 4-b] [1,4] benzothiazin-2 (3H) -one), G-clamps, eg substituted phenoxazine cytidine (eg 9- (2-aminoethoxy) -H-pyrimido [5,4-b] [1,4] benzoxazin-2 (3H) -one), carbazole cytidine (2H-pyrimido [5,4-b] indol-2-one), pyridoindole cytidine (H-pyrido [3 ′, 2 ′): 4,5] pyrrolo [2,3-d] pyrimidin-2-one). Modified nucleobases may also include those in which purine or pyrimidine bases are replaced by other heterocyclic compounds such as 7-deazaadenine, 7-deazaguanosine, 2-aminopyrimidine and 2-pyridone. Additional nucleobases are those disclosed in US Pat. No. 3,687,808, THE CONCISE ENCYCLOPEDIA OF POLYMER SCIENCE AND ENGINEERING, pages 858-859, Kroschwitz, J. et al. J. et al. , Ed. Disclosed in John Wiley & Sons, 1990, Englisch et al. And those disclosed by Angelwandte Chemie, International Edition, 1991, 30, 613, and Sanghvi, Y. et al. S. , Chapter 15, ANTIENSE RESEARCH AND APPLICATIONS, pages 289-302, Crooke, S .; T.A. and Lebleu, B .; ed. , CRC Press, 1993. Certain of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds of the invention. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to enhance the stability of nucleic acid duplexes by 0.6-1.2 ° C. (Sangvi, YS, Crooke, ST and Lebleu, B. et al. eds., ANTISENSE RESEARCH AND APPLICATIONS, CRC Press, Boca Raton, 1993, pp. 276-278), and more particularly when combined with the 2'-O-methoxyethyl sugar modification, currently preferred base substitutions It is a thing.

Other modifications of the oligonucleotides of the invention involve chemically linking the oligonucleotide with one or more moieties or conjugates that enhance the activity, cell partitioning or cellular uptake of the oligonucleotide. The compounds of the present invention may include a linking group covalently bonded to a functional group, eg, a primary or secondary hydroxyl group. The linking groups of the present invention include intercalators, polyamines, polyamides, polyethylene glycols, polyethers, groups that enhance the pharmacodynamic properties of the oligomer, and groups that enhance the pharmacokinetic properties of the oligomer. Typical linking groups include cholesterol, lipids, phospholipids, biotin, phenazine, foracin, phenanthridine, anthraquinone, acridine, rhodamine, coumarin and dyes. In the context of the present invention, groups that enhance pharmacodynamic properties include groups that improve oligomer uptake, enhance oligomer resistance to degradation, and / or enhance sequence-specific hybridization with RNA. . In the context of the present invention, groups that enhance pharmacokinetic properties include groups that improve oligomer uptake, distribution, metabolism or excretion. The binding moiety includes, but is not limited to, a lipid moiety, such as a cholesterol moiety (Letsinger et al., (1989) Proc. Natl. Acad. Sci. USA 86: 6553-6556), cholic acid (Manoharan et al. (1994) Bioorg. Med. Chem. Let. 4: 1053-1060), thioethers such as hexyl-S-trityl thiol (Manoharan et al., (1992) Ann. NY Acad. Sci. 660: 306-. 309; Manoharan et al., (1993) Bioorg. Med. Chem. Let. 3: 2765-2770), thiocholesterol (Oberhauser et al., (1992) Nucl. Acids. Res.20: 533-538), aliphatic chains such as dodecanediol or undecyl residues (Saison-Behmoaras et al., (1991) EMBO J. 10: 1111-1118; Kabanov et al., (1990) FEBS Lett 259: 327-330; Svinarchuk et al., (1993) Biochimie 75: 49-54), phospholipids such as dihexadecyl-rac-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero. -3-H-phosphonate (Manoharan et al., (1995) Tetrahedron Lett. 36: 3655-1654; Shea et al., (1990) Nucl. Acids. Res. ), Palmityl moiety (Mishra et al., (1995) Biochim. Biophys. Acta 1264: 229-237), or octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., (1996) J. Pharmacol Exo. Ther. 277: 923-937). The oligonucleotides of the present invention are also active drug substances such as aspirin, warfarin, phenylbutazone, ibuprofen, suprofen, fenbufen, ketoprofen, (S)-(+)-planoprofen, carprofen, dansylsarcosine, 2, Binds to 3,5-triiodobenzoic acid, flufenamic acid, benzothiadiazide, chlorothiadiazide, diazepine, indomethicin, barbitalate, cephalosporin, sulfa drugs, antidiabetics, antibacterials or antibiotics May be.

It need not be uniformly modified for all positions of a given compound; in fact, more than one of the modifications may be incorporated in a single compound or even a single nucleotide within an oligonucleotide. The present invention also includes antisense compounds that are chimeric compounds. In the context of the present invention, a “chimeric” antisense compound or “chimera” is a group of two or more chemically made up of at least one monomer unit, ie a nucleotide in the case of an oligonucleotide compound. Antisense compounds containing different regions, in particular oligonucleotides. These oligonucleotides are typically modified at least 1 to provide the oligonucleotide with increased resistance to nuclease degradation, increased cellular uptake and / or increased binding affinity to the target nucleic acid. Contains one region. Additional regions of the oligonucleotide may serve as substrates for enzymes that can cleave RNA: DNA or RNA: RNA hybrids. For example, RNase H is a cellular endonuclease that cleaves the RNA strand of an RNA: DNA double helix. Thus, activation of RNase H results in cleavage of the RNA target, thereby greatly enhancing the efficacy of oligonucleotide suppression of gene expression. As a result, comparable results can often be obtained with shorter oligonucleotides when chimeric oligonucleotides are used compared to phosphorothioate deoxyoligonucleotides that hybridize to the same target region. Cleavage of the RNA target can be routinely detected by gel electrophoresis and, if necessary, can be detected by related nucleic acid hybridization techniques known in the art.

The chimeric antisense compound of the present invention may be formed as a complex structure of two or more oligonucleotides, modified oligonucleotides and / or oligonucleotide mimetics as described above. Such compounds are also referred to in the art as hybrids or gapmers.

Antisense compounds used according to the present invention may be conveniently and routinely made by the well-known technique of solid phase. Such synthetic equipment is sold by several vendors including, for example, Applied Biosystems (Foster City, Calif.). Any other means for such synthesis known in the art may additionally or alternatively be used. It is well known to use similar techniques to prepare oligonucleotides such as phosphorothioates and alkylated derivatives.

The antisense compounds of the present invention are synthesized in vitro and do not include antisense compositions of biological origin or genetic vector constructs designed to direct in vivo synthesis of antisense molecules. The compounds of the present invention may also be used to facilitate uptake, distribution and / or absorption of other molecules, molecular structures or compounds, eg, as liposomes, receptor targeting molecules, oral, anal, topical or other formulations. It may be mixed, encapsulated, bound or otherwise associated with the mixture.

D. Morpholino modified oligonucleotide

A specific form of antisense technology is morpholino oligonucleotides (Summerton, J. and D. Weller (1997) Antisense Ncul. Acid Drug Dev. 7: 187-195; Nasevicius, A. and SC Ekker ( 2000) Nat.Genet.26: 216-220; Yan, YK. Et al. (2002) Development 129: 5065-5079). Morpholino oligonucleotides are nonionic DNA analogs with altered backbone bonds compared to DNA or RNA, but follow Watson-Crick base pairs with complementary sequences. Typically, the morpholino is at least about 18-25 nucleobases in length, in certain embodiments, the morpholino is at least about 25-30 nucleobases, and in yet another embodiment, the morpholino is at least Nucleobases of about 30-35 or more in length. The length of morpholino as a tool for studying vertebrate development is the Ekker S. et al. C. (2000) Yeast 17: 302-306, which is fully described in this recent disclosure, the disclosure of which is incorporated herein by reference.

Morpholino does not become a substrate for RNase H and forms an RNA-morpholino hybrid that is not degraded. Ekker and co-workers report that fluorescently labeled morpholino oligonucleotides can be injected into sphere stage zebrafish embryos and achieve a homogeneous distribution. A morpholino oligomer targeted to the start codon for green fluorescent protein (GFP) blocked GFP expression, whereas a control oligomer complementary to GFP did not. This confirmed that morpholino oligomers can clearly block gene expression in a sequence specific manner. Ekker and co-workers also reported suppression of several endogenous zebrafish genes.

The present invention provides morpholino modified oligonucleotides that target the 5 'untranslated region of CXCR4, LEC3 or EDNRA polynucleotides or the splice sites of CXCR4, LEC3 or EDNRA polynucleotides. Such morpholino modified oligonucleotides are effective in suppressing the expression of CXCR4, LEC3 or EDNRA and interfere with angiogenesis in animals treated with these modified oligonucleotides.

Morpholino is highly nonpolar. Thus, modified or unmodified morpholino oligos may be administered in combination with any known delivery carrier / vector that facilitates delivery of morpholino oligos into cells / tissues.

Morpholino (and other oligonucleotides) are also used by Multon, H. et al. M.M. et al. (2004) Bioconjugate Chem. 15: 290-299 may be conjugated with an arginine rich peptide. This is incorporated herein by reference in its entirety.

E. Delivery of nucleic acids to cells:

The nucleic acid constructs of the invention may be delivered by any means known in the art. In certain embodiments, the nucleic acid is delivered into the cell using ex vivo means. In other embodiments, the nucleic acid is delivered into the cell using in vivo means.

In ex vivo gene therapy, cells are removed from a host organism, such as a human, prior to experimental manipulation. These cells are then transfected in vitro with nucleic acids using methods well known in the art. These genetically engineered cells are then reintroduced into the host organism. On the other hand, in vivo gene therapy approaches do not require removal of target cells from the host organism. Rather, the nucleic acid may be complexed with reagents such as liposomes or retroviruses and subsequently administered to target cells in the organism using known methods. For example, Morgan et al. (1987) Science 237: 1476, 1987; Gerard et al. (1993) Nat. Genet. 3: 180.

Several different methods for transfecting cells can be used for either ex vivo or in vivo gene therapy approaches. Known transfection methods may be classified according to the agent used to deliver the selected nucleic acid into the target cell. These transfection agents include virus-dependent, lipid-dependent, peptide-dependent, and direct transfection (“naked DNA”) approaches. Other approaches used for transfection include calcium coprecipitation and electroporation.

Viral approaches use genetically engineered viruses to infect host cells, thereby “transfecting” cells with exogenous nucleic acids. Known viral vectors include recombinant viruses whose examples have already been disclosed, including poxviruses, herpesviruses, adenoviruses and retroviruses. Such recombinants can carry heterologous genes under the control of promoter or enhancer elements and can cause their expression in host cells infected with the vector. Vaccinia and other types of recombinant viruses are described in Macckett et al. (1994) J. Am. Virol. 49: 3. Also, Kotani et al. (1994) Hun. Gene Ther. See also 5:19.

Non-viral vectors such as liposomes may also be used as mediators for nucleic acid delivery in gene therapy. Compared to viral vectors, liposomes are safe, have a relatively high capacity, have a relatively low toxicity, can deliver a variety of nucleic acid-based molecules, and are relatively non-immunogenic. See Felger, P .; L. and Ringold, G .; M.M. (1989) Nature 337: 387-388. Among these vectors, cationic liposomes have been most studied due to their effectiveness in mediating mammalian cell transfection in vitro. One technique known as lipofection uses a lipoplex made from nucleic acids and cationic lipids that facilitate transfection into cells. Lipid / nucleic acid complexes fuse or otherwise disrupt the protoplasm or endosomal membrane and transport the nucleic acid into the cell. Lipofection is typically more efficient in introducing DNA into cells than the calcium phosphate transfection method. Change et al. (1988) Focus 10:66.

One known protein-dependent approach involves the use of polylysine mixed with nucleic acid. The polylysine / nucleic acid complex is then exposed to target cells for entry. See, for example, Verma and Somia (1997) Nature 389: 239; Wolff et al. (1990) Science 247: 1465.

The “naked DNA” transfection approach involves a method in which the nucleic acid is administered directly in vivo. See US Pat. No. 5,837,693 to German et al. The administration of the nucleic acid can be performed by injection into the interstitial space of tissues in organs, such as muscle or skin, by direct introduction into the bloodstream, the desired body cavity, or also by inhalation. In these so-called “naked DNA” approaches, the nucleic acid is injected or otherwise contacted with the animal without any adjuvant. It has been reported that injection of free ("naked") plasmid DNA directly into body tissues such as skeletal muscle or skin results in protein expression. See Ulmer et al. (1993) Science 259: 1745-1749; Wang et al. (1993) Proc. Natl. Acad. Sci. USA 90: 4157-4160; Raz et al. (1994) Proc. Natl. Acad. Sci. USA 91: 9519-9523.

Electroporation is another transfection method. See Szoka, Jr. U.S. Pat. No. 4,394,448 to U.S. Pat. No. 4,619,794 to Hauser. Application of short high voltage electrical pulses to various animal and plant cells results in the formation of nanometer sized pores in the plasma membrane. DNA can enter directly into the cytoplasm through these small pores or as a result of redistribution of membrane components with pore closure.

2. Host cell

Another aspect of the invention relates to a host cell into which a recombinant expression vector of the invention has been introduced. The terms “host cell” and “recombinant host cell” are used interchangeably herein. Such terms refer not only to specific cells, but also to the progeny or potential progeny of such cells. Since certain alterations may occur in subsequent generations due to mutations or environmental influences, such progeny may not actually be identical to the parent cell, but the term still used herein Is included in the range.

The host cell can be any prokaryotic or eukaryotic cell. For example, a vertebrate CXCR4, LEC3 or EDNRA polypeptide can be expressed in bacterial cells such as E. coli, insect cells, yeast or mammalian cells (eg, Chinese hamster ovary cells (CHO) or COS cells). Other suitable host cells are known to those skilled in the art.

Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. As used herein, the terms “transformation” and “transfection” refer to foreign nucleic acids in a host cell, including calcium phosphate or calcium chloride coprecipitation, DEAE-dextran mediated transfection, lipofection or electroporation. It is intended to refer to various technically recognized techniques for introducing (eg, DNA). Suitable methods for transforming or transfecting host cells are described by Sambrook et al. (MOLECULAR CLONING: A LABORATORY MANUAL 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory, Cold Spring Harbor Lab. ) And other laboratory manuals.

For stable transfection of mammalian cells, it is known that only a small portion of cells can integrate foreign DNA into their genome, depending on the expression vector and transfection technique used. In order to identify and select these integrants, a gene that encodes a stable marker (eg, resistance to antibiotics) is generally introduced into the host cells along with the gene of interest. Various selectable markers include those that confer resistance to drugs such as G418, hygromaosin and methotrexate. Nucleic acid encoding a selectable marker can be introduced into a host cell on the same vector encoding vertebrate CXCR4, LEC3 or EDNRA or can be introduced on a separate vector. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (eg, cells that have incorporated a selectable marker gene survive as other cells die). Will).

A host cell of the invention in culture, such as a prokaryotic or eukaryotic host cell, can be used to produce (ie, express) a vertebrate CXCR4, LEC3 or EDNRA polypeptide. Thus, the present invention further provides methods for producing vertebrate CXCR4, LEC3 and EDNRA polypeptides using the host cells of the present invention. In one embodiment, the method comprises recombinant expression encoding a host cell of the invention (vertebrate CXCR4, LEC3 or EDNRA) in a suitable medium such that a vertebrate CXCR4, LEC3 or EDNRA polypeptide is produced. Culturing the vector). In other embodiments, the method further comprises isolating vertebrate CXCR4, LEC3 or EDNRA from the medium or the host cell.

3. Transgenic animals:

The host cells of the present invention can also be used to create non-human transgenic animals. For example, in one embodiment, the host cell of the invention is a fertilized oocyte or embryonic stem cell into which a sequence encoding vertebrate CXCR4, LEC3 or EDNRA has been introduced. Such host cells then have an exogenous vertebrate CXCR4, LEC3 or EDNRA sequence in their genome or a homologous recombinant animal in which the endogenous vertebrate CXCR4, LEC3 or EDNRA sequence has been altered. It can be used to create introduced non-human transgenic animals. Such animals are useful for studying the function and / or activity of vertebrate CXCR4, LEC3 or EDNRA and for identifying and / or evaluating modulators of vertebrate CXCR4, LEC3 or EDNRA activity. As used herein, a “transgenic animal” is a non-human animal, in one embodiment the animal is a fish and in other embodiments the animal is a mammal (eg, such as a rat or mouse). Rodents), where one or more cells of the animal contain the transgene. Other examples of transgenic animals include non-human primates, sheep, dogs, cows, goats, chickens, amphibians and the like. A transgene is an exogenous DNA integrated into the genome of a cell from which the transgenic animal develops, and it remains in the genome of the mature animal and is encoded in one or more cell types or tissues of the transgenic animal. Guidance on the expression of gene products. As used herein, a “homologous recombinant animal” is a non-human animal, such as a mammal (eg, a mouse), in which an endogenous CXCR4, LEC3 or EDNRA gene is an endogenous gene, It has been altered by homologous recombination between exogenous DNA introduced into animal cells, eg, embryonic cells of an animal prior to animal development.

The transgenic animal of the present invention introduces a nucleic acid encoding a vertebrate CXCR4, LEC3 or EDNRA into the male pronucleus of a fertilized oocyte by, for example, microinjection or retroviral infection, It can be produced by growing oocytes in animals. Vertebrate of SEQ ID NO: 1 (human CXCR4), SEQ ID NO: 3 (human LEC3), SEQ ID NO: 5 (human EDNRA), SEQ ID NO: 7 (Danio CXCR4), SEQ ID NO: 9 (Danio LEC3), SEQ ID NO: 11 (Danio EDNRA) CXCR4, LEC3 or EDNRA cDNA sequences or artificial constructs can be introduced into the genome of non-human animals as a transgene. In some embodiments, a vertebrate CXCR4, LEC3 or EDNRA variant is introduced, wherein the vertebrate CXCR4, LEC3 or EDNRA lacks one or more domains or is wild-type Has substitutions of homologous chromosomes in the sequence. Alternatively, homologous chromosomes of vertebrate CXCR4, LEC3 or EDNRA genes may be isolated based on hybridization to vertebrate CXCR4, LEC3 or EDNRA cDNA (detailed above) and used as a transgene. Further, intron sequences and polyadenylation signals may be included in the transgene to enhance the transgene expression efficiency. A tissue-specific regulatory sequence may be operably linked to a vertebrate CXCR4, LEC3 or EDNRA transgene to direct expression of a vertebrate CXCR4, LEC3 or EDNRA polypeptide to a particular cell. Methods for producing transgenic animals, particularly animals such as mice, via embryo manipulation and microinjection are routine in the art, for example, US Pat. No. 4,736,866; 4,870. , 009 and 4,873,191; and Hogan 1986, In: MANIPULATING THE MOUSE EMBRYYO, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N .; Y. Described in. Similar methods are used for the production of other transgenic animals. A transgenic founder animal can be identified based on the presence of a vertebrate CXCR4, LEC3 or EDNRA transgene in its genome and / or expression of a vertebrate CXCR4, LEC3 or EDNRA mRNA in an animal tissue or cell. The transgenic founder animal can then be used for the breeding of additional animals carrying the transgene. Furthermore, transgenic animals carrying transgenes encoding vertebrate CXCR4, LEC3 or EDNRA may be further bred with other transgenic animals carrying other transgenes.

In order to create homologous recombinant animals, deletions, additions or substitutions were introduced to alter the vertebrate CXCR4, LEC3 or EDNRA gene, eg, functionally disrupted, of a vertebrate CXCR4, LEC3 or EDNRA gene A vector containing at least a portion is prepared. Vertebrate CXCR4, LEC3 or EDNRA genes are SEQ ID NO: 1 (human CXCR4), SEQ ID NO: 3 (human LEC3), SEQ ID NO: 5 (human EDNRA), SEQ ID NO: 7 (Danio CXCR4), SEQ ID NO: 9 (Danio LEC3), The cDNA of SEQ ID NO: 11 (Danio EDNRA) may be used. The vertebrate CXCR4, LEC3 or EDNRA may then be introduced into the genome of a mammal, or other vertebrate. In one embodiment, the vector is designed such that in homologous recombination, the endogenous vertebrate CXCR4, LEC3 or EDNRA gene is functionally disrupted (ie, no longer encodes a functional protein; "Called a vector).

Alternatively, the vector may be mutated in the endogenous vertebrate CXCR4, LEC3 or EDNRA gene or otherwise modified in homologous recombination to express endogenous vertebrate CXCR4, LEC3 or EDNRA polypeptide. May be designed to still encode functional proteins (eg, upstream regulatory regions may be altered to alter endogenous vertebrate CXCR4, LEC3 or EDNRA polypeptide expression). Is good). In homologous recombination vectors, the altered portion of the vertebrate CXCR4, LEC3 or EDNRA gene is flanked by additional vertebrate CXCR4, LEC3 or EDNRA gene nucleic acids at its 5 ′ and 3 ′ ends, and the exogenous carried on the vector. Homologous recombination occurs between the sex vertebrate CXCR4, LEC3 or EDNRA gene and the endogenous vertebrate CXCR4, LEC3 or EDNRA gene in embryonic stem cells. The additional adjacent vertebrate CXCR4, LEC3 or EDNRA nucleic acid is of sufficient length for successful homologous recombination with the endogenous gene. Typically, several kilobases of flanking DNA (at both 5 'and 3' ends) are included in the vector. For references, eg, description of homologous recombination vectors, see Thomas et al. (1987) Cell 51: 503. The vector is introduced into an embryonic stem cell line (eg, by electroporation) and the introduced vertebrate CXCR4, LEC3 or EDNRA gene is homologously recombined with the endogenous vertebrate CXCR4, LEC3 or EDNRA gene (See, eg, Li et al. (1992) Cell 69: 915). Methods for making transgenic non-human animals are well known in the art (for mice see Brinster et al., (1985) Proc. Natl. Acad. Sci. USA 82: 4438-42; US Pat. No. 4 No. 7,736,866, No. 4,870,009, No. 4,873,191, No. 6,127,598; Hogan, B., Manipulating The Mouse Embryo, Cold Spring Harbor Laboratory Press, Cold. Spring Harbor, NY, (1986); for homologous recombination, see, Capecchi (1989) Science 244: 1288-1292; Joyner et al. (1989) Nat. re338: 153-156; for particle bombardment, see US Pat. No. 4,945,050; for Drosophila, see Rubin and Spradling (1982) Science 218: 348-53, US Pat. No. 4,670. 388; see for transgenic insects, see Berghmer AJ et al. (1999) Nature 402: 370-37; for zebrafish, see Lin S. (2000) Methods Mol. Biol. 136: 375. -3830; see fish, amphibians and birds, see Houdebine and Chourout, (1991) Expertia 47: 397-905; see rats, Ham mer et al, (1990) Cell 63: 1099-1112; for embryonic stem (ES) cells, see TERATOCARCINOMAS AND EMBRYONIC STEM CELLS, A PRACTICAL APPROACH, EJ Robertson, ed., IRL Press (1987); For livestock, see, Pursel et al. (1989) Science 244: 1281-1288; for non-human animal clones, see, Wilmut, I. et al. (1997) Nature 385: 810-813, PCT Publication No. WO 97 / 07668 and WO97 / 07669; see Rekso et al., For recombinase systems for regulated transgene expression. (1992) Proc. Natl. Acad. Sci. 89: 6232-6236; U.S. Pat. No. 4,959,317 (for cre.loxP) and O'Gorman et al. (1991) Science 251: 1351-1355; US Pat. No. 5,654,182 (for FLP / FRT)). When the cre / loxP recombinase system is used to regulate transgene expression, animals containing both Cre recombinases and a transgene encoding the selected protein are required. Such animals, for example, through the construction of “double” transgenic animals, for example, contain two transgenic animals, one containing a transgene encoding the selected protein and one encoding a recombinase. Can be provided by crossing with the other containing the transgene.

The invention also provides mutant forms of dominant inhibition and constitutive activity of CXCR4, LEC3 and EDNRA transgenes. These mutant transgenes allow studies on these GPCRs and therapeutic potential. In some embodiments, an expression vector comprising a nucleic acid sequence encoding a dominant negative mutant form of CXCR4, LEC3 or EDNRA is introduced into a non-human embryo or cell, and the resulting transgenic animal or cell is Has dominant inhibitory expression of the subject GPCR.

Dominant inhibitory mutations may be introduced into a region of the protein sequence that confers a dominant inhibitory effect, for example by site-directed mutagenesis (by any means known in the art). For example, without limitation, it has been demonstrated that phenylalanine at amino acid position 303 (F303G and F303N) in the hamster α ₁ adrenergic receptor is involved in conferring a dominant inhibitory effect on this GPCR (Chen S. et al (2000) EMBO J. 19 (16): 4265-4271). Since this region of the GPCR protein is highly conserved, it will therefore generate a similar dominant negative receptor for GPCRs with conserved sequences (Chen S. et al (2000) EMBO J. 19). (16): 4265 at page 4270). This region is actually conserved in, for example, CXCR4, LEC3, EDNRA and the hamster α ₁ adrenergic receptor, histamine (H ₁ ), dopamine (D ₂ ), muscarin (M ₃ ), and angiotensin II (AT ₁ ). The Thus, mutations to this region, particularly to phenylalanine, boxed in FIG. 16, are useful for generating dominant-inhibiting mutants. Dosil M.M. et al (1998) Mol. Cell. As discovered by Biol18 (10): 5981-5991, further mutations in the extracellular domain of the GPCR may be directed to the carboxyl-terminal region of the extracellular domain of the GPCR or to Leavitt L. et al. M.M. et al (1999) Mol Gen. Genet. 261 (6): 917-932 closes in an area similar to that reported.

In some embodiments, an expression vector comprising a nucleic acid sequence encoding CXCR4, LEC3 or EDNRA is operably linked to a promoter that drives constitutive expression of the GPCR protein. These sequences are introduced into non-human embryos and the resulting transgenic animals have constitutive expression of the subject GPCR. Constitutive promoters are well known in the art and include, but are not limited to, SV40, cytomegalovirus promoter (eg, CMV5), human CMV enhancer / chicken b-actin promoter, mouse pgk promoter, and human ubiquitin C. Includes a promoter.

In other embodiments, the CXCR4, LEC3 or EDNRA protein has a substitution in the amino acid sequence leading to a constitutively active protein in a manner similar to that described in other GPCR literature. Is altered by site-directed mutagenesis. For example, but not limited to: Vischer H. F. et al. (2006) Trends Pharmacol. Sci. 27 (1): 56-63; Miura S. et al. et al. (2005) J. Org. Biol. Chem. 280 (18): 18237-18244; et al. (2005) Mol. Microbiol. 55 (2): 482-497; Zhang M.M. et al. (2005) Mol. Biol. Cell. 16 (2): 562-572; Cotecchia S .; et al. (2003) Assay Drug Dev. Technol 1 (2): 311-316; Behan D. et al. P. and D.D. T.A. Chalmers. (2001) Curr. Opin. Drug Discov. Dev4 (5): 548-560; Parnot C.I. et al. (2002) Trends Endocrinol. Metab. 13 (8): 336-343; and Egan C.I. et al. (1998) Ann. NY Acad. Sci. 861: 136-139.

In some embodiments, the promoter used to drive CXCR4, LEC3, or EDNRA expression may be tissue specific. This allows constitutive expression of the GPCR in selected tissues of the animal. Such constitutively expressed GPCRs are useful in screening for compounds that modulate GPCR activity for assessment of therapeutic potential. Tissue specific promoters are also well known in the art and include, but are not limited to, neuron-specific enolase (NSE) that promotes high expression in neurons; tubulin α1 (T-α1) that promotes high expression in neurons Glial fibrillary acidic protein (GFAP) which promotes high expression in astrocytes; myosin light chain-2 (MLC2) which promotes high expression in cardiomyocytes; preproendothelin-1 which promotes high expression in endothelial cells ( ET-1); tie (tie) that promotes high expression in endothelial cells; SM22a (SM22a) that promotes high expression in vascular smooth muscle cells; α1 antitrypsin (α1-AT) that promotes high expression in hepatocytes; Albumin (ALB) promotes high expression in hepatocytes; Including and renal proximal renal androgen responsive protein that promotes the high expression in tubular cells (KAP); side chain splitting enzymes that facilitate high expression in de-producing cells (SCC).

The present invention also provides CXCR4, LEC3 and EDNRA2, fusion proteins, where the fusion protein partners can be roughly or histologically visualized. Briefly, an expression construct is produced that has an in-frame junction between CXCR4, LEC3 or EDNRA and a protein that can be visualized or easily identified. Such fusion proteins include, for example, green fluorescent protein (GFP) and others well known in the art. Such constructs can be introduced into animals (eg, by homologous recombination) or cells (through various transfection techniques) and the G protein, subcellular localization, the physiological and pathophysiological aspects of these GPCR targets. Useful for studying molecular interactions with other signaling molecules such as and metabolic changes.

The present invention also provides a GFP fluorescent reporter construct driven by a CXCR4, LEC3 or EDNRA promoter. Such a construct is generated by operably linking a CXCR4, LEC3 or EDNRA promoter to a reporter such as GFP. Such constructs are introduced into animals (eg by homologous recombination) or into cells (through various transfection techniques) to facilitate studies of pharmaceutical or genetic regulation of the expression of these GPCRs. May be. For example, transgenic zebrafish expressing GFP under the control of promoters for CXCR4, LEC3 or EDNRA to identify therapeutic agents and gene modifiers that can regulate the expression levels of these GPCRs Useful to facilitate large scale drug screening or genetic screening. Promoters for CXCR4 are known in the art, see, eg, Haviv Y. et al. S. et al. (2004) Mol Cancer Ther. 3 (6): 687-691; Moriuchi M. et al. et al. (1999) AIDS Res. Hum. Retroviruses 15 (9): 821-827; Moriuchi M. et al. et al. (1999) J. MoI. Immunol. 162 (10): 5986-5992; also Caruz A. et al. et al. (1998) FEBS Lett. 426 (2): 271-278; Moriuchi M. et al. et al. (1997) J. MoI. Immunol. 159 (9): 4322-4329. Promoters for EDNRA are also known in the art, see, for example, Yamashita J. et al. et al. (1998) J. MoI. Biol. Chem. 273 (26): 15993-15999 and Yamashita J. et al. et al. (1995) J. MoI. Cardiovasc. Pharmacol. 26 Suppl3: S26-28.

4). Vertebrate polypeptides

The invention also provides vertebrate CXCR4, LEC3 and EDNRA polypeptides. CXCR4, LEC3 and EDNRA polypeptides, variants, fragments and antigenic portions thereof may be obtained from any vertebrate species. In certain embodiments, CXCR4, LEC3 and EDNRA polypeptides are obtained from mice or rats. In other embodiments, CXCR4, LEC3 and EDNRA polypeptides are obtained from humans. In other embodiments, CXCR4, LEC3 and EDNRA polypeptides are obtained from fish, such as zebrafish. In some embodiments, the CXCR4 polypeptide has an amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 8, or SEQ ID NO: 33. In some embodiments, the LEC3 polypeptide has the amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 10, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 27, or SEQ ID NO: 28. In some embodiments, the EDNRA polypeptide has an amino acid sequence of SEQ ID NO: 6, SEQ ID NO: 12, SEQ ID NO: 20, SEQ ID NO: 22, or SEQ ID NO: 23.

The terms “polypeptide”, “peptide” and “protein” are used interchangeably herein. “Polypeptide” refers to a polymer of amino acids without referring to a specific length. The polypeptides of the present invention include peptide fragments, derivatives and fusion proteins. The peptide fragment preferably has at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90 or 100 amino acids. Some peptide fragments of the present invention are biologically active. Biological activity includes activities related to immunogenicity, ligand binding and reference peptides. The immunogenic peptides and fragments of the present invention generate an epitope-specific immune response, where “epitope” refers to the immunogenic determinants of the peptide, and preferably at least 3, 5, 8, 9, Contains 10, 15, 20, 30, 40, 45 or 50 amino acids. Some immunogenic peptides of the present invention produce an immune response specific for that peptide. The polypeptides of the present invention include naturally occurring peptides and non-naturally occurring peptides. The term refers to modified polypeptides (in which case examples of such modifications include glycosylation, acetylation, phosphorylation, carboxylation, ubiquitination, labeling, etc.), analogs (eg, non-naturally occurring amino acids) , Substitution bonds, etc.) and functional mimetics. Various methods of labeling polypeptides are well known in the art and include radioisotopes such as ³² P or ³⁵ S, ligands that bind to labeled antiligands (eg, antibodies), fluorophores, chemiluminescent agents, Contains enzymes and anti-ligands.

As used herein, the term “amino acid” refers to a molecule that contains both an amino group and a carboxyl group. In certain embodiments, the amino acids are α-, β-, γ-, or δ-amino acids, including their stereoisomers and racemates. As used herein, the term “L-amino acid” refers to an α-amino acid having an L configuration around the α-carbon, ie, the general formula CH (COOH) (NH ₂ )-(Side chain) carboxylic acid. The term “D-amino acid” similarly refers to a carboxylic acid of the general formula CH (COOH) (NH ₂ ) — (side chain) having a D-configuration around the α-carbon. The side chain of an L-amino acid includes a naturally occurring part and a non-naturally occurring part. Non-naturally occurring (ie, unnatural) amino acid side chains are moieties that are used in place of naturally occurring amino acid side chains, for example, in amino acid analogs. Amino acid substitutes may be linked, for example, through their carbonyl group, their oxygen or carbonyl carbon, or their amino group, or a functional group present in their side chain moieties.

The amino acid sequence is shown from right to left in the amino (N) to carboxy (C) direction. N-terminal α-amino and C-terminal β-carboxy groups are not drawn in the sequence. Amino acids are represented in the manner recommended by the IUPAC-IUB Biochemical Nomenclature Commission, or amino acids are represented by their three letter code designations.

The present invention also contemplates the use of cell penetrating peptides. Cell penetrating peptides are described in neuronal degeneration (Borsello T, and C. Bonny (2004) “Use of cell-permissible peptides to prevent neurodegeneration”, Trends Mol. Med. 10 (5): 239-44). Fibrogenesis (Gordon D. J. et al. (2002) “Design and charac- terization of a membrane permeable N-methyl amino acid-containing peptide tatbet. 55) suppress the function of various pathways including It has been designed to. Methods for designing cell penetrating peptides are known in the art and are described, for example, in Du C. et al. et al. (1998) "Conformational and topological requirement of cell-permable peptide function" J. Pept. Res. 51: (3): 235-243. Cell penetrating peptides derived from CXCR4, LEC3 or EDNRA and / or VEGF are also used in the methods of the invention to inhibit angiogenesis in a subject and to treat angiogenesis related diseases and angiogenesis dependent tumors. May be. To determine which peptides are useful for such purposes, the screening techniques described herein can be used to routinely screen for functional cell penetrating peptides.

5. Various antibodies that specifically recognize vertebrate CXCR4, LEC3 or EDNRA

The present invention relates to vertebrate CXCR4, LEC3 or EDNRA, or the epitope is SEQ ID NO: 2, SEQ ID NO: 8, SEQ ID NO: 33, SEQ ID NO: 4, SEQ ID NO: 10, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 27, SEQ ID NO: 28 A compound comprising a CDR sequence that specifically recognizes a fragment of a vertebrate CXCR4, LEC3 or EDNRA comprising at least a portion of the amino acid sequence of SEQ ID NO: 6, SEQ ID NO: 12, SEQ ID NO: 20, SEQ ID NO: 22, or SEQ ID NO: 23 Antibodies (eg, monoclonal and polyclonal antibodies, single chain antibodies, chimeric antibodies, bivalent / bispecific antibodies, humanized antibodies, human antibodies and antibodies grafted with complementarity determining regions (CDRs)) provide.

Fab, Fab ', F (ab ') containing ₂ and _{F v,} antibody fragments are also provided by the present invention. When used to describe an antibody of the invention, the term “specific for” means that the variable portion of the antibody of the invention exclusively recognizes vertebrate CXCR4, LEC3 or EDNRA, and Show binding (ie in spite of possible presence of sequence identity, homology or similarity between vertebrate CXCR4, LEC3 or EDNRA and other GPCR family members in binding affinity) Vertebrate CXCR4, LEC3 or EDNRA can be distinguished from other different GPCRs due to measurable differences). “High binding affinity” refers to a binding affinity greater than about 5 × 10 ⁻⁸ M, preferably from about 5 × 10 ⁻⁸ M to about 5 × 10 ⁻¹² M, and in certain embodiments, the binding affinity is from about 5 × 10 ⁻⁹ M to about 5 × 10 M. ^-11 M, in certain embodiments, the binding affinity is from about 5 × 10 ⁻⁷ M to about 5 × 10 ⁻⁸ M, and in certain embodiments, the binding affinity is from about 5 × 10 ⁻⁸ M to about 5 × 10 ⁻⁹ M; In certain embodiments, the binding affinity is from about 5 × ^{10 −9} M to about 5 × ^{10 −10} M, and in certain embodiments, the binding affinity is from about 5 × ^{10 −10} M to about 5 × ^{10 −11} M.

Specific antibodies also interact with other proteins (eg, S. aureus protein A or other antibodies in ELISA technology) through interaction with sequences outside the variable region of the antibody, particularly in the constant region of the molecule. You can understand that Screening assays for determining the binding specificity of the antibodies of the present invention are well known and routinely performed in the art. For a comprehensive discussion of such assays, see Harlow et al. (Eds.), ANTIBODIES: A LABORATORY MANUAL; Cold Spring Harbor, N .; Y. 1988. See Chapter 6. If the antibody is specific for vertebrate CXCR4, LEC3 or EDNRA, an antibody that recognizes and binds to a fragment of vertebrate CXCR4, LEC3 or EDNRA is also contemplated. The antibodies of the present invention can be generated using any method well known in the art and routinely practiced.

In the production of polyclonal antibodies, various suitable host animals (eg, rabbits, goats, mice or other mammals) are immunized by injecting the native polypeptide, or a synthetic variant thereof, or the former derivative. May be. Suitable immunogenic preparations may include, for example, vertebrate CXCR4, LEC3 or EDNRA polypeptides expressed by recombinant techniques, or chemically synthesized vertebrate CXCR4, LEC3 or EDNRA polypeptides. The preparation further comprises an adjuvant. Various adjuvants used to enhance the immunological response include, but are not limited to, Freund's (complete and incomplete), metal gels (eg, aluminum hydroxide), surfactants (eg Lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, dinitrophenol, etc.), human adjuvants such as Bacille Calmette-Guerin and Corynebacterium parvum, or similar immunostimulants. If necessary, antibody molecules directed against vertebrate CXCR4, LEC3 or EDNRA can be isolated from mammals (eg from blood) and further well known techniques such as protein A chromatography to obtain IgG fractions. Can be purified.

As used herein, the term “monoclonal antibody” or “monoclonal antibody composition” refers to only one type of antigen binding site capable of immunoreacting with a particular epitope of vertebrate CXCR4, LEC3 or EDNRA. Refers to a population of antibody molecules containing. Thus, a monoclonal antibody composition typically displays a single binding affinity for a particular vertebrate CXCR4, LEC3 or EDNRA polypeptide with which it immunoreacts. For the preparation of monoclonal antibodies directed against a specific CXCR4, LEC3 or EDNRA polypeptide, or derivative, fragment, analog or homologue thereof, any technique provided for the production of antibody molecules by continuous cell line culture May be used. Such techniques include, but are not limited to, hybridoma technology (see, Kohler & Milstein, 1975 Nature 256: 495-497); trioma technology; human B cell hybridoma technology (see, Kozbor, et al., (1983). Immunol Today 4:72) and EBV hybridoma technology for producing human monoclonal antibodies (see Cole, et al,. Including. Human monoclonal antibodies may be utilized in the practice of the present invention, by using human hybridomas (see Cote, et al. (1983) Proc. Natl. Acad. Sci. USA 80: 2026-2030) or in vitro. Produced by transformation of human B cells with Epstein-Barr virus (see Cole, et al. (1985) In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96). Good.

In accordance with the present invention, several techniques can be applied for the production of single chain antibodies specific for vertebrate CXCR4, LEC3 or EDNRA polypeptides (see, eg, US Pat. No. 4,946,778). issue). In addition, several methods can be applied for the construction of Fab expression libraries (see, eg, Huse, et al. (1989) Science 246: 1275-1281), CXCR4, LEC3 or EDNRA polypeptides, or derivatives thereof, Allows rapid and efficient identification of monoclonal Fab fragments with the desired specificity for a fragment, analog or homolog. Non-human antibodies can be “humanized” by techniques well known in the art. See, for example, US Pat. No. 5,225,539. Antibody fragments containing idiotypes against CXCR4, LEC3 or EDNRA polypeptides include, but are not limited to: (i) F (ab ′) ₂ fragments produced by pepsin digestion of antibody molecules; (ii) F ( ab ′) Fab fragments generated by reducing disulfide bridges of ₂ fragments; (iii) Fab fragments generated by treatment of antibody molecules with papain and reducing agents; and (iv) Fv fragments. May be produced by known techniques.

In addition, recombinant and anti-vertebrate CXCR4, LEC3 or EDNRA antibodies, such as chimeric and humanized monoclonal antibodies comprising both human and non-human portions, which can be made using standard recombinant DNA techniques, are of the present invention. Is in range. Such chimeric and humanized monoclonal antibodies are described, for example, in International Application No. PTC / US86 / 02269; European Patent Application No. 184,187; European Patent Application No. 171,496; European Patent Application No. 173,494. PCT International Publication No. WO86 / 01533; US Pat. No. 4,816,567; US Pat. No. 5,225,529; European Patent Application No. 125,023; Better et al. (1988) Science 240: 1041-1043; Liu et al. (1987) Proc. Natl. Acad. Sci. USA 84: 3439-3443; Liu et al. (1987) J. MoI. Immunol. 139: 3521-3526; Sun et al. (1987) Proc. Natl. Acad. Sci. USA 84: 214-218; Nishimura et al. (1987) Cancer Res. 47: 999-1005; Wood et al. (1985) Nature 314: 446-449; Shaw et al. (1988) J. Am. Natl. Cancer Inst. 80: 1533-1559; Morrison (1985) Science 229: 1202-1207; Oi et al. (1986) BioTechniques 4: 214; Jones et al. (1986) Nature 321: 552-525; Verhoeyan et al. (1988) Science 239: 1534; and Beidler et al. (1988) J. Am. Immunol. 141: 4053-4060 can be produced in recombinant DNA technology known in the art.

In one embodiment, methods for screening for antibodies that process the desired specificity include, but are not limited to, enzyme linked immunosorbent assays (ELISA) and other immunologically mediated methods known in the art. Including technology. In certain embodiments, the selection of an antibody that is specific for a particular domain of a zebrafish CXCR4, LEC3 or EDNRA polypeptide binds to a fragment of a vertebrate CXCR4, LEC3 or EDNRA polypeptide, and This is facilitated by the generation of hybridomas that process. Also provided herein are antibodies that are specific for Ig-like domains in vertebrate CXCR4, LEC3 or EDNRA polypeptides, or derivatives, fragments, analogs or homologues thereof.

The anti-CXCR4 antibody, anti-LEC3 antibody or anti-EDNRA antibody may be used in methods known in the art for localization and / or quantification of vertebrate CXCR4, LEC3 or EDNRA polypeptides (eg, suitable physiology For use in measuring the level of vertebrate CXCR4, LEC3 or EDNRA polypeptide in an experimental sample, in diagnostic methods, for use in imaging of a polypeptide, etc.). In certain embodiments, an antibody directed to a vertebrate CXCR4, LEC3 or EDNRA polypeptide containing an antibody-derived binding domain, or a derivative, fragment, analog or homologue thereof, is a pharmacologically active compound (hereinafter: “Therapeutic agent”).

An anti-CXCR4 antibody, anti-LEC3 antibody or anti-EDNRA antibody (eg, monoclonal antibody) can be used to isolate zebrafish CXCR4, LEC3 or EDNRA by standard techniques such as affinity chromatography or immunoprecipitation. Anti-CXCR4 antibody, anti-LEC3 antibody or anti-EDNRA antibody facilitates purification of native CXCR4, LEC3 or EDNRA from cells and vertebrate CXCR4, LEC3 or EDNRA produced as recombinants expressed in host cells be able to. In addition, anti-CXCR4, anti-LEC3 or anti-EDNRA antibodies may be used to assess vertebrate CXCR4, LEC3 or EDNRA polypeptide expression levels and patterns (eg, cytolytic Can be used to detect (in objects). An anti-CXCR4 antibody, anti-LEC3 antibody or anti-EDNRA antibody can be used diagnostically to track protein levels in tissues as part of a clinical trial procedure, for example, to determine the efficacy of a particular therapy . Detection can be facilitated by coupling (ie, physically linking) the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase or acetylcholinesterase; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, Examples of luminescent materials include luminol; examples of bioluminescent materials include luciferase, luciferin and acequorin, and examples of suitable radioactive materials include iodine-125, iodine-131. , Sulfur-35 or tritium. Furthermore, the antibodies of the present invention may be conjugated to poisons such as radioisotopes, protein toxins and chemical toxins. Such poisons include, but are not limited to, lead-212, bismuth-212, astatine-211, iodine-131, scandium-47, rhenium-186, rhenium-188, yttrium-90, iodine-123, Contains iodine-125, bromine-77, indium-111, boron-10, actinides, lysine, adriamycin, calicheamicin and 5-fluorouracil.

Since LEC3 is a cell surface receptor, antibodies to LEC3 may have particular utility. Thus, the antibody can act on the outer ring of LEC3. The antibody may be raised against an isolated peptide derived from a predicted amino acid sequence that would be expected to reside on the outer surface of the cell (see FIGS. 3A-B). The portion of the LEC3 polypeptide sequence predicted to be present on the outer surface of the cell is amino acids 1-806 of SEQ ID NO: 16; amino acids 861-868 of SEQ ID NO: 16; amino acids 930-947 of SEQ ID NO: 16 Amino acids 1026-1028 of SEQ ID NO: 16;

6). Pharmaceutical composition

CXCR4, LEC3 and EDNRA nucleic acid molecules, CXCR4, LEC3 and EDNRA polypeptides (including cell-permeability modified versions of proteins) and anti-CXCR4, anti-LEC3 and anti-EDNRA antibodies (also referred to herein as “active ingredients”) ), And derivatives, fragments, analogs and homologs thereof, can be incorporated into the pharmaceutical composition in a therapeutically effective amount suitable for administration. Such compositions typically comprise a nucleic acid molecule, protein or antibody and a pharmaceutically acceptable carrier. As used herein, “pharmaceutically acceptable carrier” refers to any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption compatible with pharmaceutical administration. Intended to contain retarders and the like. Suitable carriers are described in Remington's Pharmaceutical Sciences, the latest edition of standard reference texts in the field, which is incorporated herein by reference. Suitable examples of such carriers or excipients include, but are not limited to water, saline, Ringer's solution, dextrose solution and 5% human serum albumin. Liposomes and non-aqueous media such as fixed oils may also be used. The use of such media and agents for pharmaceutically active substances is well known in the art. Except as long as any conventional medium or agent is not compatible with the active compound, its use in the composition is contemplated. Supplementary active compounds can also be incorporated into the compositions.

A pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, eg intravenous, intradermal, subcutaneous, oral (eg inhalation), transdermal (topical), transmucosal and anal administration. Solutions or suspensions used for parenteral, intradermal or subcutaneous application include the following components: sterile diluents such as water for injection, sterile saline, non-volatile oil, polyethylene glycol, glycerin, propylene glycol Or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetic acid, citric acid or phosphate, and isotonic Sex regulating agents such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (here water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL ™ (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Microbial defense can be achieved by various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, and sodium chloride in the composition. Delayed absorption of injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are active compounds (eg CXCR4, LEC3 or EDNRA polypeptide or anti-CXCR4, Anti-LEC3 or anti-EDNRA antibody) and subsequent sterile filtration. Generally, dispersions are prepared by incorporating the active compound into a sterile medium that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the methods of preparation are vacuum drying and lyophilization, which include any additional desired ingredients in addition to the active ingredient from a pre-sterilized filtered solution. Produce powder.

Oral compositions generally include an inert excipient and an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions may also be prepared using a fluid carrier for use as a mouthwash, in which case the compound in the fluid carrier is applied orally and spit or swallowed. Be defeated. Pharmaceutically compatible binding agents, and / or adjuvant materials may be included as part of the composition. Tablets, pills, capsules, lozenges, etc. may contain any of the following ingredients or compounds with similar properties: binders such as microcrystalline cellulose, gum tragacanth or gelatin; additives such as starch or lactose , Disintegrants such as alginic acid, primogel or corn starch; lubricants such as magnesium stearate or Sterotes; slip agents such as colloidal silicon dioxide; sweeteners such as sucrose or saccharin; or flavoring agents such as peppermint, methyl salicylate or orange Flavor.

For administration by inhalation, the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, eg, a gas such as carbon dioxide, or a nebulizer.

Systemic administration may also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art and include, for example, for transmucosal administration, detergents, bile salts and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams generally known in the art.

The compounds may also be prepared in the form of suppositories (eg, with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

In one embodiment, the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers may be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for producing such formulations will be apparent to those skilled in the art. Materials are also from Alza Corporation and Nova Pharmaceuticals, Inc. A commercially available product can be obtained from Liposomal suspensions (including liposomes targeted to infected cells containing monoclonal antibodies against viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in US Pat. No. 4,522,811.

It is especially advisable to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to a physically distinct unit adapted as a unit dose for the subject being treated; each unit comprises the required pharmaceutical carrier and Together, it contains a predetermined amount of active compound calculated to produce the desired therapeutic effect. Details regarding the dosage unit forms of the present invention will depend on the unique properties of the active compound and the particular therapeutic effect to be achieved, as well as the technical limitations inherent in formulating such active compounds for individual treatment. Directed and depends directly on them.

The nucleic acid molecules of the invention can be inserted into vectors and used as gene therapy vectors. Gene therapy vectors are described, for example, by intravenous injection, local administration (see, US Pat. No. 5,328,470) or stereotactic injection (see, eg, Chen et al. (1994) Proc. Natl. Acad. Sci. USA91. : 3054-3057). The gene therapy vector pharmaceutical preparation can include the gene therapy vector in an acceptable excipient or can include a sustained release matrix in which the gene delivery carrier is embedded. Alternatively, a complete gene delivery vector can be produced intact from recombinant cells, eg, in the case of a retroviral vector, the pharmaceutical preparation can include one or more cells that produce the gene delivery system.

The pharmaceutical composition may be included in a container, package or dispenser along with instructions for administration.

7). How to regulate angiogenesis

Diseases associated with angiogenesis can be diagnosed and treated using the vertebrate CXCR4, LEC3 or EDNRA proteins of the present invention as drug targets for the development of therapeutic agents. Diseases associated with angiogenesis include, but are not limited to, angiogenesis-dependent cancers, including, for example, solid tumors, tumors of blood origin such as leukemia, and tumor metastases; Eg hemangiomas, auditory neuromas, neurofibromas, trachoma and purulent granuloma; rheumatoid arthritis; psoriasis; ocular angiogenesis diseases such as diabetic retinopathy, retinopathy of prematurity, macular degeneration, corneal transplantation Rejection, neovascular glaucoma, post-lens fiber proliferation, skin flushing; Osler-Weber syndrome; myocardial neovascularization; plaque neovascularization; peripheral vasodilatation; hemophilia joint; . In certain embodiments, the ultimate goal is to promote angiogenesis in the subject. Thus, CXCR4, LEC3 or EDNRA or products thereof, without limitation, promote endothelium formation to promote wound healing, promote endothelialization in vascular graft surgery, and cure vascular injury after myocardial infarction. Can be used to promote.

The present invention provides a method of modulating angiogenesis in an animal. In certain embodiments, the method promotes angiogenesis. In certain embodiments, the method inhibits angiogenesis.

A. Promotion of angiogenesis

In certain embodiments of the invention, angiogenesis is promoted in cells by enhancing the expression of CXCR4, LEC3 or EDNRA. In the methods of the invention, a nucleic acid sequence encoding CXCR4, LEC3 or EDNRA is administered to a cell so as to promote enhanced expression of CXCR4, LEC3 or EDNRA. The nucleic acid sequence may be any CXCR4, LEC3 or EDNRA (ie may be obtained from any species) as long as the CXCR4, LEC3 or EDNRA nucleic acid molecule encodes CXCR4, LEC3 or EDNRA having biological activity. ). The polynucleotide encoding CXCR4, LEC3 or EDNRA may be part of an expression vector as described herein. Methods for introducing nucleic acids into cells and animals are known in the art. Polynucleotides that may be used encoding CXCR4, LEC3 or EDNRA include, but are not limited to, SEQ ID NO: 1, SEQ ID NO: 7, SEQ ID NO: 32, SEQ ID NO: 3, SEQ ID NO: 9, SEQ ID NO: 5, SEQ ID NO: 11, SEQ ID NO: 19 or SEQ ID NO: 35 is included. The amount of CXCR4, LEC3 or EDNRA expression may be dictated by the strength of the promoter and the tissue specific factors chosen for the appropriate system. In general, the expression level should be such that angiogenesis is promoted. In some embodiments, CXCR4, LEC3, or EDNRA alone is sufficient to activate the biological pathway, while in other embodiments, two or more of CXCR4, LEC3, or EDNRA are used. Thus, the present invention also comprises the co-expression of two or more of CXCR4, LEC3 and EDNRA for the promotion of angiogenesis. Co-expression can be achieved by any means known in the art. Nucleic acids encoding the two polypeptides may be incorporated into the same expression vector or may be present in separate expression vectors. Transfection of the vector into the cell may be simultaneous or sequential. The expression of the two subunits may be under the control of the same or different regulatory elements to allow controlled expression of one or both polypeptides. Also, the expression of either or both polypeptides may be made to be constitutive.
B. Anti-angiogenesis

In certain embodiments of the invention, angiogenesis is suppressed in the cell by decreasing the expression of CXCR4, LEC3 or EDNRA or suppressing the function of CXCR4, LEC3 or EDNRA. Angiogenesis involves the use of compounds targeted against CXCR4, LEC3 or EDNRA, such as chemical compounds including natural or synthetic small molecules, antisense CXCR4, LEC3 or EDNRA molecules or antibodies to CXCR4, LEC3 or EDNRA Then it may be suppressed. Polynucleotides that inhibit angiogenesis are those that bind to CXCR4, LEC3 or EDNRA polynucleotides and inhibit the expression of CXCR4, LEC3 or EDNRA or the correct splicing of CXCR4, LEC3 or EDNRARNA. Examples of antisense molecules that suppress CXCR4, LEC3 or EDNRA expression include, but are not limited to, 5′-cgccccattctcatacagaagaag-3 ′ (SEQ ID NO: 24) (MO-LEC3 for zebrafish gene (antisense for splicing suppression) )); 5′-taagcccatctctaaaaaactctcccc-3 ′ (SEQ ID NO: 25) (MO-CXCR4a (antisense for translation blocking) against the zebrafish gene); 5′-gccattgcagaactgggccgctct-3 ′ (SEQ ID NO: 26) MO-EDNRA (antisense for translation blocking)).

Compounds that may be administered to inhibit the function of CXCR4, LEC3 or EDNRA include chemical compounds including natural or synthetic small molecules, polyclonal and monoclonal antibodies that specifically bind CXCR4, LEC3 or EDNRA. When administered to a subject, the antibody may be any known CXCR4, LEC3 or EDNRA binding antibody, provided that the antibody specifically binds CXCR4, LEC3 or EDNRA produced by the subject's cells.
C. Anti-angiogenic combination therapy

In another embodiment of the invention, angiogenesis is modulated by manipulating CXCR4, LEC3 or EDNRA expression and / or function in combination with modulation of vascular endothelial growth factor (VEGF) expression and / or function. The In certain embodiments, angiogenesis is stimulated by administering a nucleic acid encoding CXCR4, LEC3 or EDNRA in combination with a nucleic acid encoding VEGF. CXCR4, LEC3 or EDNRA and VEGF nucleic acid may be present in a single expression vector or in separate expression vectors. VEGF and CXCR4, LEC3 or EDNRA nucleic acids may be administered simultaneously or separately. In another embodiment, angiogenesis is inhibited by administering a compound that decreases CXCR4, LEC3 or EDNRA expression or function and a compound that decreases VEGF expression or function. In certain embodiments, the compound that decreases CXCR4, LEC3, or EDNRA expression is an antisense oligonucleotide. In another embodiment, the compound that decreases CXCR4, LEC3 or EDNRA expression is a morpholino opposed to CXCR4, LEC3 or EDNRA. In certain embodiments, the compound that decreases VEGF expression is an antisense oligonucleotide. In another embodiment, the compound that decreases VEGF expression is a morpholino opposed to VEGF. In one embodiment, the compound that inhibits the function of CXCR4, LEC3 or EDNRA is an anti-CXCR4, anti-LEC3 or anti-EDNRA polyclonal or monoclonal antibody. In some embodiments, the compound that inhibits VEGF function is an anti-VEGF polyclonal or monoclonal antibody. See, for example, Ferrara, N .; Hillan, K .; J. et al. & Novotny, W.M. (2005) “Bevacizumab (Avastin), a humanized anti-VEGF monoclonal antibody for cancer therapy” Biochem. Biophys. Res. Cornun. 333: 328-335.

VEGF has been cloned and sequenced and many different VEGFs are known, including VEGF-A, VEGF-B, VEGF-C and VEGF-D. (For a review of VEGF biology, see Tammela, T., Enholm, B., Alitaro, K., Paavonen, K. (2005) "The biologic of vascular endothelial growth factors" Cardiovasc. See.) Any VEGF-encoding nucleic acid that produces biologically active VEGF may be used. For example, without limitation, nucleic acids encoding VEGF that may be used include human VEGF (SEQ ID NO: 59), (protein: SEQ ID NO: 60), human VEGF-B (SEQ ID NO: 61) ( Protein: SEQ ID NO: 62), human VEGF-C (SEQ ID NO: 69) (protein: SEQ ID NO: 70), human VEGF-D (SEQ ID NO: 71) (protein: SEQ ID NO: 72); mouse VEGF-A (SEQ ID NO: 65) (Protein: SEQ ID NO: 66), mouse VEGF-B (SEQ ID NO: 63) (protein: SEQ ID NO: 64), mouse VEGF-C (SEQ ID NO: 73) (protein: SEQ ID NO: 74), mouse VEGF-D (SEQ ID NO: 75) ) (Protein: SEQ ID NO: 76); VEG such as zebrafish VEGF (SEQ ID NO: 67) (protein: SEQ ID NO: 68) Including the coding nucleic acid.

In certain embodiments, VEGF expression and / or function is suppressed by administering an antisense sequence. Antisense sequences have the general properties described for CXCR4, LEC3 or EDNRA antisense sequences. In certain embodiments, for example, the antisense sequence has the sequence of SEQ ID NO: 77 and SEQ ID NO: 79 sequence target zebrafish VEGF. The human VEGF sequence has the accession number: NM_003376, Unigene cluster: Hs. It is found under 73793 (Ensembl gene ID: ENSG0000012715).

In certain embodiments, VEGF function is inhibited by administration of anti-VEGF polyclonal or monoclonal antibodies. Examples of anti-VEGF monoclonal antibodies include AVASTINTIN ™ (Genentech) and BioCarta Catalog Nos. 09-06-16460 and 09-06-11335. The VEGF signaling pathway may also be suppressed by any tyrosine kinase inhibitor known in the art that disrupts VEGF signaling, or any other type of inhibitor for VEGF known in the art. These inhibitors may be used in combination with the strategies described herein for modulating CXCR4, LEC3 or EDNRA expression or activity.

In embodiments for inhibiting the function of CXCR4, LEC3 or EDNRA and VEGF, the compounds used to inhibit the function or biological activity are small molecule compounds, polynucleotides (eg, antisense and morpholinos, ribozymes). Antibodies against VEGF and CXCR4, LEC3 or EDNRA; cell penetrating peptides that block the interaction of CXCR4, LEC3 or EDNRA with other receptors (upstream receptors), G protein partners and downstream effectors; and Includes a combination of these approaches.

8). Screening for compounds that promote or inhibit angiogenesis:

Another aspect of the invention is to contact CXCR4, LEC3 or EDNRA or a nucleic acid molecule encoding CXCR4, LEC3 or EDNRA with a test compound, wherein the test compound encodes CXCR4, LEC3 or EDNRA or CXCR4, LEC3 or EDNRA. It relates to a method of identifying a compound that binds to any of CXCR4, LEC3 or EDNRA or a nucleic acid molecule encoding CXCR4, LEC3 or EDNRA comprising determining whether or not to bind to a nucleic acid molecule.

Binding is not limited to gel-shift assays, Western blots, radiolabeled competition assays, phage-based expression cloning, chromatographic co-fractionation, coprecipitation, cross-linking, interaction trap / It can be determined by any binding assay known in the art, including 2-hybrid analysis, Southwestern analysis, ELISA, and the like, which are described, for example, in Current Protocols In Molecular Biology, 1999, John Wiley & Sons, N .; Y. It is described in. The CXCR4, LEC3 or EDNRA polypeptide or polynucleotide used in such a test may be free in solution, adhered to a solid support, localized in a cell, May be associated with a fraction. In one embodiment of the invention, high-throughput screening (“HTS”) for compounds with appropriate binding affinity for CXCR4, LEC3 or EDNRA is used. A number of test compounds may be exposed to immobilized CXCR4, LEC3 or EDNRA. Bound CXCR4, LEC3 or EDNRA is then detected by methods well known in the art.

Other methods for identifying ligands for target proteins are described in Wieboldt et al. , Anal. Chem. 69: 1683-1691 (1997). This technique screens a combinatorial library of 20-30 agents simultaneously in solution phase for binding to the target protein. Agents that specifically bind to the target protein retained on the filter are subsequently released from the target protein and analyzed by HPLC and pneumatic electrospray (ion spray) ionization mass spectrometry. This approach selects library components with the greatest affinity for the target protein and is particularly useful for small molecule libraries.

Other embodiments of the invention include the use of antibody-based competitive screening assays. In one embodiment, a specific neutralizing antibody that binds CXCR4, LEC3 or EDNRA specifically competes with a test compound for binding to CXCR4, LEC3 or EDNRA. In this manner, the antibody can be used to determine the presence of any peptide that shares one or more antigenic determinants with CXCR4, LEC3 or EDNRA. Such binding studies may use labeled antibodies and / or labeled test compounds. Examples of such techniques are described in, for example, Lin, A. et al. H. et al. (1997) Antimicrobial Agents and Chemotherapy 41 (10): 2127-2131.

Other aspects of the invention are directed to methods for identifying compounds that modulate (ie, increase or decrease) the biological activity of CXCR4, LEC3 or EDNRA. Such methods contact a test compound with CXCR4, LEC3 or EDNRA, and the compound is positive (agonist) or negative (antagonist) compared to the activity of CXCR4, LEC3 or EDNRA in the absence of the test compound. Determining whether to affect the biological activity of CXCR4, LEC3 or EDNRA in a manner.

In certain embodiments, the compound that modulates CXCR4, LEC3 or EDNRA expression or biological activity is a cell wherein the test compound expresses a CXCR4, LEC3 or EDNRA polypeptide or has a CXCR4, LEC3 or EDNRA polynucleotide. Incubated with and may be identified in an in vitro cellular assay to determine the effect of a test compound on CXCR4, LEC3 or EDNRA expression or biological activity. Modulators of CXCR4, LEC3 or EDNRA activity will be therapeutically useful in the treatment of diseases associated with angiogenesis. Compounds identified as modulating biologically active CXCR4, LEC3 or EDNRA expression in vitro may be further tested in vivo to confirm the associated effective activity.

Test compounds contemplated by the present invention include compounds from chemical libraries, including natural products and / or synthetic products from combinatorial chemical synthesis. Such compounds may include random peptides, oligonucleotides or organic molecules.

In certain embodiments, a transgenic zebrafish carrying a GFP transgene directed by a CXCR4, LEC3 or EDNRA promoter is a CXCR4, LEC3, wherein the compound, small molecule or gene product results in altered expression of GFP. Alternatively, it is used to detect whether or not it has a regulatory activity on the promoter of the EDNRA gene. Increases or decreases in activity can be detected by fluorescent signals generated in living zebrafish, thus providing an in vivo model for screening biologically active drugs, small molecules and gene products. .

The compounds identified by this screening method may be used in the methods of the invention to promote or inhibit angiogenesis, alone or in combination with compounds that promote or inhibit VEGF.

The following examples are for illustrative purposes only and should not be construed as limiting the invention in any way.

Example Example 1

Preserved fish: Danio rerio: Florida wild species (Lancaster, Pennsylvania) and Longfin species (Scientific Hatcheries, Huntington Beach, California).

Zebrafish antisense CXCR4, LEC3 and EDNRA morpholino oligonucleotides (synthesized at Gene Tools, LLC, Philomath, Oregon) had the following sequence: CXCR4 translation-inhibited antisense morpholino: 5'-taagccactctataacactact 3 ′ (SEQ ID NO: 25); LEC3 splicing antisense morpholino: 5′-cgccccgactcatcatacagaagaag-3 ′ (SEQ ID NO: 24);

For LEC3, the portion of the human Lec3 protein spanning the splice site was identified as shown below:

Human Lec3 (also called Latrophilin 3) protein, NP — 056051, 1240 aa (SEQ ID NO: 4 (boldface amino acids and underlined amino acids are adjacent exons corresponding to zebrafish exons)> gi, | 14149967 | Ref | NP_056051.1 | Lec3 or latrophilin3 [human]

The portion of the protein spanning the splice site in human Lec3 was compared to the same portion of the zebrafish Lec3 sequence and found to be highly homologous (conservative substitutions are shown below as “+”) :

An alignment of the complementary DNA (SEQ ID NO: 29) and amino acid (SEQ ID NO: 10) sequences of zebrafish Lec3 shows introns, exons and spliced portions:

A splice inhibitory morpholino was designed to suppress splicing of two exons. In the following, the zebrafish Lec3 genomic DNA sequence showing one exon (bold) and the adjacent exon (underline) is shown. The sequence of the morpholino targeting sequence is shown in uppercase italics.

Thus, the morpholino for the targeted region is the reverse complement of the sequence shown in italics, ie 5'-cgcccgcatcatcatacaagaagag-3 '(SEQ ID NO: 24).

For CXCR4 translation-inhibited morpholinos, morpholinos were designed to span the translation start site. The targeted sequence for morpholino is shown below in bold (starting methionine is shown in capital letters):

Zebrafish CXCR4a (SEQ ID NO: 32) polynucleotide (NM — 131882) and translation (SEQ ID NO: 33) (1..56th 5′UTR; 57-1139th open reading frame, start codon is uppercase, boldface = morpholino target Sequence):

Thus, the morpholino for the targeted region is the reverse complement of the sequence shown in bold, ie 5'-taagcccatctctaaaagacttctcc-3 '(SEQ ID NO: 25).

For EDNRA translation-inhibited morpholinos, morpholinos were designed to span the translation start site. The targeted sequence for morpholino is shown below in bold font (starting methionine is shown in capital letters):

Zebrafish EDNRA polynucleotide (SEQ ID NO: 35) (acc. # CK141648) and translation product (SEQ ID NO: 12): (1.242th 5′UTR; 243-677 reading frame, start codon is uppercase, bold Font = Sequence targeted by morpholino)

Thus, the morpholino for the targeted region is the reverse complement of the sequence shown in bold, ie 5'_gccattgcagaactggggccctct-3 '(SEQ ID NO: 26).

Microinjection of mRNA and morpholino antisense oligo:

The cap-structured sense RNA was synthesized using SP6 RNA polymerase and mMESSAGEmMACHINE system (Ambion). For microinjection of mRNA or morpholino antisense oligos, zebrafish embryos are injected with 0.25-0.5 nl in the early 1-cell stage yolk followed by 0.3 × Danieau's at 28.5 ° C. Incubated in medium. Embryos are maintained under the above conditions until they reach the rodent stage or 24 hours after fertilization and then harvested for RNA preparation or in 4% paraformaldehyde for whole mount in situ hybridization fixed.

In situ hybridization:

The flk in situ construct is Kindly provided by Brant Weinstein (Lawson, ND et al. (2003) Genes Dev. 17 (11): 1346-1351). In situ hybridization methods are described in Leung, T .; et al. (2003) Development 130 (6): 3639-3649. Briefly, embryos are hybridized overnight at 68 ° C., then washed with 66% Hyb / 33% 2XSSC for 30 minutes, then with 33% Hyb / 66% 2XSSC for 30 minutes at 68 ° C. and then with 2XSSC at 68 ° C. For 15 minutes followed by 0.2XSSC for 1 hour at 68 ° C. Hybridized probe was detected by NBT / BCIP staining (Roche). After color staining, embryos were washed for 1 hour in 100% ethanol.

Identification of zebrafish lect3, cxcr4 and ednra genes

As a first step, we sought to identify lect3, cxcr4 and ednra that were specifically expressed in the developing vasculature of zebrafish embryos. Using human and / or mouse protein sequences to probe the zebrafish sequence database, we identified the zebrafish lect3, cxcr4 and ednra genes (SEQ ID NOs: 9, 7 and 11 respectively). The expression patterns of zebrafish rec3, cxcr4 and ednra genes were very dynamic during embryonic development (FIGS. 9-11). Lec3 was expressed in the dorsal artery of 1dpf zebrafish embryos. It is where intersegmental blood vessels are formed by collapse of angiogenesis at 24 and 28 hours after conception (FIG. 12).

Similarly, cxcr4a was expressed in 1dpf developing blood vessels. cxcr4a was expressed in the dorsal artery, intersegmental and cranial vessels of zebrafish embryos 28 hours after conception (FIG. 13).

Expression of ednra was found in the axial vasculature and developing blood vessels. Like lec3, ednra was expressed in the dorsal artery, where intersegmental blood vessels are formed by collapse of angiogenesis in zebrafish embryos 24 and 28 hours after conception (Figure 14).

Targeted knockdown of zebrafish lect3, cxcr4 and ednra suppresses angiogenesis

To elucidate the role that lect3, cxcr4 and / or ednra may play in angiogenesis, we used the morpholino antisense knockdown method in zebrafish embryos (Nasevicius A. and SC Ecker (2000) Nat Genet. 26: 216-220). When injected into zebrafish embryos, morpholinos induced angiogenesis defects, as revealed by molecular markers of vascular endothelial cells (see below).

These intersegmental vessels develop by angiogenic collapse from the dorsal aorta and main vein, and the process is very similar to tumor angiogenesis (Bergers G, and LE Benjamin (2003) Nature Rev, Cancer 3). : 401-410), so intersegmental blood vessels are particularly interesting.

Molecular analysis of lec3, cxcr4 and ednra knockdown phenotypes

To test the nature of the angiogenesis defect in lec3, cxcr4 and ednra knockdown embryos, we used the endothelium specific marker flk1 encoding the VEGFR-2 receptor (Flk-1 / KDR) The developing vasculature was visualized (Liao W et al. (1997) Development 124: 381-389). The flk1 transcript germinates in the cranial blood vessels, the axial vasculature (including the dorsal and main veins) and more importantly from the dorsal aorta, as shown by in situ hybridization of 1 dpf control embryos It was abundantly expressed in intersegmental blood vessels (FIG. 8). In situ hybridization analysis of lec3, cxcr4 and ednra knockdown embryos in synchrony revealed flk1 transcript expression along the dorsal aorta and main vein, which is deficient in endothelial cell differentiation along the axial vasculature It was not. However, since lect3, cxcr4 and ednra knockdown embryos showed very reduced staining of intersegmental vessels sprouting from the axial vasculature (FIGS. 12-14), this defect is specific to the angiogenic process. It suggests that there was.

cxcr4, rec3 and ednra interact developmentally with the vegf pathway

Vascular endothelial growth factor (VEGF) is a major mediator for embryonic and tumor angiogenesis (Tammela et al., 2005; Ferrara et al., 2005). The lect3, cxcr4 and ednra knockdown phenotypes share a surprising similarity with that of the vegf knockdown embryo (Nasevicius A. et al. (2000) Yeast 17: 294-301; Children S. et al. (2002). ) Development 129: 973-982), which suggests that they may function in a common or converging pathway. Using the zebrafish model, we discovered that GPCRs interact with vegf signaling and that both GPCRs and VEGF may function in a common or converging pathway in angiogenesis. Here we provide an example to demonstrate a novel synergistic interaction between the cxcr4a-dependent and vegf-dependent pathways that regulate the angiogenic process in the whole animal model (FIG. 15). Sub-effective doses of either morpholino alone had no effect on angiogenesis (FIGS. 15E-F, vegf knockdown; FIGS. 15C-D, cxcr4a knockdown). However, simultaneous inhibition of both vegf and cxcr4a at the same dose significantly eliminated the process of angiogenic collapse in the zebrafish model (FIGS. 15G-H). Our findings provide a novel opportunity to simultaneously target this GPCR- and VEGF-dependent pathway to synergistically block pathological angiogenesis, which is safer to fight cancer It will lead to a more effective treatment regimen.

Example 2
Anti-angiogenic therapy for retinopathy

Recently, a mouse model of oxygen-induced retinopathy recapitulates the main points of human disease, retinopathy of prematurity (ROP), and newly connected diseases of the retina affecting premature infants Used to do. Using this mouse model to test new anti-angiogenic compounds developed in our laboratory offers several important advantages over other species: (1) Oxygen The mouse model of retinopathy induced by retinal is a highly effective and highly reproducible method that leads to abnormal development of blood vessels in the retina; (2) the retina monitors abnormal blood vessel growth by fundus examination Is an excellent organ to examine this process, and the entire retinal vasculature can be observed in a flat retinal tissue specimen; and (3) limitations on the retina Delivery enables the effectiveness of these novel anti-angiogenic compounds to be evaluated, while at the same time limiting any potential toxicity of these compounds to the retina .
A mouse model of oxygen-induced retinopathy

This model serves as a paradigm for precocious human state retinopathy. On day P7 after birth, up to 2 breeders and their litters were placed in animal containers in sealed containers and mixed with oxygen and compressed air for ventilation to a final oxygen concentration of 75% ± 2% ( ProOx110 System, Biosphere, Ltd). Such exposure to excess oxygen prevents blood vessel growth in the retina. On the 12th day after birth, the animals were returned to room air. Returning to room air induces new blood vessel growth (neovascularization) in the retina due to relatively hypoxic conditions. To observe oxygen-induced changes in the retina, these animals were buried by a lethal dose of sodium pentobarbital (120 mg / kg) at P7 prior to exposure to oxygen; at P12, immediate oxygen exposure And at P17 this is the time of maximum retinal neovascularization. Eyes from one litter are processed for histological analysis to confirm abnormal blood vessel growth in the retina, while eyes from the other litter identify phenotypic changes in the retina To be processed for RNA and protein analysis. This test was repeated three times to evaluate the reproducibility of the results.
Absorption of fluorescent phosphoramidite morpholino oligomers (PMO)

Comparisons were made with respect to the absorption of fluorescently labeled PMO alone or PMO bound to peptide (PMO-pep) into retinal cells. On day P12 after birth, one eye of a newborn mouse was given an intraocular injection of PMO alone or a PMO-pep in the other eye. Three hours after injection, the animals were euthanized by injection of a lethal dose of sodium pentobarbital (120 mg / kg). A frozen section of the eye was prepared to visualize the location of the injected material by fluorescence microscopy. This test was repeated three times to evaluate the reproducibility of the results. The results of this study will indicate whether peptide pairing provides a delivery advantage compared to PMO alone.
Effectiveness of PMO and PMO pairing

Studies were conducted to evaluate the efficacy of PMO and PMOpep-dependent inhibition of lect3, ednra or cxcr4 expression to suppress retinal neovascularization in a mouse model of oxygen-induced retinopathy. As described above, retinopathy can be created by exposing newborn mice to 75% oxygen for 5 days. On the 12th day of life, the day when oxygen exposure ends, these mice with bilateral retinopathy will receive intraocular injections of PMO or PMO-pep targeting lect3, edra or cxcr4 on one eye . Since intraocular injection itself can inhibit neovascularization to some extent, an additional 6 base mismatch, control PMO or PMO-pep intraocular injection is placed on the contralateral eye for these mice. At postnatal day P17, corresponding to the period of maximum neovascularization, these mice are euthanized and their eyes are processed for histological analysis. Neovascularized, ischemic and normal vasculature areas are quantified by observers who were unaware of the treatment. The effectiveness of the treatment will be measured as the difference between the areas of neovascularization in the treated and control eyes. The discovery that targeted knockdown of lec3, ednra or cxcr4 can suppress pathological neovascularization without impeding physiological angiogenesis has led to these genes for various vascular-related retinopathy Would be identified as a potential therapeutic target.
Combination therapy with vegf

To suppress retinal neovascularization, studies were conducted to compare the relative effectiveness of PMO-dependent inhibition of gng2, lect3, edra or cxcr4 with gegf, or the expression of gng2, rec3, edra or cxcr4 / vegf. These tests will be performed as described above. The discovery that targeted knockdown of gng2, rec3, ednra or cxcr4 is as effective or better than the corresponding knockdown of vegf identifies another potential target for drug development Will. This is important because not all retinopathy will show a similar dependence on the vegf pathway. Thus, the identification of additional targets for drug development will expand therapeutic options for various patients and retinal disorders. Similarly, the discovery that simultaneously knocking down rec3, ednra or cxcr4 with vegf provides greater efficacy and lower side effects than either treatment alone would allow optimization of treatment options. I will.

FIG. 1 shows the membrane structure of CXCR4 (SEQ ID NO: 13). Legend: O, amino acid outer membrane; M, transmembrane amino acid; i, amino acid inner membrane compartment. FIG. 2 shows the retained amino acid contrast between CXCR4 of human (SEQ ID NO: 15) and zebrafish (SEQ ID NO: 14). 3A and 3B show the membrane structure of human LEC3 (SEQ ID NO: 16). Legend: O, amino acid outer membrane; M, transmembrane amino acid; i, amino acid inner membrane compartment. 3A and 3B show the membrane structure of human LEC3 (SEQ ID NO: 16). Legend: O, amino acid outer membrane; M, transmembrane amino acid; i, amino acid inner membrane compartment. FIG. 4 shows the retained amino acid contrast between LEC3 of human (SEQ ID NO: 18) and zebrafish (SEQ ID NO: 17). FIG. 5 shows a sequence alignment of the zebrafish EDNRA nucleic acid (SEQ ID NO: 19) and the deduced amino acid (SEQ ID NO: 20). FIG. 6 shows the membrane structure of human EDNRA (SEQ ID NO: 21). Legend: O, amino acid outer membrane; M, transmembrane amino acid; i, amino acid inner membrane compartment. FIG. 7 shows the retained amino acid contrast between human (SEQ ID NO: 23) and zebrafish (SEQ ID NO: 22) EDNRA. FIG. 8 shows the expression of CXCR4 in the growing blood vessel and its essential function. FIG. 9 shows the expression of lectmedin-3 (lec3) in the dorsal artery of zebrafish embryos one day after conception (dpf: day-postfertilization). By using in situ hybridization and RNA probes for zebrafish lect3, we have developed intersegmental blood vessels due to angiogenesis collapse in 24 and 28 hour (hpf) hour-postfertilization (zebrafish) embryos. Reveal that ec3 is expressed in the dorsal artery that is formed (scale bar is 100 um). FIG. 10 shows the expression of C—X—C chemokine receptor type 4 (cxcr4a) in blood vessels growing at 1 dpf. By using in situ hybridization and RNA probes for zebrafish cxcr4a, we demonstrate that cxcr4a is expressed in the dorsal artery, intersegmental and cranial vessels in zebrafish embryos 28 hours after conception. Legend: d. a, dorsal artery; c. v, cranial blood vessels; s. v, erupted blood vessels. (Scale bar is 100 um). FIG. 11 shows the expression of endothelin-1 receptor (ednra) in the axial vasculature and growing blood vessels. By using in situ hybridization and RNA probes on zebrafish ednra, we have introduced ednra into the dorsal artery where intersegmental vessels are formed by collapse of angiogenesis in zebrafish embryos at 24 and 28 hours after conception. It is revealed that is expressed. (Scale bar is 100 um). Legend: c. v, cranial blood vessels; a. v, axial vasculature. FIG. 12 shows that using the flk-1 marker, targeted lect3 knockdown disrupts the collapse of angiogenesis of intersegmental blood vessels. Targeted lek3 knockdown using morpholino antisense oligos in zebrafish embryos disrupts the collapse of intersegmental blood vessel angiogenesis as revealed by the vascular endothelium-specific marker flk-1. Legend: IS, intersegmental blood vessel. (Scale bar is 100 um). FIG. 13 shows that targeted cxcr4a knockdown specifically blocks flk-1 expression in erupting blood vessels. Targeted cxcr4a knockdown using morpholino antisense oligos in zebrafish embryos disrupts the collapse of intersegmental vascularization as demonstrated by the vascular endothelium-specific marker flk-1. Legend: IS, intersegmental blood vessel. (Scale bar is 100 um). FIG. 14 shows that using flk-1 marker, targeted edra knockdown disrupts the collapse of angiogenesis of intersegmental blood vessels. Targeted ednra knockdown using morpholino antisense oligos in zebrafish embryos disrupts the collapse of intersegmental vascularization as revealed by the vascular endothelium-specific marker flk-1. Legend: IS, intersegmental blood vessel. (Scale bar is 100 um). FIG. 15 shows synergistic suppression by CXCR4 and VEGF-dependent pathways for angiogenesis using the flk-1 marker. A and B: wild type control (B, high magnification); C and D: targeted cxcr4a knockdown at sub-effective dose (D, high magnification), eruption of intersegmental blood vessels (IS) E and F: Targeted vegf knockdown at sub-effective doses (F, high magnification) against eruption of intersegmental blood vessels (IS) G and H: Co-targeting both cxcr4a and vegf with morpholino antisense oligos (H, high magnification) in zebrafish embryos, due to the vascular endothelium-specific marker flk-1 As revealed, it showed a synergistic inhibition against the collapse of angiogenesis of intersegmental blood vessels. The scale bar is 100 um. FIG. 16 shows the sequence contrast of various GPCR regions homologous to the hamster adrenergic receptor (α1B-AR), which shows a dominant negative effect when phenylalanine is replaced with glycine (G) or asparagine (N) (Chen S Et al. (2000) EMBO J. 19 (16): 4265-4271). Shown are cognate regions from: human CXCR4 (SEQ ID NO: 80); human LEC3 (SEQ ID NO: 81); human EDNRA (SEQ ID NO: 82); hamster α1B-AR (SEQ ID NO: 83) Hamster α2C2-AR (SEQ ID NO: 84); hamster β2-AR (SEQ ID NO: 85); hamster H1 (SEQ ID NO: 86); hamster D2 (SEQ ID NO: 87); hamster M3 (SEQ ID NO: 88); 89); Hamster rhodopsin (SEQ ID NO: 90). Retained phenylalanine (hamster α1B-AR Phe303) is placed in the box.

Claims (54)

A method of promoting angiogenesis in an animal comprising administering to the animal an isolated expression vector comprising a polynucleotide selected from:
(A) SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 11, and
(B) A sequence encoding a polypeptide having the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10 and SEQ ID NO: 12. An antibody that specifically binds to a polypeptide comprising the amino acid sequence of SEQ ID NO: 10 with high affinity. An antibody that specifically binds with high affinity to the extracellular portion of a polypeptide comprising the amino acid sequence of SEQ ID NO: 4. The antibody of claim 2, wherein said antibody is monoclonal. The antibody of claim 2, wherein the antibody is a polyclonal. 4. The antibody of claim 3, wherein the antibody is monoclonal. 4. The antibody of claim 3, wherein the antibody is a polyclonal. A method for producing all or part of zebrafish LEC3, CXCR4 or EDNRA,
A host cell comprising an expression vector comprising the sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 or SEQ ID NO: 11 operably linked to an expression control sequence is transformed into zebrafish LEC3, CXCR4. Or cultured under culture conditions suitable for the expression of all or part of EDNRA,
Isolating all or part of the LEC3, CXCR4 or EDNRA;
A method comprising: A therapeutic composition comprising an isolated polynucleotide comprising a nucleic acid sequence encoding all or part of LEC3, CXCR4 and EDNRA, and a pharmaceutically acceptable carrier. The therapeutic composition of claim 9, wherein the nucleic acid sequence encodes the polypeptide of SEQ ID NO: 4 or SEQ ID NO: 10. A therapeutic composition comprising the antibody of claim 3 and a pharmaceutically acceptable carrier. A polypeptide comprising at least one amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10 and SEQ ID NO: 12, and a pharmaceutically acceptable carrier. A therapeutic composition comprising. A method of treating a disease associated with angiogenesis, comprising administering to a patient in need of the treatment an amount of a polynucleotide that inhibits the expression of LEC3 sufficient to inhibit angiogenesis. The angiogenesis-related diseases include angiogenesis-dependent cancers; benign tumors; rheumatoid arthritis; psoriasis; visual angiogenesis diseases; Austler-Weber syndrome; myocardial angiogenesis; lytic angiogenesis; 14. The method of claim 13, selected from the group consisting of amphiphilic arthropathy; fibroangioma; wound granulation; intestinal adhesions, atherosclerosis, scleroderma, hyperplastic scar, cat scratch disease and Helicobacter pylori ulcer . 14. The method of claim 13, wherein the angiogenesis-related disease is an angiogenesis-dependent cancer. 14. The method of claim 13, wherein the angiogenesis-related disease is an angiogenesis-dependent tumor and the polynucleotide is administered in an amount sufficient to cause tumor regression. A method of promoting angiogenesis in an animal in need, comprising administering to the animal an effective amount of a polynucleotide encoding all or part of LEC3, CXCR4 and EDNRA polypeptides. 18. The method of claim 17, wherein the LEC3, CXCR4 or EDNRA polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10 and SEQ ID NO: 12. 18. The method of claim 17, wherein the LEC3 polypeptide comprises the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 10. A method for treating an angiogenesis-related disease, comprising: a first polynucleotide that suppresses expression of all or part of lect3, cxcr4, and ednra in a patient in need of such treatment; and expression of vegf And a second polynucleotide that inhibits angiogenesis, wherein the first polynucleotide and the second polynucleotide are provided sufficient to inhibit angiogenesis. 21. The nucleic acid sequence of claim 20, wherein the first polynucleotide comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, and combinations thereof. Method. The second polynucleotide is a group consisting of SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 65, SEQ ID NO: 63, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 67, and combinations thereof. 21. The method of claim 20, comprising a nucleic acid sequence selected from. 21. The method of claim 20, wherein the angiogenesis-related disease is an angiogenesis-dependent cancer. A method of treating a patient with an angiogenesis-dependent tumor, the first compound suppressing the expression or function of all or part of lect3, cxcr4 and ednra for a patient in need of the treatment, and expression of vegf Or a method comprising administering a second compound that inhibits function, wherein the first compound and the second compound are provided in an amount sufficient to cause tumor regression. A method for promoting angiogenesis in an animal in need, wherein the animal encodes a VEGF polypeptide with an effective amount of a first polynucleotide encoding all or part of the LEC3, CXCR4 and EDNRA polypeptides. Administering an effective amount of a second polynucleotide. 26. The method of claim 25, wherein said LEC3, CXCR4 and / or EDNRA polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 12 and SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO: 22. The VEGF polypeptide is an amino acid sequence selected from the group consisting of SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 66, SEQ ID NO: 64, SEQ ID NO: 74, SEQ ID NO: 76, and SEQ ID NO: 68. 26. The method of claim 25 comprising: The LEC3 polypeptide comprises the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 10, and the VEGF polypeptide is SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 66, SEQ ID NO: 64, SEQ ID NO: 74. 26. The method of claim 25, comprising the amino acid sequence of SEQ ID NO: 76 and SEQ ID NO: 68. 25. The method of claim 24, wherein the second compound is an antibody that specifically binds to VEGF. 25. The method of claim 24, wherein the first compound is an antibody that specifically binds to LEC3. A method for treating a disease associated with angiogenesis, comprising: a first compound that inhibits all or part of the functions of LEC3, CXCR4, and EDNRA for a patient in need of the treatment; and a method that inhibits the function of VEGF. And a method wherein the first compound and the second compound are provided sufficient to inhibit angiogenesis. 32. The method of claim 31, wherein the first compound is an antibody that specifically binds to LEC3. 32. The method of claim 31, wherein the second compound is an antibody that specifically binds to VEGF. 35. The method of claim 32, wherein the antibody binds to at least a portion of the extracellular portion of the LEC3 protein. A method of identifying a compound that inhibits LEC3, CXCR4 and / or EDNRA activity, wherein the test compound is contacted with a LEC3, CXCR4 and / or EDNRA polypeptide, said test compound inhibiting LEC3, CXCR4 and / or EDNRA activity A test compound that inhibits the activity of LEC3, CXCR4 and / or EDNRA is identified as an antagonist of LEC3, CXCR4 and / or EDNRA. 36. The method of claim 35, wherein the biological activity of LEC3, CXCR4 and / or EDNRA is measured by binding the test compound to LEC3, CXCR4 and / or EDNRA. 36. The method of claim 35, wherein the biological activity of LEC3, CXCR4 and / or EDNRA is measured by inhibition of angiogenesis in a model system. 38. The method of claim 37, wherein the model system is a zebrafish generation system. 38. The method of claim 37, wherein the model system is a transgenic animal system. 38. The method of claim 37, wherein the model system is an in vitro cell system. A method for inhibiting angiogenesis, comprising administering to a cell an effective amount of a cell-penetrating peptide that inhibits the biological function of LEC3, CXCR4 and / or EDNRA. 42. The method of claim 41, further comprising administration of a compound that inhibits VEGF expression or biological function. A method of promoting angiogenesis comprising administering to a subject an effective amount of a compound selected from the group consisting of:
(A) an isolated expression vector comprising a polynucleotide having the nucleotide sequence of SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO:
(B) a sequence encoding a polypeptide having the amino acid sequence of SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56 and SEQ ID NO: 58; An isolated expression vector comprising a polynucleotide having
(C) A polypeptide comprising the amino acid sequences of SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, and SEQ ID NO: 58. A method of treating an angiogenesis-related disease, comprising administering to a patient in need of such treatment an antibody that binds to SDF-1 or ET-1 in an amount sufficient to inhibit angiogenesis. Method. The angiogenesis-related diseases include angiogenesis-dependent cancers; benign tumors; rheumatoid arthritis; psoriasis; visual angiogenesis diseases; Austler-Weber syndrome; myocardial angiogenesis; lytic angiogenesis; 45. The method of claim 44, selected from the group consisting of amphipathic arthropathy; fibroangioma; wound granulation; intestinal adhesions, atherosclerosis, scleroderma, hyperplastic scar, cat scratch disease and Helicobacter pylori ulcer . 45. The method of claim 44, wherein the angiogenesis-related disease is an angiogenesis-dependent cancer. 45. The method of claim 44, wherein the angiogenesis-related disease is an angiogenesis-dependent tumor and the polynucleotide is administered in an amount sufficient to cause tumor regression. 45. The method of claim 44, wherein the SDF-1 comprises the amino acid sequence of SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48 or SEQ ID NO: 50. 45. The method of claim 44, wherein the ET-1 comprises an amino acid sequence of SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56 or SEQ ID NO: 58. A method for preventing or treating retinopathy, wherein CXCR4, LEC3 and / or EDNRA polypeptide, which inhibits the expression or function of a protein selected from the group consisting of LEC3, EDNRA and CXCR4, is vascularized to the eye. Administering a sufficient amount to inhibit the disease. 51. The method of claim 50, wherein the polynucleotide is an antisense oligonucleotide or a phosphoramidite morpholino oligomer. 52. The method of claim 51, wherein said polynucleotide binds to a peptide to promote cellular uptake of said polynucleotide. 51. The method of claim 50, wherein the retinopathy is age-related macular degeneration, diabetic retinopathy or retinopathy of prematurity. 51. The method of claim 50, wherein the antibody is an antibody that specifically binds to CXCR4, LEC3 and / or EDNRA polypeptide.

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