WO2003005034A2 - Procedes d'identification d'agents therapeutiques de traitement de maladies impliquant le gene - Google Patents

Procedes d'identification d'agents therapeutiques de traitement de maladies impliquant le gene Download PDF

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Publication number
WO2003005034A2
WO2003005034A2 PCT/CA2002/001004 CA0201004W WO03005034A2 WO 2003005034 A2 WO2003005034 A2 WO 2003005034A2 CA 0201004 W CA0201004 W CA 0201004W WO 03005034 A2 WO03005034 A2 WO 03005034A2
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WIPO (PCT)
Prior art keywords
fzd4
polypeptide
disease
seq
amino acid
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PCT/CA2002/001004
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English (en)
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WO2003005034A3 (fr
Inventor
Marcia L. Macdonald
Jutta M. Zeisler
Mark Samuels
Yigal P. Goldberg
Johanne M. Robataille
Michael R. Hayden
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Xenon Genetics, Inc.
University Of British Columbia
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Publication of WO2003005034A2 publication Critical patent/WO2003005034A2/fr
Publication of WO2003005034A3 publication Critical patent/WO2003005034A3/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
    • A61K38/179—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/566—Immunoassay; Biospecific binding assay; Materials therefor using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/16—Ophthalmology
    • G01N2800/164—Retinal disorders, e.g. retinopathy

Definitions

  • the present invention relates to the field of mutations in selected genes, such as the Frizzled-4 gene ("fzd4') disclosed herein, which are causative agents in hereditary human visual disorders, such as familial exudative itreoretinopathy ("FEVR") and the uses of such mutations in the diagnosis of the underlying diseases as well as the use of the Frizzled-4 Gene and/or protein in the development of assays for therapeutic agents useful in treating diseases related to Frizzled-4 expression or activity, as can now be extrapolated from the phenotypic effects of mutations in fzd4.
  • fzd4' Frizzled-4 gene
  • FEVR familial exudative itreoretinopathy
  • This locus was further refined to approximately 200 kb on 11q14 in a recent report involving seven small families of Japanese origin. (Kondo, H. et al. 2001. Delineation of the critical interval for the familial exudative vitreoretinopathy gene by linkage and haplotype analysis. Hum Genet. May;108(5):368-75). A bioinformatics analysis of this region did not reveal any positional candidate genes, suggesting that an unlinked family was used to define the distal boundary by the polymorphic marker GATA30G01. Locus EVR2 was designated based on the finding, in the X-linked form, of mutations in the gene for Norrie disease at Xp11.4-p11.3 (Chen et al 1993z; Shastry et al. 1995). A new locus, EVR3, was recently designated in the report of Bamashmus et al. (2000 Br. J. Opthalmol. 84:358-363) at 11 p12-13, located about 30 cM from EVR1.
  • retinopathy of prematurity ROP
  • diabetic retinopathy ROP
  • other diseases related to a deficiency/excess of angiogenesis and vasculogenesis not the least of which is cancer and neoplasia.
  • ROP retinopathy of prematurity
  • the cellular processes that underlie vascularization of the human retina, as they are known, are set out in Hughes, S. et al. 2000. Invest. Opthalmol. Vis. Sci.
  • the present invention relates to a process for identifying an FZD4 modulating agent comprising:
  • said biological activity is the binding of a ligand.
  • the FZD4 polypeptide is a human FZD4 polypeptide, most preferably a polypeptide comprising the sequence of SEQ ID NO: 2.
  • Such assay may be conducted in vitro and may involve a cell-based procedure, employing whole cells, such as mammalian cells, preferably human cells, most preferably recombinant cells, such as those engineered to express the polypeptide or may involve an assay wherein the polypeptide is merely part of a membrane, such as where the polypeptide is part of a liposome.
  • the FZD4 polypeptide is a recombinant polypeptide.
  • the ligand which may be any ligand that binds to FZD4 protein, is a Wnt protein, most preferably Wnt11
  • the present invention relates to a process for identifying an FZD4 modulating agent comprising: (a) contacting a compound with a cell that expresses FZD4 polypeptide under conditions promoting expression of said FZD4 polypeptide,
  • the cell useful in such an assay may comprise a DNA construct that encodes an FZD4 polypeptide, such as the human FZD4 polypeptide disclosed herein, and may further employ a polypeptide that comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 2 and wherein any difference of said polypeptide from SEQ ID NO: 2 is only by conservative amino acid substitutions.
  • the sequence identity may be at least 95%, at least 98%, or may comprise the amino acid sequence of SEQ ID NO: 2.
  • the DNA construct comprises a nucleotide sequence at least 95% identical to the sequence of SEQ ID NO: 1 , preferably at least 97% identical to the sequence of SEQ ID NO: 1 , and most preferably the nucleotide sequence of SEQ ID NO: 1.
  • the construct may be a cDNA and may comprise a mutation, such as that of SEQ ID NO: 3 or 5.
  • the difference in expression is the amount of FZD4 polypeptide produced by said cell or the amount of FZD4 messenger RNA produced by said cell.
  • the present invention also includes the isolated polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1 , 3 or 5.
  • the present invention relates to an isolated polynucleotide comprising a polynucleotide selected from the group consisting of:
  • the present invention further relates to vectors comprising the polynucleotides disclosed herein as well as to recombinant cells expressing said polynucleotides.
  • the present invention relates to a process for diagnosing the presence of an FZD4-linked disease in a patient suspected of being afflicted therewith comprising detecting a mutation in the FZD4 gene in the genome of said patient.
  • the sample of DNA is taken from a patient.
  • the mutation to be determined is located on chromosome 11 at map location 11q14.2 (UCSC designation), especially where the mutation is that of SEQ ID NO: 3 or 5.
  • the detecting is commonly performed using probes of varying lengths of contiguous amino acids, all of which probes serve to locate the mutation.
  • the disease to be detected is vascular disease, angiogenic disorders, retinopathies or cancer, preferably familial exudative vitreoretinopathy (FEVR).
  • FEVR familial exudative vitreoretinopathy
  • the disease may also be one of retinopathy of prematurity (ROP), Coat's disease, retinal angiomatosis, diabetic retinopathy, neovascular inflammatory vitreoretinopathy (VRNI), persistent hyperplastic primary vitreous (PHPV), and congenital retinal folds.
  • ROP retinopathy of prematurity
  • Coat's disease Coat's disease
  • retinal angiomatosis retinal angiomatosis
  • diabetic retinopathy diabetic retinopathy
  • VRNI neovascular inflammatory vitreoretinopathy
  • PGPV persistent hyperplastic primary vitreous
  • congenital retinal folds congenital retinal folds.
  • the present invention relates to a process for determining a patient's risk of developing an FZD4-linked disease where said patient is suspected to be at risk thereof, comprising detecting a mutation in the FZD4 gene in the genome of said patient.
  • the mutation may be detected in a sample taken from a patient or may be conducted in situ.
  • Such mutation is a mutation in the sequence of SEQ ID NO: 1 and the disease is selected from the group consisting of vascular disease, angiogenic disorders, retinopathies and cancer and may include neurological diseases.
  • familial exudative vitreoretinopathy (FEVR) familial exudative vitreoretinopathy
  • the present invention relates to a process for identifying an agent having therapeutic activity in ameliorating the symptoms of an FZD4-linked disease comprising :
  • the disease is selected from among vascular disease, angiogenic disorders, diseases of the eye, including retinopathies and age-related macular degeneration, cancer, neoplasia and a neurological disease.
  • the disease is familial exudative vitreoretinopathy (FEVR).
  • the present invention relates to a process for identifying an agent having therapeutic activity in ameliorating the symptoms of an FZD4-linked disease comprising :
  • the disease may be one of vascular disease, angiogenic disorders, retinopathies and cancer, or may include a neurological disease or disease of the eye.
  • the disease is familial exudative vitreoretinopathy (FEVR).
  • the present invention relates to a process for treating an FZD4-linked disease in a patient afflicted therewith comprising administering to said patient an effective amount of an agent that modulates the biological activity of FZD4, wherein such agent was first identified as having such activity by the processes of the invention.
  • the present invention also relates to a method for producing a product comprising identifying an agent according to the processes of the invention wherein said product is the data collected with respect to said agent as a result of said process and wherein said data is sufficient to convey the chemical structure and/or properties of said agent.
  • the present invention also relates to antibodies that react with a polypeptide that comprises the amino acid sequence of SEQ ID NO: 2, 4 or 6.
  • such antibody is a monoclonal antibody, including recombinant and synthetic antibodies.
  • Figure 1 sets forth a large part of the family tree of the FEVR proband.
  • Figure 2 shows the effects of a number of mutations in the FZD4 sequence.
  • Figure 2A shows the effects of a 6 bp deletion producing superimposed sequences; 2B a 2 bp deletion producing superimposed sequences; 2C a 1033 A>G mutation (meaning "A” is replaced by "G” at position 1033 of the nucleotide sequence of FZD4); 2D a502 C>T mutation; 2E a 97 C>T mutation; 2F a 766 A>G mutation.
  • Figure 3A sets forth a comparison of known members of the human frizzled gene family. The FZD4 M493-W494del mutation found in FEVR patients is included for comparison.
  • Figure 4A sets forth a comparison of known wild-type FZD4 homologs from a variety of eukaryotes.
  • the FZD4 M493-W494del mutation found in FEVR patients is included for comparison.
  • the highly conserved nature of the region where the mutation is found is self-evident conserved amino acids are outlined in black and functionally conserved amino acids are in gray. Variable amino acids are not outlined.
  • the mutation in FEVR patients is identified as a two amino acid mutation at site M493-W494del.
  • Figure 4B is an alignment of the FZD4 M342V variation with sequences from FZD4 homologs from other eukaryotes.
  • the FZD4 homologs shown are human FEVR FZD4 (FEVR), human (hFZD4), mouse (mFZD4), rat (rFZD4), chicken (gallus gallus) frizzled- 4 (gFz-4), Xenopus laevis (XFz4), zebrafish (Danio rerio) frizzled protein) (Zfz4), zebrafish (Danio rerio), Frizzled X (Zfzx), Caenorhabditis elegans wingless protein receptor (Cfz2), and Drosophila (Dfz2)
  • Figure 5A shows a sequence alignment for the P33S mutation (proline replaced by serine at residue 33) for a number of eukaryotes.
  • Figure 5B shows an alignment the P168S mutation.
  • Figure 5C is an alignment for the I256V change for the ROP5 mutation.
  • Figure 6 is a schematic of the domain structure of FZD4 with the cysteine-rich domain near the N-terminus and the M493-W494 deletion.
  • Figure 7 illustrates the two exon genomic structure of FZD4. Domains marked in black encode open reading frames. Shown at the top is the genomic arrangement of exons. The middle panel illustrates a potential alternative transcript of FZD4 resulting from alternative splicing. The bottom panel illustrates the approximate location of a mutation directly associated with FEVR.
  • Figure 8 shows that the 2 nd ATG is closer to the optimal Kozak consensus C-C-[AG]-C-C-A-T-G-G context for initiation of translation and is the first ATG in mouse and rat FZD4 mRNA. Putative start codons are indicated by italics.
  • Figure 9 shows a proposed mechanism of action of FZD4.
  • This invention relates to the discovery that mutations in the Frizzled-4 gene (“fzd4') are responsible for a hereditary human disorder, familial exudative vitreoretinopathy ("FEVR").
  • fzd4' mutations in the Frizzled-4 gene
  • FEVR familial exudative vitreoretinopathy
  • Frizzled-4 protein (“FZD4") is involved in FEVR and in many pathologically related forms of retinopathy, such as retinopathy of prematurity (ROP), diabetic retinopathy, and others, and other pathologically related diseases involving a deficiency/excess of angiogenesis and vasculogenesis in neurological and/or other tissues, not the least of which is cancer and neoplasia.
  • this invention now teaches therapeutic targets and agents for a wide variety of diseases including FEVR, retinopathies, angiogenic and vascularization disorders in neurological tissues and throughout the body, including cancer and neoplasia.
  • These therapeutic agents may be the FZD4 protein or gene (and fragments or analogs thereof), or they may be compounds, such as small organic molecules, which modulate FZD4 activity or expression and thereby provide therapeutic benefit to a person in need thereof.
  • the present invention relates to a process for identifying an FZD4 modulating agent comprising:
  • said biological activity is the binding of a ligand.
  • the process comprises:
  • the source of FZD4 biological activity is a polynucleotide comprising the FZD4 gene, preferably a polynucleotide comprising a polynucleotide that encodes the FZD4 gene product or where said polynucleotide that encodes the FZD4 gene product is a cDNA, most preferably wherein said cDNA is the cDNA of SEQ ID NO: 1.
  • the source of FZD4 biological activity is a polynucleotide comprising a mutant FZD4 gene, preferably a polynucleotide comprising a mutated FZD4 cDNA, most preferably wherein said mutated FZD4 cDNA is the cDNA of SEQ ID NO: 3.
  • the source of FZD4 biological activity is a mRNA encoded by the FZD4 gene.
  • the source of FZD4 biological activity is a polypeptide encoded by the FZD4 gene.
  • the change in biological activity to be measured is a change in the expression of the FZD4 gene, preferably a change in the amount of mRNA transcribed by the FZD4 gene, or where the change in biological activity is a change in the amount of polypeptide produced by expression of the FZD4 gene, preferably a change in the activity of a polypeptide encoded by the FZD4 gene, most preferably where the polypeptide comprises the amino acid sequence of SEQ ID NO: 2.
  • the present invention specifically contemplates cell-based assays, such as where the source of FZD4 biological activity is a cell expressing FZD4 gene activity.
  • the cell may be a cell that normally expresses this gene or may be a cell, such a recombinant cell, that does not otherwise express this gene but has been engineered to express this gene, such as where this gene is an exogenous DNA that has been inserted into the cell, such as through transfection or by use of a vector, including both viral vectors and plasmids.
  • the recombinant cell contains and expresses a cDNA as disclosed herein, especially the cDNA of SEQ ID NO: 1 , such as where the cell expresses a polypeptide comprising the amino acid sequence of SEQ ID NO: 2.
  • the cell may express a mutant form of the FZD4 gene.
  • SEQ ID NO: 3 represents the full length sequence of a mutant FZD4 cDNA found in members of the same family who were diagnosed with FEVR. (1479-- 1484del).
  • SEQ ID NO: 4 is the amino acid sequence of a mutant FZD4 protein formed from the cDNA (M493-W494del).
  • SEQ ID NO: 10 shows cDNA nucleotide sequence of human FZD4 with a CT deletion at residues 1501 - 1502 (1501 --1502del).
  • the first 9 nucleotides represent an alternative translation initiation site for FZD4 protein.
  • SEQ ID NO: 6 is the sequence of a mutated polypeptide formed from the cDNA sequence of SEQ ID NO: 5 (L501fsX533).
  • the first 3 amino acids represent an alternative translation initiation site for hFZD4 protein.
  • FZD4 polypeptide is a human FZD4 polypeptide, such as wherein the FZD4 polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 2 and wherein any difference of said polypeptide from SEQ ID NO: 2 is only by conservative amino acid substitutions.
  • sequence identity is at least 95%, most preferably wherein the sequence identity is at least 98%, and especially preferred is wherein the polypeptide has the amino acid sequence of SEQ ID NO: 2.
  • the present invention involves such processes as disclosed herein where the FZD4 polypeptide, which in prefered embodiments may be a recombinant or synthetically prepared polypeptide, is part of a lipid bilayer, such as wherein the FZD4 polypeptide is part of a liposome or other experimentally reconsitituted membrane or wherein said FZD4 polypeptide is part of a cell membrane.
  • the FZD4 polypeptide is part of an intact cell, such as where the cell expresses FZD4 polypeptide on the surface of said cell.
  • said cell is a recombinant cell, such as where the recombinant cell express FZD4 polypeptide on its surface as a result of engineering, especially genetic engineering, most preferably wherein said recombinant cell does not express FZD4 polypeptide absent said engineering.
  • the present invention further relates to a process for identifying an
  • FZD4 modulating agent comprising:
  • the cell comprises a DNA construct that encodes an FZD4 polypeptide, preferably wherein said FZD4 polypeptide is a human FZD4 polypeptide.
  • the FZD4 polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 2 and wherein any difference of said polypeptide from SEQ ID NO: 2 is only by conservative amino acid substitutions, preferably wherein said sequence identity is at least 95%, most preferably wherein said sequence identity is at least 98%, and especially wherein said polypeptide has the amino acid sequence of SEQ ID NO: 2.
  • the DNA construct comprises a nucleotide sequence at least 95% identical to the sequence of SEQ ID NO: 1 , preferably wherein said DNA construct comprises a nucleotide sequence at least 97% identical to the sequence of SEQ ID NO: 1 , most preferably wherein said DNA construct comprises the nucleotide sequence of SEQ ID NO: 1.
  • the DNA construct is a cDNA.
  • the DNA construct comprises a mutation, preferably wherein said DNA construct has the nucleotide sequence of SEQ ID NO: 3 or wherein said DNA construct has the nucleotide sequence of SEQ ID NO: 5.
  • the difference in expression is a difference in the amount of FZD4 polypeptide produced by said cell, especially wherein said difference in expression is a difference in the amount of FZD4 messenger RNA produced by said cell.
  • the present invention relates to an isolated polynucleotide comprising a polynucleotide at least 95% identical to the polynucleotide of SEQ ID NO: 1 or its complement, preferably an isolated polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1.
  • the present invention relates to an isolated polynucleotide comprising the nucleotide sequence of SEQ ID NO: 3 or SEQ ID NO: 5.
  • the present invention relates to an isolated polynucleotide comprising a polynucleotide encoding a polypeptide having the amino acid sequence of SEQ ID NO: 2 or its complement.
  • the present invention further relates to an FZD4 reporter gene nucleic acid construct comprising up to about 2000 nt of genomic DNA upstream (5') of the translation initiation site of the FZD4 gene (SEQ. ID NO: 7) fused to a reporter gene, for use in identifying modulators of FZD4 gene expression.
  • a process for identifying such modulators comprises
  • Reporter genes are typically genes having gene products which are relatively easy to assay in experimental systems.
  • Well known reporter genes include luciferase, chloramphenicol-acetyl transferase (CAT), beta- galactosidase, alkaline phosphatase, placental alkaline phosphatase, glucruonide synthetase, green fluorescence protein (GFP), and others.
  • the present invention also contemplates vectors comprising the native and mutant forms of the FZD4 gene.
  • the present invention relates to a vector comprising a polynucleotide that encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 2, preferably wherein said polynucleotide is the polynucleotide of SEQ ID NO: 1.
  • the present invention contemplates a vector comprising a polynucleotide that encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 4, preferably wherein said polynucleotide is the polynucleotide of SEQ ID NO: 3, or wherein the polynucleotide is that of SEQ ID NO: 5 and the expressed polypeptide is that of SEQ ID NO: 6.
  • Such vectors may comprise any of the novel polynucleotides disclosed herein, including all or a portion of the polynucleotide sequence that is used to generate antisense trancripts.
  • the present invention also relates to cells, such as mammalian, including human cells, especially recombinant cells, that comprise the vectors as disclosed herein or that comprise, such as in their genome, a novel polynucleotide as disclosed herein, especially a human FZD4, most preferably a recombinant human FZD4 polynucleotide.
  • Such cells may express an FZD4 polypeptide, such as a recombinant human FZD4 polypeptide, encoded by a novel polynucleotide of the invention.
  • vectors and cells as contemplated by the present invention may readily be prepared by those skilled in the art and specific methods of preparing such need not be described further herein except in such manner as is further disclosed below.
  • the present invention further relates to processes diagnosing the presence of such a disease, especially at its earliest stages, or the risk to a patient of developing such diseases.
  • the present invention relates to a process for diagnosing the presence of a disease in a patient suspected of being afflicted therewith comprising detecting a mutation in the FZD4 gene in the genome of said patient.
  • the present invention relates to such diagnostic processes where the mutation is the mutation in the nucleotide sequence as depicted in SEQ ID NO: 3.
  • the mutation is detected in a sample of DNA taken from said patient.
  • a sample may be obtained in any manner commonly used to obtain such sample, such as by blood sample, biopsy or other methods.
  • detecting may be accomplished in vivo, such as where an in situ procedure is used to detect the presence of a mutated FZD4 gene or nucleotide sequence in a patient.
  • the diseases that can be so detected are not limited to FEVR but include all manner of neurological diseases and syndromes, especially those involving the visual pathway.
  • the disease is one or more of a group consisting of vascular disease, angiogenic disorders, retinopathies and cancer.
  • the disease is a neurological disease, or a disease of the eye, preferably a retinopathy, most preferably familial exudative vitreoretinopathy (FEVR).
  • FEVR familial exudative vitreoretinopathy
  • the present invention relates to a process for diagnosing the presence of an FZD4-linked disease in a patient suspected of being afflicted therewith comprising detecting a mutation in the FZD4 gene in the genome of said patient.
  • the invention relates to such a process wherein said mutation is detected in a sample of DNA taken from the patient, most preferably wherein the mutation is the mutation of SEQ ID NO: 3 or SEQ ID NO: 5.
  • said detecting is accomplished by determining the ability of a nucleic acid probe comprising at least 15 contiguous nucleotides that are complementary to a mutated portion of the sequence of SEQ ID NO: 1 , most preferably wherein said probe comprises at least 30 contiguous nucleotides, especially where the probe comprises at least 50 contiguous nucleotides. In an especially preferred embodiment, the probe comprises at least 100 nucleotides contiguous nucleotides.
  • the processes disclosed herein are also useful in determining the risk of a patient in developing one or more of the above diseases, so that the present invention also relates to a process for determining a patient's risk of developing a disease where said patient is suspected to be at risk thereof, comprising detecting a mutation in the FZD4 gene in the genome of said patient.
  • detection may, in keeping with the foregoing, be by either in vitro or in vivo means.
  • said disease is a retinopathy, most preferably familial exudative vitreoretinopathy (FEVR).
  • FEVR familial exudative vitreoretinopathy
  • the mutation is a mutation in the gene corresponding to SEQ ID NO: 1.
  • the present invention relates to processes for identifying agents with therapeutic activity in treating diseases related to FZD4 gene activity, such as any of the diseases disclosed elsewhere herein.
  • the present invention relates to a process for identifying an agent having therapeutic activity in ameliorating the symptoms of an FZD4-linked disease comprising :
  • the present invention relates to processes for identifying agents with therapeutic activity in treating diseases related to FZD4 gene activity, such as any of the diseases disclosed elsewhere herein. Once such agents are identified through high throughput screening or other techniques, the compounds may be further tested in animals, such as humans. In one such embodiment, the present invention relates to a process for identifying an agent having therapeutic activity in ameliorating the symptoms of a disease comprising:
  • said disease is a retinopathy, most preferably familial exudative vitreoretinopathy (FEVR).
  • FEVR familial exudative vitreoretinopathy
  • the present invention relates to a process for treating a disease in a patient afflicted therewith comprising administering to said patient an effective amount of an agent that modulates the biological activity of FZD4, wherein such agent was first identified as having such activity by process as disclosed herein.
  • Such disease may be any of those as disclosed herein, such as vascular disease, angiogenic disorders, retinopathies and cancer, preferably a retinopathy, most preferably familial exudative vitreoretinopathy (FEVR).
  • vascular disease angiogenic disorders
  • retinopathies preferably a retinopathy, most preferably familial exudative vitreoretinopathy (FEVR).
  • FEVR familial exudative vitreoretinopathy
  • the present invention relates to a process for identifying an agent having therapeutic activity in ameliorating the symptoms of a disease comprising:
  • the animal model demonstrates the effect of
  • Preferred rodent models include an ROP mouse model (Smith LE, Wesolowski E, McLellan A, Kostyk SK, D'Amato R, Sullivan R, D'Amore PA. Oxygen-induced retinopathy in the mouse. Invest Ophthalmol Vis Sci. 1994 Jan;35(1):101 -11); an ROP rat model (Reynaud X, Dorey CK. Extraretinal neovascularization induced by hypoxic episodes in the neonatal rat. Invest Ophthalmol Vis Sci.
  • the present invention also finds use in inducing ocular angiogenesis.
  • Preferred rodent models for tumor growth and angiogenesis include mouse tumour xenografts (Lyden D, Young AZ, Zagzag D, Yan W, Gerald W, O'Reilly R, Bader BL, Hynes RO, Zhuang Y, Manova K, Benezra R. Id1 and Id3 are required for neurogenesis, angiogenesis and vascularization of tumour xenografts. Nature. 1999 Oct 14;401 (6754):670-7. and O'Reilly MS, Boehm T, Shing Y, Fukai N, Vasios G, Lane WS, Flynn E, Birkhead JR, Olsen BR, Folkman J. Endostatin: an endogenous inhibitor of angiogenesis and tumor growth. Cell. 1997 Jan 24;88(2):277-85.)
  • such a therapeutic agent is a gene product encoded by the FZD4 gene, preferably a polypeptide, such as one that comprises an amino acid sequence at least 95% identical to the sequence of SEQ ID NO: 2, especially where said polypeptide differs from the polypeptide of SEQ ID NO: 2 by a conservative amino acid substitution, most preferably wherein said polypeptide comprises the amino acid sequence of SEQ ID NO: 2.
  • amino acid substitutions can be made anywhere within the sequence of the peptide epitopes and such substitutions are by no means, limited to the sequences disclosed herein. Such substitutions may be of a conservative nature, for example, where one amino acid is replaced by an amino acid of similar structure and characteristics, such as where a hydrophobic amino acid is replaced by another hydrophobic amino acid. Even more conservative would be replacement of amino acids of the same or similar size and chemical nature, such as where leucine is replaced by isoleucine.
  • amino acids may be described chemically as hydrophobic, polar, acidic, or basic. Less conservative substitutions might involve the replacement of one amino acid by another that has similar characteristics but is somewhat different in size, such as replacement of an alanine by an isoleucine residue. Highly nonconservative replacements might involve substituting an acidic amino acid for one that is polar, or even for one that is basic in character.
  • radical substitutions cannot be dismissed as potentially ineffective since chemical effects are not totally predictable and radical substitutions might well give rise to serendipitous effects not otherwise predictable from simple chemical principles.
  • substitutions may involve structures other than the common L-amino acids.
  • D-amino acids might be substituted for the L- amino acids commonly found in the antigenic peptides of the invention and yet still be encompassed by the disclosure herein.
  • amino acids possessing non-standard R groups i.e., R groups other than those found in the common 20 amino acids of natural proteins may also be used for substitution purposes to produce immunogens and immunogenic polypeptides according to the present invention.
  • compositions useful herein also contain a pharmaceutically acceptable carrier, including any suitable diluent or excipient, which includes any pharmaceutical agent that does not itself induce the production of antibodies against the polypeptides disclosed herein where such is other than an intended therapeutic effect or where such would be otherwise harmful to the individual receiving the composition, and which may be administered without undue toxicity.
  • Pharmaceutically acceptable carriers include, but are not limited to, liquids such as water, saline, glycerol and ethanol, and the like.
  • the present invention also relates to a process that comprises a method for producing a product comprising identifying an agent according to one of the disclosed processes for identifying such an agent (i.e., the therapeutic agents identified according to the assay procedures disclosed herein) wherein said product is the data collected with respect to said agent as a result of said identification process, or assay, and wherein said data is sufficient to convey the chemical character and/or structure and/or properties of said agent.
  • identifying an agent i.e., the therapeutic agents identified according to the assay procedures disclosed herein
  • said product is the data collected with respect to said agent as a result of said identification process, or assay, and wherein said data is sufficient to convey the chemical character and/or structure and/or properties of said agent.
  • the present invention specifically contemplates a situation whereby a user of an assay of the invention may use the assay to screen for compounds having the desired enzyme modulating activity and, having identified the compound, then conveys that information (i.e., information as to structure, dosage, etc) to another user who then utilizes the information to reproduce the agent and administer it for therapeutic or research purposes according to the invention.
  • information i.e., information as to structure, dosage, etc
  • the user of the assay may screen a number of test compounds without knowing the structure or identity of the compounds (such as where a number of code numbers are used the first user is simply given samples labeled with said code numbers) and, after performing the screening process, using one or more assay processes of the present invention, then imparts to a second user (user 2), verbally or in writing or some equivalent fashion, sufficient information to identify the compounds having a particular modulating activity (for example, the code number with the corresponding results).
  • This transmission of information from user 1 to user 2 is specifically contemplated by the present invention.
  • Homolog H.sapiens FZD4 100 % / 536 aa; M.musculus SMOH 27 % /
  • Tissue Expression cDNA sources:Adipose, Aorta, Bone, Brain, Breast, Gall bladder, Germ Cell, Heart, Kidney, Lung, Lymph, Parathyroid, Placenta, Prostate, Skin, Smooth muscle, Stomach, Uterus, Whole embryo, breast, colon, colon-normal, head-neck, head-normal, heart, kidney, lung, lung- normal, lung-tumor, muscle (skeletal), ovary, small intestine.
  • FZ 'frizzled'
  • FZD4 encodes a deduced 537-amino acid protein that has a cysteine-rich domain in the N-terminal extracellular region, 2 cysteine residues in the second and third extracellular loops, 2 N-linked glycosylation extracellular sites, and the S/T-X-V motif in the C terminus. Amino acid sequence identity with other FZD proteins ranged from 39 to 52% in the N terminus to 42 to 69% in the transmembrane domains.
  • Northern blot analysis revealed expression of a 7.7-kb transcript of FZD4 in large amounts in adult heart, skeletal muscle, ovary, and fetal kidney, in moderate amounts in adult liver, kidney, pancreas, spleen, and fetal lung, and in small amounts in placenta, adult lung, prostate, testis, colon, fetal brain, and liver. Expression was also strong in HeLa S3 cells but not in several other cancer cell lines. Kirikoshi et al. (1999) determined that the FZD4 gene contains 2 exons. By FISH, they mapped the FZD4 gene to chromosome 11q14-q21.
  • FZD4 has not previously been linked to human diseases, either by mutation or by aberrant expression or biological activity.
  • a human EST fragment of FZD4 was said to have a proposed utility in diagnosis of certain cancers, e.g., breast cancer and colorectal cancer in PCT publication no. WO 99/26960, although no experimental data was provided.
  • large deletions of chromosome 7 that encompass Fzd4 and possibly many other genes resulted in defects in thymus gland development and cardiac disorders. Both disorders were said to result from defects in the cardiac neural crest during embryogenesis, and did not relate to retinopathy of any kind. DeRossi et al. 2000. genesis. 27:64-75.
  • Table 1 sets out corrections to the previously disclosed sequence of the FZD4 gene (GenBank Accession NM-012193) and resulting amino acid changes where applicable.
  • Figure 8 illustrates an alternate methionine translation initiation site that may be active in some cells of the body.
  • the 2nd ATG is closer to the optimal Kozak consensus C-C-AG-C-C-A-T-G-G context for initiation of translation.
  • the second ATG is the basis for numbering the FZD4 protein in GenBank (for example, Accession No. NM-012193). For consistency, the same numbering is used in the figures of this specification.
  • Figure 7 illustrates the 2 exon genomic structure of FZD4. ' Domains marked in black encode open reading frames.
  • Figure 7 (top) sets out the basic genomic arrangement of exons. Intron 1 is completely spliced out of the translated mRNA for the full length active protein.
  • Figure 4b illustrates a potential alternative transcript of FZD4 resulting from alternative splicing. In the alternate transcript, a failure of the splicing event at the Exon 1 /Intron 1 boundary leads to an open reading frame from Exon 1 that continues briefly into Intron 1 before terminating. The resulting gene product encodes a portion of the extra-cellular domain of FZD4 but terminates prior to the transmembrane domain. It is believed that this alternative transcript could be involved in regulating expression or activity of FZD4.
  • Figure 7 (bottom) illustrates the approximate location of the mutation directly associated with FEVR.
  • the present invention includes screening assays, such as where a large number of compounds are to be screened for activity in modulating FZD4 biological activity.
  • modulation may include either an increase or a decrease in FZD4 biological activity.
  • FZD4 biological activity as used herein is a very broad term that relates to all the directly or indirectly measurable and identifiable biological activities of the FZD4 gene and protein.
  • FZD4 biological activity includes, but is not limited to, all those biological processes, interactions, or binding of ligands, proteins, membrane components or other compounds (such as small organic compounds), binding behavior, binding-activity relationships, pKa, pD, enzyme kinetics, stability, and functional assessments of the protein.
  • Relating to FZD4 biological activity in cell fractions, reconstituted cell fractions or whole cells, these activities include, but are not limited to the ligand or antibody binding behavior and all measurable consequences of this effect, such measurement of any signaling cascade, membrane composition and behavior, cell growth, development or behavior and other direct or indirect effects of FZD4 activity.
  • FZD4 biological activity includes the rate, scale or scope of transcription of genomic DNA to generate FZD4 mRNA or its alternate transcripts; the effect of regulatory proteins on such transcription, the effect of modulators of such regulatory proteins on such transcription; plus the stability and behavior of mRNA transcripts, post-transcription processing, mRNA amounts and turnover, and all measurements of translation of the mRNA into polypeptide sequences.
  • FZD4 biological activity in organisms this includes but is not limited biological activities which are identified by their absence or deficiency in disease processes or disorders caused by aberrant FZD4 biological activity in those organisms. Broadly speaking, FZD4 biological activity can be determined by all these and other means for analyzing biological properties of proteins and genes that are known in the art.
  • FZD4 screening assays Those skilled in the art are able to identify measurable biological activities of FZD4 which can be usefully incorporated into low or high throughput screening assays. Some possible assays are described herein for illustration purposes. Based on these teachings, other embodiments of FZD4 screening assays will be directly reduced to practice.
  • an assay for activity of FZD4 comprising using members of the Wnt family as ligands to measure binding of radiolabeled ligands to cell membrane fragments containing the receptor.
  • Wnt11 is the ligand.
  • Other Wnt family ligands may also be used.
  • GPCRs G-protein coupled receptors
  • SPA sintillation proximity assay
  • Protein-protein interactions of FZD4 can also provide a fruitful source of assays. Compounds which modulate these interactions are potential therapeutic agents according to this invention.
  • the extra-cellular domain of FZD4 in a nucleic acid construct could be transfected into yeast, and tested in a standard yeast 2 hybrid assay. Drosophila wingless protein or other Wnt ligands would be useful controls.
  • a further exemplary assay was set out in Hsieh, et al. 1999. PNAS USA 96:3546-3551 where Wnt/Frizzled interactions were studied using IgG fusion proteins containing the extracellular domain of frizzled 8.
  • a sepharose was used for affinity purification of the frizzled-lgG fusion protein and Wnt8 coprecipitated with the frizzled-lgG fusion protein in a solution binding assay.
  • a fizzled-lgG fusion protein can also be used in a solid phase enzyme-linked binding assay to measure affinity for Wnt-alkaline phosphatase fusion proteins.
  • An important aspect of the instant invention is that the mutation identified in FEVR patients, namely the M493-W494del, occurs at the same site as the mutation reported in the human Smoothened (SMO - see OMIM *601500)) gene homolog reported in Xie, J. et al. 1998. Nature 391 :90-92.
  • the SMO mutation appears to be a gain of function mutation whereby the mutation prevents the inhibition of this protein by a regulating protein, leading to constitutive signaling.
  • the mutation may disrupt the formation of a polar pocket that keeps the receptor in a latent state!
  • the inventors hereto recognize that the FEVR mutation may also be a "gain of function" mutation. As such, it remains to be determined whether agonists or antagonists of normal FZD4 biological activity are preferred for treatment of any one specific disease. Additionally, it may be useful to employ the mutant FZD4 gene/protein in screening assays to identify agonists or antagonists thereof.
  • FZD4 The putative intracellular signaling pathways of FZD4 also suggests measurable FZD4 biological activities that are suitable for low or high throughput screening assays.
  • FZD4 may stimulate one or more of three known signaling pathways, or other pathways. Since the activated FZD4 intracellular domain may bind to Dishevelled protein, dishevelled expression, binding or behavior may indicate the effect of modulation of FZD4. Other dishevelled interacting proteins such as beta-catenin, Gsk3 or GBP may also respond to modulation of FZD4 activity. In another pathway, FZD4 modulates Protein Kinase C (PKC) activity.
  • PKC Protein Kinase C
  • PKC activation can be measured by various ways, including auto (trans) phosphorylation of PKC or by migration of PKC from the cytoplasm to the plasma membrane.
  • auto (trans) phosphorylation of PKC or by migration of PKC from the cytoplasm to the plasma membrane.
  • phosphorylation of a synthetic peptide by CAMKII can be measured as an outcome of FZD4 modulation. See Sheldahl et al. 1999. Curr. Biol.
  • FZD4 with intracellular proteins thus suggests a process for identifying a FZD4 modulating agent comprising contacting a compound with an FZD4 polypeptide in the presence of an intracellular FZD4- interacting protein, preferably a protein containing a PDZ domain, and determining a difference in binding of said intracellular protein to said FZD4 polypeptide compared to when said compound is not present, thereby identifying an FZD4 modulating agent.
  • the inventors have recognized that the protein Wnt11 is implicated in the related ocular disease known as neovascular inflammatory vitreoretinopathy (VRNI), by virtue of its location in a locus identified for autosomal dominant neovascular inflammatory vitreoretinathy (VRNI [MIM 193235] (Sheffield, V. C; Kimura, A. E.; Folk, J. O; Bennett, S. R.; Streb, L. M.; Nichols, B. E.; Stone, E. M. The gene for autosomal dominant neovascular inflammatory vitreoretinopathy maps to 11 q13. (Abstract) Am. J. Hum. Genet.
  • the VRNI locus contains WNT11 , a potential ligand for FZD4.
  • a_process for identifying an agent having therapeutic activity in ameliorating Wnt11 -linked diseases comprising :
  • the Wnt11 -linked disease may be one of vascular disease, angiogenic disorders, retinopathies and cancer, or may include a neurological disease or disease of the eye, including age related macular degeneration.
  • the disease is neovascular inflammatory vitreoretinopathy (VRNI).
  • the Wnt11 biological activity being measured is any measurable biological activity of wnt11 , but preferably its FZD4 binding activity, as measured in a protein/protein binding assay.
  • FZD4 assays can also be developed based on standard assays well known to those in the art including RNA expression or stability assays, amount or level of FZD4 protein in a cell, and the like. Many other types of screening assays can also be employed. Some of these may not actually use human FZD4 genes/proteins, but may use orthologs or variants of orthologs found in other species.
  • modulators of FZD4 should be useful in treating a wide variety of diseases, herein sometimes called FZD4-linked diseases.
  • diseases include any disease that is related to the cellular processes that underlie vascularization of the human retina (set out in Hughes, S. et al. 2000. Invest. Opthalmol. Vis. Sci. 41 (5): 1271 - 1228) and which are now treatable by modulators of FZD4.
  • the therapeutic applications of this invention have implications substantially beyond the rare clinical disorder of FEVR.
  • This invention now teaches therapeutic targets and agents for a wide variety of diseases including FEVR, retinopathies, angiogenic and vascularization disorders in neurological tissues and throughout the body, including cancer and neoplasia.
  • ROP Retinopathy of Prematurity
  • Coat's disease Norrie disease
  • Retinal angiomatosis Ocular toxocariasis
  • retinoblastoma Diabetic retinopathy
  • Sickle cell retinopathy Eales disease
  • Incontinentia pigmenti other ischemic eye conditions (generally including arterial occlusive disease, venous occlusive disease, and vasculitis)
  • Neovascular inflammatory vitreoretinopathy (VRNI) retinitis pigmentosa
  • persistent hyperplastic primary vitreous (PHPV) congential retinal folds
  • congenital retinal nonattachment Retinal dysplasia
  • microphthalmia Anophthalmia
  • other abnormalities associated with some FEVR patients such as abnormalities of platelet aggregation with arachidonic acid, collagen and epinephrine and abnormal bleeding times (see Chaudhuri et al.
  • FZD4-linked diseases may include ALS, stroke, acute spinal cord ischemia, and ischemic neuropathy affected by vascular growth or function. Other related diseases are set out in Jampol et al. 1994. Surv. Ophthalmol. 38:519-540.
  • modulators of FZD4 may also be useful in treating any of the other angiogenic disorders, for examples those set out in Liau, G. et al. 2001. DDT 6(13): 689- 696.
  • FZD4 modulating agents are useful for treating other diseases involving the physiological and pathological processes of neovascularization.
  • these modulators are useful for treating diseases, conditions and disorders including the following neovascularization indications: 1) Physiological neovascularization, including female estrus cycle, pregnancy, wound healing, collateral formation, exercise-induced hypertrophy; 2) Indications that may require stimulation of neovascularization, including induction of collateral vessel formation (including myocardial ischemia, peripheral ischemia, cerebral ischemia), coronary artery disease, peripheral vascular disease, wound healing, engraftment subsequent to organ transplantation such as islet cell tranplantation, fracture and tendon repair, reconstructive powery, tissue engineering, restenosis, hair loss, decuitus and sasis ulcers, gastrointestinal ulcers, placental insufficiency, aseptic necrosis,.
  • neovascularization indications including myocardial ischemia, peripheral ischemia, cerebral ischemia), coronary artery disease, peripheral
  • vascular dementia Alzheimer's Disease, cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL); thyroid psuedocyst and lymphoedema; 3) Indications that may require inhibition of neovascularization such as cancer tumour growth and metastasis, ocular neovascularization (including age-related macular degeneration, diabetic retinopathy, retinopathy of prematurity, choroidal neovascularization), rehomatoid arthritis, synovitis, bone and cartilage destruction, pannus growth, osteophyte formation, psoriasis, obesity, haemangiomas, Kaposi's sarcoma, atherosclerosis (including atherosclerotic plaque rupture), endometriosis, warts, excess hair growth, scar keloids, allergic oedema, dysfunctional uterine bleeding, follicular cysts, ovarian
  • the present invention also relates to therapeutic agents, regardless of molecular size or weight, effective in treating and/or diagnosing and/or preventing any of the diseases disclosed herein, preferably where such agents have the ability to modulate activity and/or expression of the FZD4 disclosed herein, and most preferably where said agents have been determined to have such activity through at least one of the screening assays disclosed according to the present invention.
  • test compounds are conveniently compiled into libraries of such compounds, and a key object of the screening assays of the invention is to select which compounds are relevant from libraries having hundreds of thousands, or millions of compounds having unknown therapeutic efficacy.
  • test extracts or compounds are not critical to the screening procedure(s) of the invention. Accordingly, virtually any. number of chemical extracts or compounds can be screened using the exemplary methods described herein. Examples of such extracts or compounds include, but are not limited to, plant-, fungal-, prokaryotic- or animal-based extracts, fermentation broths, and synthetic compounds, as well as modification of existing compounds. Numerous methods are also available for generating random or directed synthesis (e.g., semi-synthesis or total synthesis) of any number of chemical compounds, including, but not limited to, saccharide-, lipid-, peptide-, and nucleic acid-based compounds.
  • Synthetic compound libraries are commercially available from Brandon Associates (Merrimack, NH) and Aldrich Chemical (Milwaukee, Wl).
  • libraries of natural compounds in the form of bacterial, fungal, plant, and animal extracts are commercially available from a number of sources, including Biotics (Sussex, UK), Xenova (Slough, UK), Harbor Branch Oceangraphics Institute (Ft. Pierce, FL), and PharmaMar, U.S.A. (Cambridge, MA).
  • natural and synthetically produced libraries are produced, if desired, according to methods known in the art, e.g., by standard extraction and fractionation methods.
  • any library or compound is readily modified using standard chemical, physical, or biochemical methods.
  • the present invention relates to agents capable of modulating the activity and/or expression of the FZD4 receptor as disclosed herein, such as where said receptor is present on a cell, especially where said modulating ability was first identified using an assay of comprising FZD4 or a gene encoding FZD4, or an assay which measures FZD4 activity according to this disclosure.
  • the term "capable of modulating” refers to the characteristic of such an agent whereby said agent has the effect of changing the overall biological activity of FZD4, either by increasing or decreasing said activity, under suitable conditions of temperature, pressure, pH and the like so as to facilitate such modulation to a point where it can be detected either qualitatively or quantitatively and wherein such modulation may occur in either an in vitro or in vivo environment.
  • the term “modulation” is used herein to mean a change in activity, more specifically either an increase or decrease in such activity
  • the term “activity” is not to be limited to specific enzymatic activity alone (for example, as measured in units per milligram or some other suitable unit of specific activity) but includes other direct and indirect effects of the protein, including increases in enzyme activity due not to changes in specific enzyme activity ' but due to changes (i.e., modulation) of expression of polynucleotides encoding and expressing said FZD4 enzyme.
  • FZD4 activity may also be influenced by agents which bind specifically to substrates of FZD4. Thus, the term .
  • modulation means a change in FZD4 activity regardless of the molecular genetic level of said modulation, be it an effect on the enzyme per se or an effect on the genes encoding the enzyme or on the RNA, especially mRNA, involved in expression of the genes encoding said enzyme.
  • modulation by such agents can occur at the level of DNA, RNA or enzyme protein and can be determined either in vivo or ex vivo.
  • First identified means the first establishment, confirmation or proof of utility, in this case as a modulator of the FZD4 target gene or protein.
  • said assay is any of the assays disclosed herein according to the invention.
  • the agent(s) contemplated by the present disclosure includes agents of any size or chemical character, either large or small molecules, including proteins, such as antibodies, nucleic acids, either RNA or DNA, and small chemical structures, such as small organic molecules.
  • the present invention contemplates agents wherein said agent is useful in treating, preventing and/or diagnosing a disease or condition which is identified as being FZD4 related according to this invention.
  • the present invention contemplates a process for identifying an agent having therapeutic activity in ameliorating a Wnt11 -related disease comprising:
  • a screening assay such as a high throughput screening assay, will identify several or even many compounds which modulate the activity of the assay protein.
  • the compound identified by the screening assay may be further modified before it is used in humans as the therapeutic agent.
  • combinatorial chemistry is performed on the modulator, to identify possible variants that have improved absorption, biodistribution, metabolism and/or excretion, or other important therapeutic aspects.
  • the essential invariant is that the improved compounds share a particular active group or groups which are necessary for the desired modulation of the target protein.
  • Many combinatorial chemistry and medicinal chemistry techniques are well known in the art.
  • therapeutic compounds identified using an FZD4 screening assay of the invention include actual compounds so identified, and any analogs or combinatorial modifications made to a compound which is so identified which are useful for treatment of the disorders claimed herein.
  • FZD4 activity can be administered to a patient at therapeutically effective doses to treat or ameliorate the diseases suggested herein.
  • a therapeutically effective dose refers to that amount of the compound sufficient to result in treatment or amelioration of symptoms of the disease, as specifically determined by those skilled in the art.
  • Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD 50 (the dose lethal to 50% of the population) and the ED 50 (the dose therapeutically effective in 50% of the population).
  • the dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD 50 / ED 5 o- Compounds which exhibit large therapeutic indices are preferred. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.
  • the data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans.
  • the dosage of such compounds lies preferably within a range of circulating concentrations that include the ED 5 o with little or no toxicity.
  • the dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.
  • the therapeutically effective dose can be estimated initially from cell culture assays.
  • a dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC 50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture.
  • IC 50 i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms
  • levels in plasma may be measured, for example, by high performance liquid chromatography.
  • compositions for use in accordance with the present invention may be formulated in conventional manner using one or more physiologically acceptable carriers or excipients.
  • the compounds and their physiologically acceptable salts and solvates may be formulated for administration by inhalation or insufflation (either through the mouth or the nose) or oral, buccal, parenteral or rectal administration.
  • the pharmaceutical compositions may take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulphate).
  • binding agents e.g., pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose
  • fillers e.g.,
  • Liquid preparations for oral administration may take the form of, for example, solutions, syrups or suspensions, or they may be presented as a dry product for constitution with water or other suitable vehicle before use.
  • Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid).
  • the preparations may also contain buffer salts, flavoring, coloring and sweetening agents as appropriate.
  • Preparations for oral administration may be suitably formulated to give controlled release of the active compound.
  • compositions may take the form of tablets or lozenges formulated in conventional manner.
  • the compounds for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • the dosage unit may be determined by providing a valve to deliver a metered amount.
  • Capsules and cartridges of e.g. gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
  • the compounds may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion.
  • Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi- dose containers, with an added preservative.
  • the compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
  • the compounds may also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.
  • the compounds may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection.
  • the compounds may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
  • compositions may, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the active ingredient.
  • the pack may for example comprise metal or plastic foil, such as a blister pack.
  • the pack or dispenser device may be accompanied by instructions for administration.
  • Biological modulators of FZD4 biological activity include FZD4 antibodies and anti-idiotypic antibodies (including Fab fragments), nucleotide constructs that inhibit expression of FZD4 gene (transcription factor inhibitors, antisense and ribozyme molecules, or gene or regulatory sequence replacement constructs), or promote expression of FZD4 (e.g., expression constructs in which FZD4 coding sequences are operatively associated with expression control elements such as promoters, promoter/enhancers, etc.).
  • the invention also relates to host cells and animals genetically engineered to express human FZD4 (or mutants thereof) that may be administered to patients either in vivo or ex vivo.
  • compositions are suggested in PCT Publication WO 99/26960 (Inventor Shyjan, A.); however the instant disclosure now provides the correct full length nucleotide and protein sequences for use in these techniques, and additionally provides important new uses for these compositions that were not previously known or understood.
  • FZD4 protein fragments themselves may be used as therapeutic agents.
  • FZD4 protein products especially soluble derivatives
  • FZD4 protein products such as peptides corresponding to functional domains of FZD4 and fusion protein products (especially FZD4-lg fusion proteins, i.e., fusions of FZD4 or a domain of the FZD4 to an IgFc)
  • antibodies and anti-idiotypic antibodies including Fab fragments
  • antagonists or agonists including compounds that modulate signal transduction which may act on downstream targets in the FZD4 signal transduction pathway
  • an effective amount of soluble FZD4, a functional domain of FZD4 or FZD4 functional domain-lgFc fusion protein or an anti-idiotypic antibody (or its Fab) that mimics a FZD4 functional domain would "mop up” or “neutralize” endogenous ligands such as the Wnt family proteins, and prevent or reduce binding and receptor activation, leading to treatment or amelioration of the diseases disclosed herein.
  • Antibodies and antibody fragments that specifically recognize one or more epitopes of FZD4, or epitopes of conserved variants of FZD4, or peptide fragments of the FZD4 are also encompassed by the invention.
  • Such antibodies include but are not limited to polyclonal antibodies, monoclonal antibodies (mAbs), humanized or chimeric antibodies, single chain antibodies, Fab fragments, F(ab')2 fragments, fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies, and epitope-binding fragments of any of the above.
  • the method includes immunizing a transgenic non-human animal, e.g., a transgenic mouse, having a genome comprising a human heavy chain transgene and a human light chain transgene, with a purified or enriched preparation of the FZD4 protein, or a fragment thereof.
  • B cells e.g., splenic B cells
  • myeloma cells to form immortal, hybridoma cells that secrete human monoclonal antibodies against the FZD4 protein.
  • Humanized antibodies may also be produced, for example by replacing an immunogenic portion of an antibody with a corresponding, but non- immunogenic portion (i.e. chimeric antibodies)
  • a corresponding, but non- immunogenic portion i.e. chimeric antibodies
  • various host animals may be immunized by injection with the FZD4, an FZD4 peptide (e.g., one corresponding the a functional domain of the receptor), truncated FZD4 polypeptides (FZD4 in which one or more domains has been deleted), functional equivalents of the FZD4 or mutants of the FZD4.
  • FZD4 peptide e.g., one corresponding the a functional domain of the receptor
  • FZD4 polypeptides FZD4 in which one or more domains has been deleted
  • functional equivalents of the FZD4 or mutants of the FZD4 may include but are not limited to rabbits, mice, goats and rats, to name but a few.
  • adjuvants may be used to increase the immunological response, depending on the host species, including but not limited to Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and Corynebacterium parvum.
  • BCG Bacille Calmette-Guerin
  • Corynebacterium parvum BCG (bacille Calmette-Guerin) and Corynebacterium parvum.
  • Polyclonal antibodies are heterogeneous populations of antibody molecules derived from the sera of the immunized animals. There are various expression systems that can be used for the production of whole antibodies and antibody fragments.
  • bacterial or mammalian cell culture and transgenic animals or plants include bacterial or mammalian cell culture and transgenic animals or plants.
  • the expression system of choice is determined by the intended application and the desired yield, as is known by those skilled in the art. For example, animal cell culture and transgenic expression systems are desirable if glycosylation of the antibody is required, whereas bacterial expression systems are more efficient for production of unglycosylated antibodies, Fab fragments and the like. See Chad, HE and Chamow, SM. 2001. Curr. Opin. Biotech. 12: 188- 193.
  • Nucleic acid sequencing of FZD4 gene in a patient can provided valuable information for predictive diagnosis of FEVR. Based on the disclosure herein, those skilled in the art can develop nucleic acid sequencing/analysis compositions methods and kits that are suitable for diagnosis of FEVR. Any method of determining the FZD4 gene sequence can be used in the method of this invention, including full length or partial gene sequencing, probe based assays, RFLP and all other techniques known to those in the art.
  • fzd4 ocular diseases, angiogenic diseases and other disorders
  • ROP Retinopathy of Prematurity
  • Coat's disease Norrie disease
  • Retinal angiomatosis Diabetic retinopathy
  • VRNI Neovascular inflammatory vitreoretinopathy
  • PGPV persistent hyperplastic primary vitreous
  • congenital retinal folds diagnostic gene sequence of fzd4 can then be used to confirm or reject such a diagnosis.
  • sequence analysis of the FZD4 gene can also be used to predict drug responsiveness, susceptibility to side-effects of drugs, and, importantly, it is useful for designing clinical trials.
  • the FZD4 polymorphisms or mutations disclosed herein can be correlated to a patient response database in order to generate a prognostic database for aiding selection of an appropriate therapeutic regime for a patient.
  • FZD4 proteins or peptides, FZD4 fusion proteins, FZD4 nucleotide sequences (oligonucleotides for probes, sequencing primers or PCR primers), antibodies, antagonists and agonists can be useful for the detection of mutant FZD4s or inappropriately expressed FZD4s for the diagnosis of the diseases disclosed herein.
  • Single nucleotide polymorphisms (SNPs) in FZD4 are related to drug responsiveness, drug side effects, and are implicated in disease and disorders processes.
  • SNPs Single nucleotide polymorphisms
  • a representative sample of possible polymorphisms of FZD4 are set forth in Table 2.
  • the sequences of Table 2 are numbered SEQ ID NO: 7-54 (top to bottom of the table). These sequences are all relative to the sequence found in GenBank entry NM-012193.1).
  • nucleotide sequences are useful in developing probes for use in the diagnostic methods disclose elsewhere herein.
  • probes comprise nucleotide sequences of at least 15, possibly at least 30, preferably at least 50 and most preferably at least 100 nucleotides in length that are specific for mutated portions of the FZD4 gene sequence, such as the sequence of SEQ ID NO: 4, and which hybridize to sequences obtained from a patient suspected of having an FZD4-related disease, or otherwise being at risk thereof, so that such hybridization shows the presence of a mutation in the FZD4 gene of said patient.
  • the antibodies of the invention may be used in the detection of the FZD4 in a biological sample and may, therefore, be utilized as part of a diagnostic or prognostic technique whereby patients may be tested for abnormal amounts of FZD4. Such antibodies may also be utilized in conjunction with, for example, compound screening schemes, as described, above, for the evaluation of the effect of test compounds on expression and/or activity of the FZD4 gene product.
  • FZD4 A mutation in FZD4 associated with autosomal dominant familial exudative vitreoretinopathy (FEVR)
  • genomic DNA was extracted from blood samples from the 81 subjects by a standard salt extraction method.
  • DNA was genotyped using fluorescently-labeled repeat-containing microsatellite markers.
  • Original primer sequences for the markers were obtained from the Genome Database.
  • GTTTCTT SEQ ID NO: 133
  • Each reaction mixture (15uL) contained 10ng genomic DNA, 50mM KCI, 10mM Tris-HCL, pH 8.0, 2.5mM MgCI 2 , 0.25 ⁇ M dNTPs, 0.33 ⁇ M each primer, and 0.6U AmpliTaq Gold DNA polymerase (Applied Biosystems).
  • the thermal cycling profile consisted of 30 cycles of 94 Q C for 15s, 55 S C for 15s, and 72 Q C for 15s.
  • PCR products were resolved using an Applied Biosystems Prism 3100 Genetic Analyzer running GeneMapper software (Applied Biosystems) for allele identification.
  • Forward and reverse primer sequences are set out on Table 3 (SEQ ID NO: 55-120) and for the exons in Table 4 (SEQ ID NO: 121 -130) wherein the forward primers have odd sequence identification numbers and the reverse primers have even sequence identification numbers.
  • the interval between D11S1887 and D11S873 corresponds to a genetic size of -5cM, according to the Marshfield Center for Medical Genetics map. To substantiate this finding and increase the resolution of the interval, we performed fine mapping with an additional 24 markers, including several new dinucleotide-repeat markers identified from repeats on BAC clones.
  • New repeat markers relevant for fine mapping were AP000654CA1 ; CA2AP001528; AC024304CA1 ; AC072050CA1 ; AP000756CA1 ; AP000676CA1 ; CA2AC023888; CA1AC011088; AC018775CA1. All primer sequences are set out on Tables 3 and 4. The physical locations of the markers were evaluated by the UCSC Golden Path sequence mapping database and the Marshfield Center for Medical Genetics genetic mapping database. Definition of the FEVR minimal genomic region by haplotype analysis
  • Haplotype construction using the 33 polymorphic markers revealed five proximal and four distal recombinations. Of the nine individuals, five were affected and four were unaffected. The additional data narrowed the boundaries to approximately 1.55MB (based on the April freeze UCSC Golden Path Human Genome Browser) and indicated that the FEVR gene was located in the genomic region delimited by markers CA2AP001528 and CA2AC023888.
  • the FEVR interval contains only two known genes, FZD4 and
  • FLJ22104 FLJ22104, and numerous expressed sequence tags. All exons of FZD4 and FLJ22104 were analyzed for mutations in 2 patients with FEVR and 1 unaffected family member by sequence analysis of PCR-amplified fragments.
  • Tryptophan 494 is conserved in putative FZD4 orthologs ( Figure 4) and other members of the human frizzled/smoothened membrane receptor family ( Figure 3). Its position in the seventh transmembrane domain is analogous to the position of the activating Smoothened W535L mutation reported in sporadic basal-cell carcinoma.(Xie et al. 1998).
  • exons and splice-site junctions were amplified from 16 ng genomic DNA in a standard 60 ⁇ L PCR reaction volume using Platinum Taq (Invitrogen). PCR conditions were 35 cycles of 30s each of 94 S C, 57 Q C, and 72 a C.
  • the PCR-amplification products were purified on Qiagen spin columns according to the manufacturer's instructions. The purified samples were sequenced in both directions with dye terminators (Applied Biosystems). Sequence reactions were electrophoresed on ABI automated sequencers (Applied Biosystems), and the resultant traces were analyzed by the Staden software package and compared against normal sequences (Genbank). The mutation was described according to the nomenclature established by den Dunnen and Antonarakis (1996), and numbering was started from the initiating ATG codon as annotated in the GenBank reference sequence.
  • the genomic DNA was amplified by PCR using a 6-FAM-labeled forward primer (for fluorescence detection) or an unlabeled forward primer (for PCR-RFLP).
  • the forward primer was 5'- TGGTGGGCATCACTTCAGG-3' (SEQ ID NO: 131), and the reverse primer was 5'-GCCTTTTCCAGGCTTCACC-3' (SEQ ID NO: 132).
  • the PCR product was incubated with ⁇ /spl (5U) in a total volume (15 ⁇ L) for 1 h at 37 S C then resolved on 3% agarose gels. The presence of the deletion removes an ⁇ /spl site. Alternately, the PCR products were resolved without digestion using an Applied Biosystems Prism 3100 Genetic Analyzer. The wild-type allele is 140bp and the mutant allele is 134bp. (Data not shown). The results indicated that the mutation segregated with disease and was absent in the 59 unaffected chromosomes in the family and 94 chromosomes from other Caucasian individuals.
  • PLC PLC C
  • Vertebrate frizzled proteins signal through three known pathways: the canonical Wnt pathway, which stabilizes ⁇ -catenin and activates target genes in the nucleus (Wodarz et al. Annu. Rev. Cell Dev. Biol. 14, 59-88, 1998); the Wnt/Ca 2+ pathway, which activates CAMKII and PKC in a G-protein-dependent manner (Kuhl et al. Trends Genet. 16, 279-83, 2000); and the planar cell polarity pathway, which activates mitogen-activated protein kinase 8 (MAPK8 or J ⁇ //0 1 (Boutros et al. Ce// 94, 109-18, 1998).
  • frizzled-4 FEVR mutations were tested using in vitro assays. Both mutant and wild-type frizzled-4 proteins were tagged at their mature amino termini with an HA- epitope and expressed in 293T cells. Western blotting revealed that all three frizzled-4 proteins are expressed. To assess subcellular localization, COS7 cells were transfected with HA-tagged wild-type frizzled-4 or its mutants.
  • Permeabilized cells stained for the HA-epitope in the secretory pathway consistent with the western blot data showing that the mutant and wild-type frizzled-4 proteins are expressed.
  • the HA-epitope is only detected on the outside of the membrane in cells expressing wild-type and M493-W494del, but not in cells expressing L501fsX533.
  • wild-type and M493W494del accumulate at the plasma membrane, while L501fsX533 is defective in membrane localization.
  • frizzled-4 mutants are functional in signaling assays.
  • Human frizzled-4 signaling has not previously been studied, but the mouse ortholog has been observed to activate the Ca 2+ pathway in assays conducted using explants of Xenopus embryos (Sheldahl et al. Curr. Biol. 9, 695-8, 1999; Kuhl et al. J. Biol. Chem. 275, 12701 -12711 , 2000).
  • pTOS is a derivative of pDNA5/TO (Invitrogen) which contains the murine Ig kappa chain leader sequence (Invitrogen) followed by an HA-epitope tag and a 6XHis tag.
  • COS7 cells plated in 12 well dishes on coverslips were transfected via lipofectamine plus reagent (Invitrogen) at 50% confluence. 24 hours after transfection, the cells were washed with PBS, fixed with 4% formaldehyde pH 7.4 for 15 min. at RT, left unpermeabilized, or permeabilized with 0.5% digitonin for 15 min. at RT. Cells were stained with a mouse monoclonal anti- HA antibody (12CA5) diluted 1 :1000 in PBS/2% CS for 1 hr. at RT and a secondary Alexa488 conjugated goat anti-mouse antibody (Molecular Probes) diluted 1 :1000 in PBS/2% CS for 1 hr at RT. Cells were washed and mounted on slides with Vectashield (Vectalabs). Alexa488 fluorescence was detected via confocal microscopy.
  • a mouse monoclonal anti- HA antibody (12CA5) diluted 1 :1000 in PBS/2%
  • HA western blotting was performed as described Kaykas et al. (EMBO J. 20, 2641 -54, 2001 ) with the following modifications.
  • the primary antibody was used at a 1 :4000 dilution
  • the secondary antibody was HRP conjugated and was used at a 1 :5000 dilution
  • the blots were developed via ECL.
  • An HA-tagged version of EGFPZEO was used as a loading and transfection control.
  • HuMab mice can be immunized with a preparation of FZD4 or a fragment thereof, which has been purified or enriched according to any standard method.
  • HuMab mice contain a human immunoglobulin gene miniloci that encodes unrearranged human heavy (mu and gamma) and kappa light chain immunoglobulin sequences, together with targeted mutations that inactivate the endogenous mu and kappa chain loci (Lonberg, N. et al. (1994) Nature. 368(6474): 856- 859.). These mice exhibit reduced expression of mouse IgM or kappa, and in response to immunization, the introduced human heavy and light chain transgenes undergo class switching and somatic mutation to generate high affinity human IgGkappa monoclonals.
  • mice are 6 - 16 weeks of age upon the first immunization.
  • IP intraperitoneal
  • IP immunizations up to a total of 6
  • the immune response can be monitored over the course of the immunization protocol with plasma samples obtained by retroorbital bleeds.
  • the plasma can be screened, for example by ELISA or flow cytometry, and mice with sufficient titers of anti-FZD4 human immunoglobulin can be used for fusions. Mice can be boosted intravenously with antigen 3 days before sacrifice and removal of the spleen. It is expected that 2-3 fusions for each antigen are needed for success.
  • the mouse splenocytes are first isolated and fused with PEG to a mouse myeloma cell line based upon standard protocols. The resulting hybridomas are then screened for the production of antigen-specific antibodies. For example, single cell suspensions of splenic lymphocytes from immunized mice are fused to one-sixth the number of P3X63-Ag8.653 nonsecreting mouse myeloma cells (ATCC, CRL 1580) with 50% PEG.
  • Cells are plated at approximately 2 X 10 5 in flat bottom microtiter plate, followed by a two week incubation in selective medium containing 20% fetal calf serum, 18% "653" conditioned media, 5% origen (IGEN), 4 mM L-glutamine, 1 mM L- glutamine, 1 mM sodium pyruvate, 5 mM HEPES, 0.055 mM 2- mercaptoethanol, 50 units/ml penicilliin, 50 mg/ml streptomycin, 50 mg/ml gentamycin and 1 X HAT (Sigma; the HAT is added 24 hours after the fusion). After two weeks, cells are cultured in medium in which the HAT is replaced with HT.
  • selective medium containing 20% fetal calf serum, 18% "653" conditioned media, 5% origen (IGEN), 4 mM L-glutamine, 1 mM L- glutamine, 1 mM sodium pyruvate, 5 mM H
  • selected hybridomas can be grown in two-litre spinner-flasks for monoclonal antibody purification.
  • Supernatants can be filtered and concentrated before affinity chromatography with protein A-sepharose. (Pharmacia, Piscataway, NJ).
  • Eluted IgG can be checked by gel electrophoresis and high performance liquid chromatography to ensure purity.
  • the buffer solution can be exchanged into PBS, and the concentration can be determined by OD 280 using 1.43 extinction co-efficient.
  • the monolconal antibodies can be aliquoted and stored at -80°C until required for use according to the methods of the invention.

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Abstract

L'invention porte sur des mutations de gènes sélectionnés tels que le gène 4 de Frizzled ('fzd4'), qui sont les agents responsables de troubles héréditaires de la vue chez l'homme, tels que la vitréorétinopathie exudative familiale ('FEVR'), ainsi que leurs méthodes d'utilisation y compris dans le criblage d'agents thérapeutiques traitant ces maladies et/ou en améliorant les effets, ainsi que sur des procédés de diagnostic de l'état des atteintes à la vue causées par les mutations de ces gènes.
PCT/CA2002/001004 2001-07-05 2002-07-04 Procedes d'identification d'agents therapeutiques de traitement de maladies impliquant le gene WO2003005034A2 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005025590A2 (fr) * 2003-09-15 2005-03-24 Develogen Aktiengesellschaft Utilisation d'un produit proteique dg280 dans la prevention et le traitement de troubles metaboliques
WO2006040163A1 (fr) * 2004-10-13 2006-04-20 Eberhard-Karls-Universität Tübingen Universitätsklinikum Anticorps monoclonaux contre les recepteurs de la famille des frizzled
EP1836224A1 (fr) * 2004-12-23 2007-09-26 Laboratoires Serono S.A. Polypeptides de bcma et leur utilisation
WO2011050096A2 (fr) 2009-10-21 2011-04-28 Retinal Solutions Llc Méthodes et compositions utilisées pour le diagnostic et le traitement d'une rétinopathie génétique

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WO1997039357A1 (fr) * 1996-04-12 1997-10-23 The Board Of Trustees Of The Leland Stanford Junior University COMPOSITIONS A BASE DU RECEPTEUR DE Wnt ET PROCEDES D'UTILISATION DE CELLES-CI
WO1999026960A2 (fr) * 1997-11-25 1999-06-03 Millennium Pharmaceuticals, Inc. Genes codant des proteines ressemblant a de la frisee
EP1001023A2 (fr) * 1998-09-09 2000-05-17 Smithkline Beecham Plc Frizzled-4, un possible récepteur pour Wnt

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WO1997039357A1 (fr) * 1996-04-12 1997-10-23 The Board Of Trustees Of The Leland Stanford Junior University COMPOSITIONS A BASE DU RECEPTEUR DE Wnt ET PROCEDES D'UTILISATION DE CELLES-CI
WO1999026960A2 (fr) * 1997-11-25 1999-06-03 Millennium Pharmaceuticals, Inc. Genes codant des proteines ressemblant a de la frisee
EP1001023A2 (fr) * 1998-09-09 2000-05-17 Smithkline Beecham Plc Frizzled-4, un possible récepteur pour Wnt

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KIRIKOSHI H ET AL: "MOLECULAR CLONING AND CHARACTERIZATION OF HUMAN FRIZZLED-4 ON CHROMOSOME 11Q14-Q21" BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, ACADEMIC PRESS INC. ORLANDO, FL, US, vol. 264, no. 3, 2 November 1999 (1999-11-02), pages 955-961, XP001057078 ISSN: 0006-291X cited in the application *

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005025590A2 (fr) * 2003-09-15 2005-03-24 Develogen Aktiengesellschaft Utilisation d'un produit proteique dg280 dans la prevention et le traitement de troubles metaboliques
WO2005025590A3 (fr) * 2003-09-15 2005-11-03 Develogen Ag Utilisation d'un produit proteique dg280 dans la prevention et le traitement de troubles metaboliques
WO2006040163A1 (fr) * 2004-10-13 2006-04-20 Eberhard-Karls-Universität Tübingen Universitätsklinikum Anticorps monoclonaux contre les recepteurs de la famille des frizzled
US7659116B2 (en) 2004-10-13 2010-02-09 Eberhard-Karls-Universitaet Tuebingen Universitaetsklinikum Monoclonal antibody against frizzled receptor 4
EP1836224A1 (fr) * 2004-12-23 2007-09-26 Laboratoires Serono S.A. Polypeptides de bcma et leur utilisation
WO2011050096A2 (fr) 2009-10-21 2011-04-28 Retinal Solutions Llc Méthodes et compositions utilisées pour le diagnostic et le traitement d'une rétinopathie génétique
EP2491143A2 (fr) * 2009-10-21 2012-08-29 Retinal Solutions LLC Méthodes et compositions utilisées pour le diagnostic et le traitement d'une rétinopathie génétique
EP2491143A4 (fr) * 2009-10-21 2013-03-20 Retinal Solutions Llc Méthodes et compositions utilisées pour le diagnostic et le traitement d'une rétinopathie génétique

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