WO2002076467A1 - Compositions et procedes de regulation du metabolisme osseux et mineral - Google Patents

Compositions et procedes de regulation du metabolisme osseux et mineral Download PDF

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Publication number
WO2002076467A1
WO2002076467A1 PCT/US2002/008846 US0208846W WO02076467A1 WO 2002076467 A1 WO2002076467 A1 WO 2002076467A1 US 0208846 W US0208846 W US 0208846W WO 02076467 A1 WO02076467 A1 WO 02076467A1
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fgf
cell
gene
expression
phosphate
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PCT/US2002/008846
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Susan Schiavi
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Genzyme Corporation
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • 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
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1825—Fibroblast growth factor [FGF]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/50—Fibroblast growth factor [FGF]
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6887—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids from muscle, cartilage or connective tissue
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/475—Assays involving growth factors
    • G01N2333/50—Fibroblast growth factors [FGF]

Definitions

  • This invention relates to an isolated polynucleotide encoding an oncogenic osteomalacia-related gene and to methods of using this gene to modulate the activity of cells and diagnose and treat diseases characterized by abnormal phosphate metabolism or bone mineralization.
  • Oncogenic osteomalacia is an acquired hypophosphatemic syndrome associated with a variety of mesenchymal tumors and is characterized by low serum levels of phosphate and calcitriol, inappropriate phosphaturia secondary to inhibition of proximal renal tubular phosphate reabsorption, and defective bone demineralization.
  • OOM is caused by a humoral factor, termed phosphatonin (PTN), that is produced by these mesenchymal tumors.
  • PTN humoral factor
  • Tumor extracts can inhibit phosphate transport in vitro and produce phosphaturia and hypophosphatemia in vivo.
  • surgical removal of tumor tissue results in normalization of serum phosphorous and calcitriol, reverses phosphaturia and eventually results in remineralization of bone.
  • the mesenchymal tumors that are associated with OOM are characteristically slow-growing, complex, polymorphous neoplasms. Many of these mesenchymal tumors contain areas of dystrophic calcification, osseous metaplasia, poorly differentiated foci of chondroid tissue and osteoclast-like giant cells. The presence of these bony elements and calcification suggest these tumors express genes that have important functions in bone formation and mineralization.
  • OOM is clinically and biochemically similar to inherited disorders of phosphate wasting, X-linked Hypophosphatemic Rickets (XLH) and Autosomal Dominant Hypophosphatemic Rickets (ADHR).
  • XLH is caused by haploinsufficency of PHEX, an endopeptidase whose substrate is yet undefined.
  • FGF-23 a novel member of the fibroblast growth factor family
  • PTHrP parathyroid hormone related-pep tide
  • Efforts to identify and isolate the phosphaturic humoral factor produced by some of these mesenchymal tumors have been hampered by difficulty propagating the tumors. Moreover, the tumors often cease making the phosphaturic factor with passage in culture.
  • This invention provides methods for modulating phosphate homeostasis and/or renal phosphate transport by delivering agents that alter the expression of the FGF 23 gene or alter the activity of the FGF 23 protein.
  • the methods of the invention are useful for one or more of modulating bone mineralization, modulating renal phosphate transport, alleviating oncogenic osteomalacia-associated symptoms and treating phosphate homeostasis-related disease.
  • the invention further provides methods for reducing phosphate re-absorption by delivering to a subject FGF 23 protein or polynucleotides that encode the FGF 23 protein.
  • the invention provides methods for detecting and monitoring expression of the FGF 23 gene.
  • the invention provides methods for modulating the phenotype of a neoplastic cell associated with oncogenic osteomalacia by delivering an agent that alters the expression of the FGF 23 gene. Additionally, the invention provides methods for screening candidate agents to identify compositions that modify the activity of the FGF 23 gene and protein.
  • the invention provides for methods of modulating the activity of cells (e.g., responsiveness to a hormone or activator), by delivering to the cell the FGF 23 protein or polynucleotides that encode the FGF 23 protein.
  • This invention provides isolated polynucleotides useful in the methods identified herein.
  • the polynucleotides encode FGF 23 protein and are intended to include DNA, cDNA, RNA and genomic DNA.
  • Expression systems, including gene delivery vehicles such as liposomes and vectors, and host cells containing the polynucleotides are further provided by this invention.
  • the present invention also provides proteins encoded by the polynucleotides.
  • nucleic acid probes and primers that hybridize to invention polynucleotides are provided, as well as isolated nucleic acids comprising unique, expressed gene sequences.
  • the present invention further includes antisense oligonucleotides, antibodies, hybridoma cell lines and compositions containing same.
  • the present invention also provides methods of monitoring gene expression using invention polynucleotides.
  • the methods of monitoring gene expression are useful for detecting a cell expressing oncogenic osteomalacia-related polypeptide and for detecting a neoplastic cell associated with oncogenic osteomalacia.
  • This invention further provides methods for modulating the expression of the inventive polynucleotides, for altering the activity of the proteins encoded by the polynucleotides, and for treating symptoms of phosphate transport related diseases and diseases characterized by abnormal bone mineralization.
  • diseases include but are not limited to, oncogenic osteomalacia, X-linked hypophosphataemia rickets, rhabdomyolysis, osteoporosis, cardiamyopathy, tumoral calcinosis, renal failure, bone mineralization and low turnover bone disease.
  • This invention also provides a method for screening for candidate agents that modulate the expression of a polynucleotide of the invention or its complement, by contacting a test agent with a neoplastic cell associated with oncogenic osteomalacia and monitoring expression of the polynucleotide, wherein the test agent which modifies the expression of the polynucleotide is a candidate agent.
  • Figure 1 shows Sequence ID No. 1 ("SEQ ID NOT”), a polynucleotide encoding FGF 23. The sequence is reproduced from GenBank Accession No. NM_020638, that was submitted in connection with White, K.E., et al. (2000) Nat. Genetics 26(3):345-348.
  • Figure 2 shows the amino acid sequence containing 251 amino acid residues is SEQ ID NO:2.
  • An alternative sequences encoding human FGF23 is available under GenBank Accession No. XP_007056.
  • a cell includes a plurality of cells, including mixtures thereof.
  • compositions and methods include the recited elements, but not excluding others.
  • Consisting essentially of when used to define compositions and methods shall mean excluding other elements of any essential significance to the combination.
  • a composition consisting essentially of the elements as defined herein would not exclude trace contaminants from the isolation and purification method and pharmaceutically acceptable carriers, such as phosphate buffered saline, preservatives, and the like.
  • Consisting of shall mean excluding more than trace elements of other ingredients and substantial method steps for administering the compositions of this invention. Embodiments defined by each of these transition terms are within the scope of this invention.
  • polynucleotide and “nucleic acid molecule” are used interchangeably to refer to polymeric forms of nucleotides of any length.
  • the polynucleotides may contain deoxyribonucleotides, ribonucleotides, and/or their analogs.
  • Nucleotides may have any three-dimensional structure, and may perform any function, known or unknown.
  • polynucleotide includes, for example, single-, double-stranded and triple helical molecules, a gene or gene fragment, exons, introns, mRNA, tRNA, rRNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers.
  • a nucleic acid molecule may also comprise modified nucleic acid molecules.
  • a “gene” refers to a polynucleotide containing at least one open reading frame that is capable of encoding a particular polypeptide or protein after being transcribed and translated.
  • FGF 23 gene is a polynucleotide comprising an ordered sequence of nucleotides located in a particular position on a particular chromosome that encodes a polypeptide, such as the amino acid sequence shown in SEQ. ID NO 2 ( Figure 2).
  • the term gene is intended to include contiguous polynucleotide sequences such as promoters and enhancers that modulate expression.
  • FGF 23 gene refers to all orthologous sequences from divergent species, ie. homologous sequences encoding polypeptides that have the same activity in different species. It is particularly intended to include the FGF 23 genes of humans, simians and rodents.
  • FGF 23 polynucleotide means any ordered sequence of polynucleotides that encode polypeptide, a portion of such a peptide, or a portion of the FGF 23 gene.
  • An "FGF 23 polynucleotide” thus include cDNA's, probes, primers, and other molecules comprising polynucleotide sequences derived from the complete FGF 23 gene, eg. biologically equivalent polynucleotides.
  • Biologically equivalent polynucleotides are polynucleotides which differ from the polynucleotides described above, but produce the same phenotypic effect, such as the allele, splice variant and homolog. These altered, but phenotypically equivalent polynucleotides are referred to as "biologically equivalent polynucleotide” and "equivalent nucleic acids.”
  • the methods of the invention also encompasses polynucleotides characterized by changes in non-coding regions that do not alter the phenotype of the polypeptide produced therefrom when compared to the polynucleotide herein. This invention further envisions the use of polynucleotides, which hybridize to the polynucleotides of the subject invention under conditions of moderate or high stringency.
  • Bioly equivalent polynucleotides useful in the methods of this invention are identified using sequence homology searches.
  • Several embodiments of biologically equivalent polynucleotides are within the scope of this invention, e.g., those characterized by possessing at least 75%, or at least 80%, or at least 90% or at least 95% sequence homology as determined using a sequence alignment program under default parameters correcting for ambiguities in the sequence data, changes in nucleotide sequence that do not alter the amino acid sequence because of degeneracy of the genetic code, conservative amino acid substitutions and corresponding changes in nucleotide sequence, and variations in the lengths of the aligned sequences due to splicing variants or small deletions or insertions between sequences that do not affect function.
  • Non-limiting examples of these programs are BLAST family programs including BLASTN, BLASTP, BLASTX, TBLASTN, and TBLASTX (BLAST is available from the worldwide web at http://www.ncbi.nlm.nih.gov/BLAST/), FastA, Compare, DotPlot, BestFit, GAP, FrameAlign, ClustalW, and PileUp. These programs can be obtained commercially in a comprehensive package of sequence analysis software such as GCG Inc.'s Wisconsin Package. Other similar analysis and alignment programs can be purchased from various providers such as DNA Star's MegAlign, or the alignment programs in GeneJockey.
  • sequence analysis and alignment programs can be accessed through the world wide web at sites such as the CMS Molecular Biology Resource at http://www.sdsc.edu ResTools/cmshp.html.
  • Any sequence database that contains DNA or protein sequences corresponding to a gene or a segment thereof can be used for sequence analysis. Commonly employed databases include but are not limited to GenBank, EMBL, DDBJ, PDB, SWISS-PROT, EST, STS, GSS, and HTGS. Sequence similarity can be discerned by aligning the tag sequence against a DNA sequence database.
  • the tag sequence can be translated into six reading frames; the predicted peptide sequences of all possible reading frames are then compared to individual sequences stored in a protein database such as s done using the BLASTX program.
  • Parameters for determining the extent of homology set forth by one or more of the aforementioned alignment programs are well established in the art. They include but are not limited to p value, percent sequence identity and the percent sequence similarity. P value is the probability that the alignment is produced by chance. For a single alignment, the p value can be calculated according to Karlin et al. (1990) PNAS 87: 2246. For multiple alignments, the p value can be calculated using a heuristic approach such as the one programmed in BLAST. Percent sequence identify is defined by the ratio of the number of nucleotide or amino acid matches between the query sequence and the known sequence when the two are optimally aligned.
  • the percent sequence similarity is calculated in the same way as percent identity except one scores amino acids that are different but similar as positive when calculating the percent similarity.
  • conservative changes that occur frequently without altering function such as a change from one basic amino acid to another or a change from one hydrophobic amino acid to another are scored as if they were identical.
  • a tag sequence is considered to lack substantial homology with any known sequences when the regions of alignment of comparable length exhibit less than 30% of sequence identity, more preferably less than 20% identity, even more preferably less than 10% identity.
  • polynucleotides useful for practicing the methods of the invention can comprise additional sequences, such as additional coding sequences within the same transcription unit, controlling elements such as promoters, ribosome binding sites, and polyadenylation sites, additional transcription units under control of the same or a different promoter, sequences that permit cloning, expression, and transformation of a host cell, and any such construct as may be desirable to provide embodiments of this invention.
  • FGF 23 polypeptide is a molecule comprising an ordered sequence of amino acids specified by translation of a FGF 23 cDNA, such as is shown in SEQ. ID NO 2.
  • the term is used to refer to the complete FGF 23 amino acid sequence of FGF 23 (SEQ. ID NO 2) as well as to alternatively spliced polypeptide molecules, and other portions of the complete molecule, such as protease cleavage products and synthetic peptides derived from the complete sequence. It also refers to orthologous FGF 23 polypeptides derived from various species including, but not limited to humans, simians, and rodents.
  • a “gene product” refers to the amino acid (e.g., peptide or polypeptide) generated when a gene is transcribed and translated.
  • a “sequence tag” or “tag” or “SAGE tag” is a short oligonucleotide containing defined nucleotide sequence that occurs in a certain position of a gene transcript.
  • the length of a tag is generally under about 20 nucleotides, preferably between 9 to 15 nucleotides, and more preferably 10 nucleotides.
  • the tag can be used to identify the corresponding transcript and gene from which it was transcribed.
  • a tag can further comprise exogenous nucleotide sequences to facilitate the identification and utility of the tag.
  • auxiliary sequences include, but are not limited to, restriction endonuclease cleavage sites and well known primer sequences for sequencing and cloning.
  • a sequence is the complement or is complementary to another sequence if they are related by the base-pairing rules. For example, in DNA, a sequence A-G-T in one strand is complementary to T-C-A in the other strand. A given sequence defines the complementary sequence.
  • module means to alter or modify a process or biological function associated with bone or mineral metabolism or any of the diseases described herein.
  • peptide is used in its broadest sense to refer to a compound of two or more subunit amino acids, amino acid analogs, or peptidomimetics.
  • the subunits may be linked by peptide bonds. In another embodiment, the subunit may be linked by other bonds, e.g. ester, ether, etc.
  • amino acid refers to either natural and/or unnatural or synthetic amino acids, including glycine and both the D or L optical isomers, and amino acid analogs and peptidomimetics.
  • a peptide of three or more amino acids is commonly called an oligopeptide if the peptide chain is short. If the peptide chain is long, the peptide is commonly called a polypeptide or a protein.
  • cDNAs refers to complementary DNA, that is mRNA molecules present in a cell or organism made in to cDNA with an enzyme such as reverse transcriptase.
  • a "cDNA library” is a collection of all of the mRNA molecules present in a cell or organism, all turned into cDNA molecules with the enzyme reverse transcriptase, then inserted into “vectors”.
  • a "probe” when used in the context of polynucleotide manipulation refers to an oligonucleotide that is provided as a reagent to detect a target potentially present in a sample of interest by hybridizing with the target.
  • a probe will comprise a label or a means by which a label can be attached, either before or subsequent to the hybridization reaction.
  • Suitable labels include, but are not limited to radioisotopes, fluorochromes, chemiluminescent compounds, dyes, and proteins, including enzymes.
  • a “primer” is a short polynucleotide, generally with a free 3' -OH group that binds to a target or "template” potentially present in a sample of interest by hybridizing with the target, and thereafter promoting polymerization of a polynucleotide complementary to the target.
  • a “polymerase chain reaction” (“PCR”) is a reaction in which replicate copies are made of a target polynucleotide using a "pair of primers” or a “set of primers” consisting of an "upstream” and a “downstream” primer, and a catalyst of polymerization, such as a DNA polymerase, and typically a thermally-stable polymerase enzyme.
  • PCR A PRACTICAL APPROACH
  • All processes of producing replicate copies of a polynucleotide, such as PCR or gene cloning, are collectively referred to herein as "replication.”
  • a primer can also be used as a probe in hybridization reactions, such as Southern or Northern blot analyses. Sambrook et al, supra.
  • a “promoter” is a region on a DNA molecule to which an RNA polymerase binds and initiates transcription.
  • the promoter is usually located at the operator end, adjacent but external to the operator.
  • the nucleotide sequence of the promoter determines both the nature of the enzyme that attaches to it and the rate of RNA synthesis.
  • genetically modified means containing and/or expressing a foreign gene or nucleic acid sequence which in turn, modifies the genotype or phenotype of the cell or its progeny.
  • Form nucleic acid includes, but is not limited to promoters, enhancers and gene activators.
  • a genetically modified cell includes a cell that contains a polynucleotide encoding FGF 23 polypeptide in its native environment but not expressed and expression has been turned on or the level of expression has been enhanced or lowered by the upstream insertion of a gene activator.
  • expression refers to the process by which polynucleotides are transcribed into mRNA or by which transcription is enhanced.
  • the RNA is translated into peptides, polypeptides, or proteins. If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA, if an appropriate eukaryotic host is selected.
  • Hybridization refers to a reaction in which one or more polynucleotides react to form a complex that is stabilized via hydrogen bonding between the bases of the nucleotide residues.
  • the hydrogen bonding may occur by Watson-Crick base pairing, Hoogstein binding, or in any other sequence-specific manner.
  • the complex may comprise two strands forming a duplex structure, three or more strands forming a multi-stranded complex, a single self-hybridizing strand, or any combination of these.
  • a hybridization reaction may constitute a step in a more extensive process, such as the initiation of a PCR reaction, or the enzymatic cleavage of a polynucleotide by a ribozyme.
  • Hybridization reactions can be performed using traditional hybridization techniques under different stringency.
  • a low stringency hybridization reaction is carried out at about 40°C in 10 X SSC or a solution of equivalent ionic strength/temperature.
  • a moderate stringency hybridization is typically performed at about 50°C in 6 X SSC, and a high stringency hybridization reaction is generally performed at about 60°C in 1 X SSC.
  • TMAC hybridization technology can be used for hybridization reactions probed with pooled oligonucleotides such as the SAGE tags. The advantage of using TMAC hybridization is that the reaction condition is not dependent on the G+C content of the oligonucleotide, and the melting temperature is determined only by the length of the oligomers to be used.
  • a double-stranded polynucleotide can be “complementary” or “homologous” to another polynucleotide, if hybridization can occur between one of the strands of the first polynucleotide and the second.
  • “Complementarity” or “homology” is quantifiable in terms of the proportion of bases in opposing strands that are expected to form hydrogen bonding with each other, according to generally accepted base-pairing rules.
  • a polynucleotide that is 100% complementary to a second polynucleotide are understood to be “complements" of each other.
  • composition is intended to mean a combination of active agent and another compound or composition, inert (for example, a detectable agent or label or a pharmaceutically acceptable carrier) or active, such as an adjuvant.
  • a “pharmaceutical composition” is intended to include the combination of an active agent with a carrier, inert or active, making the composition suitable for diagnostic or therapeutic use in vitro, in vivo or ex vivo.
  • the term "pharmaceutically acceptable carrier” encompasses any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water, and emulsions, such as an oil/water or water/oil emulsion, and various types of wetting agents.
  • the compositions also can include stabilizers and preservatives.
  • stabilizers and adjuvants see Martin, REMINGTON'S PHARM. SCI., 15th Ed. (Mack Publ. Co., Easton (1975)).
  • an “effective amount” is an amount sufficient to effect beneficial or desired results.
  • An effective amount can be administered in one or more administrations, applications or dosages.
  • a “subject,” “individual” or “patient” is used interchangeably herein, which refers to a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, murines, simians, humans, farm animals, sport animals, and pets.
  • a “control” is an alternative subject or sample used in an experiment for comparison purpose.
  • a control can be "positive” or “negative".
  • the purpose of the experiment is to determine a correlation of an altered expression level of a gene with a particular type of cancer, it is generally preferable to use a positive control (a subject or a sample from a subject, carrying such alteration and exhibiting syndromes characteristic of that disease), and a negative control (a subject or a sample from a subject lacking the altered expression and clinical syndrome of that disease).
  • a “gene delivery vehicle” is defined as any molecule that can carry inserted polynucleotides into a host cell.
  • Examples of gene delivery vehicles are liposomes, cationic liposomes, viruses, such as baculovirus, adenovirus, adeno-associated virus, and retrovirus, bacteriophage, cosmid, plasmid, fungal vectors and other recombination vehicles typically used in the art which have been described for expression in a variety of eukaryotic and prokaryotic hosts, and may be used for gene therapy as well as for simple protein expression.
  • a "viral vector” is defined as a recombinantly produced virus or viral particle that comprises a polynucleotide to be delivered into a host cell, either in vivo, ex vivo or in vitro.
  • viral vectors include retro viral vectors, adenovirus vectors, adeno-associated virus vectors and the like.
  • a vector construct refers to the polynucleotide comprising the retroviral genome or part thereof, and the inserted polynucleotide.
  • retroviral mediated gene transfer or “retroviral transduction” carries the same meaning and refers to the process by which a gene or nucleic acid sequences are stably transferred into the host cell by virtue of the virus entering the cell and integrating its genome into the host cell genome.
  • the virus can enter the host cell via its normal mechanism of infection or be modified such that it binds to a different host cell surface receptor or ligand to enter the cell.
  • retroviral vector refers to a viral particle capable of introducing exogenous nucleic acid into a cell through a viral or viral-like entry mechanism.
  • Retroviruses carry their genetic information in the form of RNA; however, once the virus infects a cell, the RNA is reverse-transcribed into the DNA form that integrates into the genomic DNA of the infected cell.
  • the integrated DNA form is called a provirus.
  • a vector construct refers to the polynucleotide comprising the viral genome or part thereof, and a polynucleotide to be inserted.
  • Ads adenoviruses
  • Ads are a relatively well characterized, homogenous group of viruses, including over 50 serotypes. (see, e.g., WO 95/27071). Ads are easy to grow and do not require integration into the host cell genome.
  • Ad-derived vectors particularly those that reduce the potential for recombination and generation of wild-type virus, have also been constructed, (see, WO 95/00655; WO 95/11984). Wild-type AAV has high infectivity and specificity integrating into the host cells genome. (Hermonat and Muzyczka (1984) PNAS USA 81 :6466-6470; Lebkowski, et al. (1988) Mol. Cell. Biol. 8:3988-3996).
  • Vectors that contain both a promoter and a cloning site into which a polynucleotide can be operatively linked are well known in the art. Such vectors are capable of transcribing RNA in vitro or in vivo, and are commercially available from sources such as Stratagene (La Jolla, CA) and Promega Biotech (Madison, WI). In order to optimize expression and/or in vitro transcription, it may be necessary to remove, add or alter 5' and/or 3' untranslated portions of the clones to eliminate extra, potential inappropriate alternative translation initiation codons or other sequences that may interfere with or reduce expression, either at the level of transcription or translation. Alternatively, consensus ribosome binding sites can be inserted immediately 5' of the start codon to enhance expression.
  • Gene delivery vehicles also include several non- viral vectors, including DNA/liposome complexes, and targeted viral protein DNA complexes. Liposomes that also comprise a targeting antibody or fragment thereof can be used in the methods of this invention.
  • the nucleic acid or proteins of this invention can be conjugated to antibodies or binding fragments thereof which bind cell surface antigens, e.g., TCR, CD3 or CD4.
  • Polynucleotides are inserted into vector genomes using methods well known in the art.
  • insert and vector DNA can be contacted, under suitable conditions, with a restriction enzyme to create complementary ends on each molecule that can pair with each other and be joined together with a ligase.
  • synthetic nucleic acid linkers can be ligated to the termini of restricted polynucleotide. These synthetic linkers contain nucleic acid sequences that correspond to a particular restriction site in the vector DNA.
  • an oligonucleotide containing a termination codon and an appropriate restriction site can be ligated for insertion into a vector containing, for example, some or all of the following: a selectable marker gene, such as the neomycin gene for selection of stable or transient transfectants in mammalian cells; enhancer/promoter sequences from the immediate early gene of human CMV for high levels of transcription; transcription termination and RNA processing signals from SV40 for mRNA stability; SV40 polyoma origins of replication and ColEl for proper episomal replication; versatile multiple cloning sites; stabilizing elements 3' to the inserted polynucleotide, and T7 and SP6 RNA promoters for in vitro transcription of sense and antisense RNA.
  • a selectable marker gene such as the neomycin gene for selection of stable or transient transfectants in mammalian cells
  • enhancer/promoter sequences from the immediate early gene of human CMV for high levels of transcription
  • “Host cell” is intended to include any individual cell or cell culture that can be or have been recipients for vectors or the incorporation of exogenous polynucleotides, polypeptides and/or proteins. It also is intended to include progeny of a single cell, and the progeny may not necessarily be completely identical (in morphology or in genomic or total DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation.
  • the cells may be prokaryotic or eukaryotic, and include but are not limited to bacterial cells, yeast cells, plant cells, insect cells, animal cells, and mammalian cells, e.g., murine, rat, simian or human.
  • an “antibody” is an immunoglobulin molecule capable of binding an antigen.
  • the term encompasses not only intact immunoglobulin molecules, but also anti-idiotypic antibodies, mutants, fragments, fusion proteins, humanized proteins and modifications of the immunoglobulin molecule that comprise an antigen recognition site of the required specificity.
  • expression refers to the process by which polynucleotides are transcribed into mRNA and translated into peptides, polypeptides, or proteins. If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA, if an appropriate eukaryotic host is selected. Regulatory elements required for expression include promoter sequences to bind RNA polymerase and transcription initiation sequences for ribosome binding.
  • a bacterial expression vector includes a promoter such as the lac promoter and for transcription initiation the Shine-Dalgarno sequence and the start codon AUG (Sambrook, et al. (1989) supra).
  • an eukaryotic expression vector includes a heterologous or homologous promoter for RNA polymerase ⁇ , a downstream polyadenylation signal, the start codon AUG, and a termination codon for detachment of the ribosome.
  • a heterologous or homologous promoter for RNA polymerase ⁇ for example, the methods described below for constructing vectors in general.
  • a “subject” is a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, murines, simians, humans, farm animals, sport animals, and pets.
  • a “control” is an alternative subject or sample used in an experiment for comparison purpose. A control can be "positive” or "negative”.
  • cytokine refers to any one of the numerous factors that exert a variety of effects on cells, for example, inducing growth or proliferation.
  • Non- limiting examples of cytokines which may be used alone or in combination in the practice of the present invention include, interleukin-2 (IL-2), stem cell factor (SCF), interleukin 3 (IL-3), interleukin 6 (IL-6), interleukin 12 (IL-12), G-CSF, granulocyte macrophage-colony stimulating factor (GM-CSF), interleukin- 1 alpha (IL-1), interleukin- 11 (IL-11), MIP-1, leukemia inhibitory factor (LIF), c-kit ligand, thrombopoietin (TPO) and flt3 ligand.
  • IL-2 interleukin-2
  • SCF stem cell factor
  • IL-3 interleukin 6
  • IL-12 interleukin 12
  • G-CSF granulocyte macrophage-colony stimulating factor
  • the present invention also includes culture conditions in which one or more cytokine is specifically excluded from the medium.
  • Cytokines are commercially available from several vendors such as, for example, Genentech (South San Francisco, CA), Amgen (Thousand Oaks, CA), R&D Systems (Minneapolis, MN) and Immunex (Seattle, WA). It is intended, although not always explicitly stated, that molecules having similar biological activity as wild-type or purified cytokines (e.g., recombinantly produced or muteins thereof) are intended to be used within the spirit and scope of the invention.
  • culture refers to the in vitro propagation of cells or organisms on or in media of various kinds. It is understood that the descendants of a cell grown in culture may not be completely identical (i.e., morphologically, genetically, or phenotypically) to the parent cell. By “expanded” is meant any proliferation or division of cells.
  • cancer refers to cells that have undergone a malignant transformation that makes them pathological to the host organism.
  • Primary cancer cells that is, cells obtained from near the site of malignant transformation
  • the definition of a cancer cell includes not only a primary cancer cell, but any cell derived from a cancer cell ancestor. This includes metastasized cancer cells, and in vitro cultures and cell lines derived from cancer cells.
  • a "clinically detectable" tumor is one that is detectable on the basis of tumor mass; e.g., by such procedures as CAT scan, magnetic resonance imaging (MRI), X-ray, ultrasound or palpation. Biochemical or immunologic findings alone may be insufficient to meet this definition.
  • Tumor cells often express antigens which are tumor specific.
  • the term "tumor associated antigen” or “TAA” refers to an antigen that is associated with or specific to a tumor.
  • Solid phase support is not limited to a specific type of support. Rather a large number of supports are available and are known to one of ordinary skill in the art. Solid phase supports include silica gels, resins, derivatized plastic films, glass beads, cotton, plastic beads, alumina gels. A suitable solid phase support may be selected on the basis of desired end use and suitability for various synthetic protocols.
  • solid phase support may refer to resins such as polystyrene (e.g., PAM-resin obtained from Bachem Inc., Peninsula Laboratories, etc.), POLYHIPE ® resin (obtained from Aminotech, Canada), polyamide resin (obtained from Peninsula Laboratories), polystyrene resin grafted with polyethylene glycol (TentaGel , Rapp Polymere, Tubingen, Germany) or polydimethylacrylamide resin (obtained from Milligen/Biosearch, California).
  • solid phase support refers to polydimethylacrylamide resin.
  • transgenic animal refers to a genetically engineered animal or offspring of genetically engineered animals.
  • the transgenic animal may contain genetic material from at least one unrelated organism (such as from a bacteria, virus, plant, or other animal) or may contain a mutation which interferes with expression of a gene product.
  • oncogenic osteomalacia refers to a tumor-acquired syndrome characterized mainly by hypophosphatemia, hyperphosphaturia, abnormally low serum level of 1,25-dihydroxyvitamin D, and osteomalacia.
  • Tumors associated with OOM are mainly of mesenchymal origin such as hemangiopericytomas, although carcinoma of prostate and lung, fibrous dysplasia of bone, linear sebaceous naevus syndrome, neurofibromatosis, and oat cell carcinoma are also associated with OOM.
  • the OOM syndrome can be described as having a paraneoplastic etiology. Surgical removal of the tumor in a patient often results in a complete or near-complete resolution of biochemical and clinical defects associated with OOM.
  • Oncogenic osteomalacia-related genes include genes that have been identified to be over-expressed or under-expressed relative to control tumors (histologically similar tumors that are not associated with OOM). Genes that are up-regulated or down-regulated in oncogenic osteomalacia may encode proteins involved in several distinct biochemical pathways. These include phosphate regulation, bone mineralization, and protein synthesis, processing and secretion.
  • the regulation of phosphate metabolism plays a central role in mediating the symptoms of oncogenic osteomalacia.
  • Genes whose expression is altered in OOM tumors can effect phosphate metabolism through a variety of mechanisms.
  • the tumor may directly produce increased amounts of phosphatonin, a secreted humoral factor whose activity includes inhibition of phosphate re-absorption in the kidney.
  • the OOM tumor cells could produce a factor or factors that alter the expression in the kidney of accessory polypeptides required for mediating the effects of phosphatonin such as the phosphatonin receptor and intracellular proteins responsible for eliciting the effects of phosphatonin.
  • FGF 23 is a gene of the second type.
  • OOM tumor cells can also alter phosphate metabolism by more complex mechanisms.
  • tumor produced factors could up-regulate expression of genes normally controlled directly by phosphatonin or in response to phosphatonin.
  • OOM tumor produced factors could increase expression of phosphate transport molecules and other cellular proteins necessary for regulating either phosphate uptake or secretion of phosphate.
  • OOM tumor factors could alter expression of extracellular regulators or carriers of phosphate or phosphatonin.
  • OOM-related genes that modulate phosphate metabolism are useful candidates for developing therapeutic agents for a variety of disease conditions related to abnormal phosphate metabolism.
  • renal conditions such as renal osteodystrophy, changes in phosphate homeostasis after kidney transplant, end stage renal disease (ESRD), and acute renal disease, bone defects, hypophosphataemia, hyperphosphataemia, hypoparathyroidism, and pseudohypoparathyroidism.
  • ESRD end stage renal disease
  • Phosphate metabolism related factors could provide useful mediators of disease conditions through a variety of alternative mechanisms. For example, during ESRD, phosphatonin or other proteins in its pathway may inhibit absorption of phosphate in the small intestine. Such factors may also enhance phosphate uptake in the proximal tubules of the kidney. Modulation of the activity of these factors could therefore be used to control the symptoms of this disease.
  • any protein that is involved in lowering serum phosphate levels or inhibiting its functions could be an effective therapy.
  • This type of therapy could be useful for a range of conditions including hype arathyroidism, X-linked hypophosphatemic rickets, vitamin D dependent rickets, Franconi Syndrome, post kidney transplant condition, and oncogenic osteomalacia.
  • Diseases characterized by increased phosphate levels or hyperphosphatemia could be affected by treatment directed towards any protein that acts in the phosphatonin pathway to lower serum phosphate levels.
  • Diseases related to hyperphosphatemia include: hypoparathyroidism (levels of PTH secreted are insufficient to maintain extracellular calcium and phosphate levels-leads to hypocalcemia and hyperphosphatemia); pseudohypoparathyroidism (a group of disorders characterized by biochemical hypoparathyroidism, hypocalcemia and hyperphosphatemia, increased secretion of PTH and resistance to the biological actions of PTH); transcellular phosphate shift from cells into the extracellular fluid caused by systemic infections, severe hyperthermia, crush injuries, non-traumatic rhabdomyoloysis, and tumor lysis syndrome after cytotoxic therapies for hematologic malignancies; and renal disease.
  • PTH parathyroid hormone
  • 1,25- dihydroxyvitamin D calcitriol
  • PTH stimulates the release of calcium and phosphate from bone.
  • PTH stimulates the re-absorption of calcium and inhibits the reabsorption of phosphate.
  • it stimulates the activity of 1 alpha- hydroxylase, thereby enhancing the synthesis of 1,25-dihydroxyvitamin D 3 .
  • 1,25- dihydroxyvitamin D 3 in turn increases intestinal abso ⁇ tion of calcium and phosphate.
  • serum calcium concentration increases and serum phosphate concentration decreases.
  • 1,25-dihydroxyvitamin D , calcium, and phosphate are important regulators of PTH secretion from the parathyroid gland. Decreasing amounts of 1,25- dihydroxyvitamin D 3 stimulate secretion of PTH. Low serum concentrations of calcium stimulate secretion of PTH, whereas increasing calcium serum concentrations repress PTH secretion. Decreased levels of serum phosphate repress PTH secretion from the parathyroid gland (E. Slatopolsky, et al; J Clin Invest 97:2534-2540). In chronic renal insufficiency, declining levels of calcitriol promote hype ⁇ arathyroidism and increased secretion of PTH.
  • Hype ⁇ arathyroidism and hype ⁇ hosphatemia leads to enhanced vascular calcification, a key factor promoting increased morbidity and mortality in chronic renal insufficiency patients (Liach, F and FV Forero; 2001, American Journal of Kidney Disease 38:1-17).
  • Therapeutic agents that control secretion of PTH and/or serum phosphate concentrations can be used to treat chronic renal insufficiency patients.
  • factors whose expression is altered in OOM tumor cells can include genes whose polypeptide products act directly on osteogenic cells to mediate bone mineralization.
  • Such proteins associated with OOM may either promote or inhibit diseases associated with defective mineralization.
  • Possible functions of proteins in the bone mineralization pathway include: inhibition of bone mineralization, regulation of the early stages of bone mineralization, and control of bone cell differentiation and bone development.
  • a variety of types of polypeptide factors may be found to modulate bone mineralization.
  • extracellular matrix proteins are an important constituent of bone. In bone, cartilage and the tissues forming the teeth, unlike those in other connective tissues, the matrices have the unique ability to become calcified.
  • OOM tumor produced ECM proteins could alter the natural process of bone mineral homeostasis by acting directly on bone cells.
  • OOM tumor cells could produce diffusable soluble factors that regulate bone cell differentiation, growth and metabolism. Such factors also provide useful targets for development of therapeutic agents to regulate bone mineralization.
  • osteoporosis a metabolic bone disease characterized by low bone mass and microarchitectural deterioration of bone tissue
  • osteomalacia a defect in bone mineralization that occurs after the cessation of growth and involves only the bone and not the growth plate
  • rickets a disorder of mineralization of the bone matrix, or osteoid, in growing bones; that involves both the growth plate (epiphysis) and newly formed traebacular and cortical bone
  • hypophosphatasis a rare heritable type of rickets or osteomalacia (1 in 100,000 births) characterized by a reduction of activity of the tissue non-specific isoenzyme of alkaline phosphatase
  • Fanconi syndrome and renal tubular acidosis a generalized defect in renal proximal tubule transport capacity that includes impaired reabso ⁇ tion of glucose, phosphate, amino acids, bicarbonate, uric acid, citrate and other organic acids
  • OOM tumor produced factors that are found to modulate fundamental processes involved in bone formation, mineralization and maintenance could provide useful targets to inhibit the progression of these diseases.
  • pathological conditions of the bone include defects in bone remodeling such as Paget's disease, osteomyeloitis, osteosarcoma, stress fracture and low turnover bone disease.
  • defects in bone remodeling such as Paget's disease, osteomyeloitis, osteosarcoma, stress fracture and low turnover bone disease.
  • polypeptide factors identified from OOM tumor cells that directly modulate bone metabolism and bone cell development are useful targets for developing novel therapeutic agents to treat diseases characterized by alternative bone pathologies.
  • expression of OOM tumor associated factors may be found to be diagnostic of bone disease making these genes useful markers for diagnostic tests to identify such conditions.
  • the present inventions also provides methods of modulating the activity of cells associated with calcium or phosphate metabolism by delivering polynucleotides encoding FGF 23 protein or FGF 23 protein to such cells.
  • cells associated with calcium or phosphate metabolism include, but are not limited to parathyroid cells, intestinal cells and smooth muscle cells.
  • modulation of the activity of such cells includes, but is not limited to, altering the responsiveness of the cell to concentrations of calcium or phosphate, or altering the levels of a cellular product (e.g., secretory product).
  • secretion of PTH by parathyroid cells is inhibited or repressed in the presence of elevated levels of calcium.
  • Contacting or delivering FGF 23 to the parathyroid cells inhibits or prevents repression of PTH secretion by the parathyroid in the presence of elevated levels of calcium.
  • Methods of contacting or delivering polynucleotides that encode the FGF 23 gene or FGF 23 protein with cells in vitro, in vivo or ex vivo may be done using conventional techniques.
  • the present invention further provides a method for modulating the phenotype of a neoplastic cell associated with oncogenic osteomalacia comprising delivering an agent that alters the expression of a polynucleotide encoding the FGF 23 polypeptide.
  • Appropriate subjects for receiving such an agent can be identified by performing the FGF 23 gene expression analysis methods described below.
  • the invention provide methods for modulating the phenotype of a cell associated with phosphate homeostasis comprising delivering an agent that alters the expression of the FGF 23 gene. This method is useful for modulating FGF 23 expression and phosphate homeostasis in a subject.
  • Agents that enhance the expression of FGF 23 are useful for reducing the re-abso ⁇ tion of phosphate in the kidney while agents that inhibit the expression of FGF 23 are useful for increasing phosphate re-abso ⁇ tion.
  • the present invention provides methods of modulating phosphate homeostasis and/or bone mineralization in a subject by altering the activity of the FGF 23 gene or by altering the activity of the FGF 23 protein.
  • phosphate homeostasis is modulated by delivering to a subject an effective amount of an agent that alters the activity of FGF 23.
  • Agents that are useful for practicing the invention include, but are not limited to small organic molecules, polypeptides and antibodies.
  • Agents that inhibit the activity of FGF 23 protein are useful for increasing phosphate re-abso ⁇ tion while agents that stimulate FGF 23 activity are useful for decreasing phosphate re-abso ⁇ tion.
  • the methods of the invention can be used for conditions characterized by either hypophosphatemia or hype ⁇ hosphatemia.
  • phosphate homeostasis is modulated by delivering an agent that alters the expression of the FGF 23 gene.
  • agents can include, but are not limited to, gene delivery vehicles comprising anti- sense RNA molecules and ribozymes, anti-sense oligonucleotides, and small organic molecules or polypeptides that specifically inhibit expression of the FGF 23 gene. Inhibition of expression of the FGF 23 gene is useful to increase the re-abso ⁇ tion of phosphate in a subject.
  • the invention also provides methods of modulating renal phosphate transport by delivering an agent that alters FGF 23 protein activity and/or by delivering an agent that alters FGF 23 gene expression.
  • the present invention encompasses methods of alleviating the symptoms of hypophosphatemia or hype ⁇ hosphatemia related diseases, such as oncogenic osteomalacia.
  • polynucleotides, polypeptides and therapeutic agents and their derivatives can be used, either alone or in conjunction with other active agents, in a pharmaceutical composition for the therapeutic treatments described herein.
  • Symptoms that can be alleviated include, but are not limited to hypophosphatemia, phosphaturia, low serum concentration of 1,25-dihydroxyvitamin D and osteomalacia.
  • a pharmaceutical composition comprising an agent identified by the screening assay described below is administered to a subject in an effective amount to treat hypophosphatemia related diseases, such as oncogenic osteomalacia, or to ameliorate the symptoms associated therewith.
  • the pharmaceutical composition is capable of modulating FGF 23 protein function in a subject with oncogenic osteomalacia; and thereby restoring normal serum phosphate levels in the subject.
  • FGF 23 gene and protein modulating agents are useful for treatment of phosphate homeostasis related diseases that include X-linked hypophosphatemia rickets, oncogenic osteomalacia, rhabdomyolysis, cardiomyopathy, tumoral calcinosis, renal failure and bone mineralization.
  • Delivery of polynucleotides encoding the FGF 23 protein or FGF 23 protein is useful for treatment of diseases involving elevated concentrations of calcium or in alleviating the symptoms of such diseases. In response to elevated concentrations of calcium, secretion of PTH by the parathyroid is suppressed. Delivery of the FGF 23 polynucleotides or FGF 23 protein prevents repression of secretion of PTH by the parathyroid.
  • subjects with low turnover bone disease have elevated levels of calcium. Delivery of an effective amount of FGF 23 polynucleotide or protein by the methods described herein, to such subjects would allow increased bone turnover in such subjects.
  • the FGF 23 polynucleotides or protein may be delivered, either alone or in conjunction with other active agents.
  • agents that alter the expression of the FGF 23 gene or protein may be administered for the treatment of disease involving elevated levels of calcium.
  • a pharmaceutical composition comprising an FGF 23 polypeptide is administered to a subject in an effective amount to reduce phosphate re-abso ⁇ tion.
  • the pharmaceutical composition contains FGF 23 polypeptide, a secreted protein, and is capable of lowering the abnormally elevated serum phosphate levels in patients with phosphate homeostasis-related disease.
  • the FGF 23 polypeptide-containing pharmaceutical composition further comprises active agents that promote the desired function in regulating phosphate homeostasis.
  • active agents include, but are not limited to, enzymes or co- factors that are involved in the post-translational modification, processing, and secretion of the mature FGF 23 protein; or factors responsible for maintaining the activated form of FGF 23 polypeptide in circulation and at the site of phosphate homeostasis.
  • Various delivery systems are known and can be used to administer a therapeutic agent, e.g., encapsulation in liposomes, microparticles, microcapsules, expression by recombinant cells, receptor-mediated endocytosis (see, e.g., Wu and Wu (1987) J. Biol.
  • Methods of delivery include but are not limited to transdermally, gene therapy, intra-arterial, intra-muscular, intravenous, intranasal, and oral routes, and include sustained delivery systems.
  • compositions identified herein as effective for their intended pu ⁇ ose can be administered to subjects or individuals susceptible to or at risk of developing diseases associated with abnormal phosphate transport in the kidney.
  • the agent When the agent is administered to a subject such as a mouse, a rat or a human patient, the agent can be added to a pharmaceutically acceptable carrier and systemically or topically administered to the subject.
  • Therapeutic amounts can be empirically determined and will vary with the pathology being treated, the subject being treated and the efficacy and toxicity of the agent.
  • Administration in vivo can be effected in one dose, continuously or intermittently throughout the course of treatment.
  • Methods of determining the most effective means and dosage of administration are well known to those of skill in the art and will vary with the composition used for therapy, the pu ⁇ ose of the therapy, the target cell being treated, and the subject being treated. Single or multiple administrations can be carried out with the dose level and pattern being selected by the treating physician. Suitable dosage formulations and methods of administering the agents can be found below.
  • the agents and compositions useful for practicing the methods of the present invention can be used in the manufacture of medicaments and for the treatment of humans and other animals by administration in accordance with conventional procedures, such as an active ingredient in pharmaceutical compositions.
  • the pharmaceutical compositions can be administered orally, intranasally, parenterally, transdermally or by inhalation therapy, and may take the form of tablets, lozenges, granules, capsules, pills, ampoules, suppositories or aerosol form. They may also take the form of gene therapy, suspensions, solutions and emulsions of the active ingredient in aqueous or nonaqueous diluents, syrups, granulates or powders.
  • the pharmaceutical compositions can be combined with other therapeutically useful agents.
  • the present invention also provides methods for detecting a cell expressing a polypeptide encoded by the FGF 23 gene by contacting a suitable sample with a suitable polynucleotide probe under conditions of moderate hybridization stringency and detecting any complementary nucleotides, thereby detecting the cell.
  • a suitable polynucleotide probe can be derived from the sequence of the FGF
  • oligonucleotide probe ranging in size from 10 or 20 nucleotides to about 50 nucleotides can be produced using an automated DNA synthesizer.
  • polynucleotide probes can be prepared from an isolated polynucleotide that comprises the FGF 23 cDNA sequence using methods well known in the art such as PCR or nick translation using various DNA polymerase enzymes. Such probes typically range in size from 50 to 500 base pairs in length. Suitable polynucleotide probes should not contain repeated DNA motifs and should not have high homology to genes other than the target FGF 23 sequence.
  • the invention further provides a method of detecting a cell expressing polypeptide encoded by the FGF 23 gene by performing RT-PCR on a suitable sample using a primer pair derived from the FGF 23 cDNA sequence.
  • RT-PCR can be performed using methods well established in the art.
  • a suitable primer pair will be oligonucleotides of similar annealing temperatures that are complementary to sequences on opposite strands of the FGF 23 cDNA.
  • the primers should amplify a portion of the FGF 23 cDNA ranging from 50 to 1,000 base pairs, preferably 250 to 750 base pairs in length.
  • Optimal conditions for performing PCR can be determined without undue experimentation by comparing a series of alternative reaction conditions in which reaction conditions such as primer concentration, magnesium concentration, annealing temperature and cycle number are varied, to identify appropriate PCR conditions.
  • Methods of detecting and monitoring FGF 23 expression are useful for detecting a neoplastic cell associated with oncogenic osteomalacia.
  • a suitable sample for such analysis can be obtained from a tissue sample removed from subject.
  • the methods are useful for localizing an osteogenic osteomalacia inducing tumor.
  • the methods of detecting FGF 23 expression levels can be used to quantitate FGF 23 expression levels.
  • it is useful to compare the level of FGF 23 expression in normal and diseased cells to determine levels of expression that are indicative of abnormal phosphate metabolism.
  • the present invention envisions using these methods to identify subjects that are appropriate candidates for treatment using the methods of this invention.
  • the methods of detecting FGF 23 gene expression are also useful for monitoring a gene delivery vehicle comprising the FGF 23 gene sequence when such a gene delivery vehicle is administered to a subject.
  • the above methods can be further modified by use of an activated cell, defined above.
  • the present invention provides methods for screening various agents that modulate the expression of the FGF 23 gene or the activity of the FGF 23 protein. These agents are useful for modulating phosphate homeostasis in a subject, for modulating renal phosphate transport, or alleviating the symptoms associated with oncogenic osteomalacia, for treating phosphate homeostasis-related disease and for altering the phenotype of a neoplastic cell associated with oncogenic osteomalacia or a cell associated with phosphate homeostasis or bone mineralization.
  • an "agent” is intended to include, but not be limited to a biological or chemical compound such as a simple or complex organic or inorganic molecule, a peptide, a protein (e.g. antibody), a polynucleotide (e.g. anti-sense) or a ribozyme.
  • a biological or chemical compound such as a simple or complex organic or inorganic molecule, a peptide, a protein (e.g. antibody), a polynucleotide (e.g. anti-sense) or a ribozyme.
  • a vast array of compounds can be synthesized, for example polymers, such as polypeptides and polynucleotides, and synthetic organic compounds based on various core structures, and these are also included in the term "agent".
  • various natural sources can provide compounds for screening, such as plant or animal extracts, and the like. It should be understood, although not always explicitly stated that the agent is used alone or in combination with another agent, having the same or different
  • small molecules are molecules having low molecular weights (MW) that are, in one embodiment, capable of binding to a protein of interest such as FGF 23 polypeptide, and thereby altering the function of the protein.
  • MW molecular weight
  • the MW of a small molecule is no more than 1,000.
  • suitable cell cultures or tissue cultures containing this type of neoplastic cell are first provided.
  • the cell can be a cultured cell or a genetically modified cell in which FGF 23, or its complement is expressed.
  • the cells can be from a tissue biopsy.
  • the cells are cultured under conditions (temperature, growth or culture medium and gas (CO )) and for an appropriate amount of time to attain exponential proliferation without density dependent constraints. It also is desirable to maintain an additional separate cell culture; one that does not receive the agent being tested as a control.
  • suitable cells may be cultured in microtiter plates and several agents may be assayed at the same time by noting genotypic changes, phenotypic changes or cell death.
  • the agent when the agent is a composition other than a DNA or RNA, such as a small molecule as described above, the agent may be directly added to the cell culture or added to culture medium for addition. As is apparent to those skilled in the art, an "effective" amount must be added which can be empirically determined.
  • the agent when it is a polynucleotide, it may be directly added by use of a gene gun or electroporation. Alternatively, it may be inserted into the cell using a gene delivery vehicle or other method as described herein.
  • Kits containing the agents and instructions necessary to perform the screen and in vitro method as described herein also are claimed.
  • the assays also can be performed in a subject.
  • the subject is an animal such as a rat, mouse or simian
  • the method provides a convenient animal model system that can be used prior to clinical testing of an agent.
  • a candidate agent is a potential drug if transcript expression is altered, i.e., upregulated (such as restoring tumor suppressor function), downregulated or eliminated as with drug resistant genes or oncogenes, or if symptoms associated or correlated to the presence of cells containing transcript expression are ameliorated, each as compared to untreated, animal having the pathological cells. It also can be useful to have a separate negative control group of cells or animals which are healthy and not treated, which provides a basis for comparison. After administration of the agent to subject, suitable cells or tissue samples are collected and assayed for altered gene expression or protein function.
  • polynucleotide encoding FGF 23 is provided by this invention.
  • Polynucleotides comprising the sequence of the FGF 23 gene are useful for practicing various embodiments of the present invention.
  • the polynucleotide also comprises a sequence.
  • These polynucleotides include, but are not limited to probes for detecting and monitoring gene expression, primers for performing polymerase chain reaction (PCR), cDNA molecules encoding FGF 23 polypeptide, gene delivery vehicles to deliver FGF 23 polynucleotides to a cell, expression vectors for the production of FGF 23 protein, and anti-sense polynucleotides and ribozymes to modulate FGF 23 expression.
  • sequence of a cDNA comprising the human FGF 23 cDNA is provided in the Figure (SEQ ID NO. 1).
  • SEQ ID NO. 1 The sequence of a cDNA comprising the human FGF 23 cDNA is provided in the Figure (SEQ ID NO. 1).
  • SEQ ID NO. 1 The sequence of a cDNA comprising the human FGF 23 cDNA is provided in the Figure (SEQ ID NO. 1).
  • SEQ ID NO. 1 The sequence of a cDNA comprising the human FGF 23 cDNA is provided in the Figure (SEQ ID NO. 1).
  • anti-sense polynucleotides e.g. antisense RNA
  • the polynucleotides can be introduced by any suitable gene delivery method or vector. They also can be expressed in a suitable host cell for generating a cell- based therapy. These methods are described in more detail below.
  • This invention can also utilize genetically modified cells that produce enhanced expression of FGF 23 polypeptide as compared to wild-type cells.
  • the genetically modified cells can be produced by insertion of upstream regulatory sequences such as promoters or gene activators (see U.S. Patent No. 5,733,761).
  • the polynucleotides and sequences identified above can be conjugated to a detectable marker, e.g., an enzymatic label or a radioisotope for detection of nucleic acid and/or expression of the gene in a cell.
  • a detectable marker e.g., an enzymatic label or a radioisotope for detection of nucleic acid and/or expression of the gene in a cell.
  • detectable markers e.g., an enzymatic label or a radioisotope for detection of nucleic acid and/or expression of the gene in a cell.
  • detectable marker e.g., an enzymatic label or a radioisotope for detection of nucleic acid and/or expression of the gene in a cell.
  • detectable markers include fluorescent, radioactive, enzymatic or other ligands, such as avidin/biotin, which are capable of giving a detectable signal.
  • a fluorescent label or an enzyme tag such as urease
  • colorimetric indicator substrates which can be employed to provide a means visible to the human eye or spectrophotometrically, to identify specific hybridization with complementary nucleic acid-containing samples.
  • this invention further provides a method for detecting a single-stranded polynucleotide identified by SEQ ID NO.
  • hybridized polynucleotide pairs are separated from un-hybridized, single-stranded polynucleotides. The hybridized polynucleotide pairs are detected using methods well known to those of skill in the art and set forth, for example, in Sambrook, et al. (19S9) supra.
  • the isolated polynucleotide encodes an oncogenic osteomalacia-related polypeptide, the polypeptide having the amino acid sequence of SEQ ID NO:2 or an analog thereof having conservative amino acid substitutions.
  • the isolated polynucleotide of this invention encodes oncogenic osteomalacia-related mutein polypeptide, the mutein polypeptide having the amino acid sequence of SEQ ID NO:2 or an analog thereof having non- conservative amino acid substitutions.
  • polynucleotides and sequences used to practice the methods of this invention can be obtained using chemical synthesis, recombinant cloning methods, PCR, or any combination thereof. Methods of chemical polynucleotide synthesis are well known in the art and need not be described in detail herein. One of skill in the art can use the sequence data provided herein to obtain a desired polynucleotide by employing a DNA synthesizer or ordering from a commercial service.
  • compositions containing the polynucleotides and sequences encoding the FGF 23 protein, in isolated form or contained within a vector or host cell may be delivered.
  • compositions are to be used pharmaceutically, they are combined with a pharmaceutically acceptable carrier.
  • Suitable cell or tissue samples used for the methods of this invention encompass body fluid, solid tissue samples, tissue cultures or cells derived therefrom and the progeny thereof, and sections or smears prepared from any of these sources, or any other samples that may contain a neoplastic tumor tissue.
  • Polynucleotides of the invention can be isolated using the techniques described herein or replicated using PCR.
  • the PCR technology is the subject matter of United States Patent Nos. 4,683,195, 4,800,159, 4,754,065, and 4,683,202 and described in PCR: THE POLYMERASE CHAIN REACTION (Mullis et al. eds, Birkhauser Press, Boston (1994)) or MacPherson, et al. (1991) and (1994), supra, and references cited therein.
  • one of skill in the art can use the sequences provided herein and a commercial DNA synthesizer to replicate the DNA.
  • one of skill in the art can insert the polynucleotide into a suitable replication vector and insert the vector into a suitable host cell (prokaryotic or eukaryotic) for replication and amplification.
  • the DNA so amplified can be isolated from the cell by methods well known to those of skill in the art.
  • a process for obtaining polynucleotides by this method is further provided herein as well as the polynucleotides so obtained.
  • RNA can be obtained by first inserting a DNA polynucleotide into a suitable host cell.
  • the DNA can be inserted by any appropriate method, e.g., by the use of an appropriate gene delivery vehicle (e.g., liposome, plasmid or vector) or by electroporation.
  • an appropriate gene delivery vehicle e.g., liposome, plasmid or vector
  • electroporation e.g., liposome, plasmid or vector
  • the RNA can then be isolated using methods well known to those of skill in the art, for example, as set forth in Sambrook, et al. (1989) supra.
  • mRNA can be isolated using various lytic enzymes or chemical solutions according to the procedures set forth in Sambrook, et al. (1989), supra or extracted by nucleic-acid- binding resins following the accompanying instructions provided by manufactures.
  • Polynucleotides exhibiting sequence complementarity or homology to SEQ ID NO. 1 find utility as hybridization probes. Since the full coding sequence of the transcript is known, any portion of this sequence or homologous sequences, can be used in the methods of this invention.
  • a "perfectly matched" probe is not needed for a specific hybridization. Minor changes in probe sequence achieved by substitution, deletion or insertion of a small number of bases do not affect the hybridization specificity. In general, as much as 20% base-pair mismatch (when optimally aligned) can be tolerated.
  • a probe useful for detecting the aforementioned mRNA is at least about 80% identical to the homologous region More preferably, the probe is 85% identical to the conesponding gene sequence after alignment of the homologous region; even more preferably, it exhibits 90% identity.
  • probes can be used in radioassays (e.g. Southern and Northern blot analysis) to detect, prognose, diagnose or monitor various neoplastic cells or tumor tissues containing these cells.
  • the probes also can be attached to a solid support or an array such as a chip for use in high throughput screening assays for the detection of expression of the gene corresponding a polynucleotide of this invention.
  • this invention also provides a probe comprising or corresponding to a polynucleotide of SEQ ID NO. 1, or its complement, or a fragment of SEQ ID NO.l, attached to a solid support for use in high throughput screens.
  • the total size of fragment, as well as the size of the complementary stretches, will depend on the intended use or application of the particular nucleic acid segment. Smaller fragments will generally find use in hybridization embodiments, wherein the length of the complementary region may be varied, such as between at least 5 to 10 to about 100 nucleotides, or even full length according to the complementary sequences one wishes to detect.
  • Nucleotide probes having complementary sequences over stretches greater than 5 to 10 nucleotides in length are generally preferred, so as to increase stability and selectivity of the hybrid, and thereby improving the specificity of particular hybrid molecules obtained. More preferably, one can design polynucleotides having gene-complementary stretches of 10 or more or more than 50 nucleotides in length, or even longer where desired. Such fragments may be readily prepared by, for example, directly synthesizing the fragment by chemical means, by application of nucleic acid reproduction technology, such as the PCR technology with two priming oligonucleotides as described in U.S. Pat. No. 4,603,102 or by introducing selected sequences into recombinant vectors for recombinant production. A preferred probe is about 50-75 or more preferably, 50-100, nucleotides in length.
  • the polynucleotides described herein can serve as primers for the detection of genes or gene transcripts that are expressed in neoplastic cells associated with oncogenic osteomalacia.
  • amplification means any method employing a primer-dependent polymerase capable of replicating a target sequence with reasonable fidelity. Amplification may be carried out by natural or recombinant DNA-polymerases such as T7 DNA polymerase, Klenow fragment of E.coli DNA polymerase, and reverse transcriptase.
  • a preferred length of the primer is the same as that identified for probes, above.
  • a preferred amplification method is PCR.
  • PCR conditions used for each reaction are empirically determined. A number of parameters influence the success of a reaction. Among them are annealing temperature and time, extension time, Mg concentration, pH, and the relative concentration of primers, templates, and deoxyribonucleotides.
  • the resulting DNA fragments can be detected by agarose gel electrophoresis followed by visualization with ethidium bromide staining and ultraviolet illumination.
  • the methods of the invention can also employ the isolated polynucleotide encoding the FGF 23 protein operatively linked to a promoter of RNA transcription, as well as other regulatory sequences for replication and/or transient or stable expression of the DNA or RNA.
  • operatively linked means positioned in such a manner that the promoter will direct transcription of RNA off the DNA molecule.
  • promoters are SP6, T4 and T7.
  • cell-specific promoters are used for cell-specific expression of the inserted polynucleotide.
  • Vectors which contain a promoter or a promoter/enhancer, with termination codons and selectable marker sequences, as well as a cloning site into which an inserted piece of DNA can be operatively linked to that promoter are well known in the art and commercially available. For general methodology and cloning strategies, see GENE EXPRESSION TECHNOLOGY (Goeddel ed., Academic Press, Inc.
  • Expression vectors containing these nucleic acids are useful to obtain host vector systems to produce proteins and polypeptides. It is implied that these expression vectors must be replicable in the host organisms either as episomes or as an integral part of the chromosomal DNA. Suitable expression vectors include plasmids, A preferred length of the primer is the same as that identified for probes, above viral vectors, including adenoviruses, adeno-associated viruses, retroviruses, cosmids, etc. Adenoviral vectors are particularly useful for introducing genes into tissues in vivo because of their high levels of expression and efficient transformation of cells both in vitro and in vivo.
  • a suitable host cell e.g., a prokaryotic or a eukaryotic cell and the host cell replicates
  • the protein can be recombinantly produced.
  • suitable host cells will depend on the vector and can include mammalian cells, animal cells, human cells, simian cells, insect cells, yeast cells, and bacterial cells constructed using well known methods. See Sambrook, et al. (1989) supra.
  • the nucleic acid can be inserted into the host cell by methods well known in the art such as transformation for bacterial cells; transfection using calcium phosphate precipitation for mammalian cells; or DEAE-dextran; electroporation; or microinjection. See Sambrook, et al. (1989) supra for this methodology.
  • this invention also provides a host cell, e.g. a mammalian cell, an animal cell (rat or mouse), a human cell, or a procaryotic cell such as a bacterial cell, containing a polynucleotide encoding a protein or polypeptide or antibody.
  • a pharmaceutically acceptable vector such as a replication-incompetent retroviral or adenoviral vector.
  • Pharmaceutically acceptable vectors containing the nucleic acids of this invention can be further modified for transient or stable expression of the inserted polynucleotide.
  • the term "pharmaceutically acceptable vector” includes, but is not limited to, a vector or delivery vehicle having the ability to selectively target and introduce the nucleic acid into dividing cells.
  • An example of such a vector is a "replication-incompetent" vector defined by its inability to produce viral proteins, precluding spread of the vector in the infected host cell.
  • LNL6 An example of a replication-incompetent retroviral vector is LNL6 (Miller, A.D. et al. (1989) BioTechniques 7:980-990).
  • the methodology of using replication- incompetent retroviruses for retroviral-mediated gene transfer of gene markers is well established (Correll, et al. (1989) PNAS USA 86:8912; Bordignon (1989) PNAS USA 86:8912-52; Culver, K. (1991) PNAS USA 88:3155; and Rill, D.R. (1991) Blood 79(10):2694-700. Clinical investigations have shown that there are few or no adverse effects associated with the viral vectors, see Anderson (1992) Science 256:808-13.
  • compositions containing the polynucleotides of this invention, in isolated form or contained within a vector or host cell are further provided herein. When these compositions are to be used pharmaceutically, they are combined with a pharmaceutically acceptable carrier. Proteins
  • This invention provides uses for the FGF 23 protein or FGF 23 polypeptides expressed from the polynucleotides described above, which is intended to include wild-type and recombinantly produced polypeptides and proteins from prokaryotic and eukaryotic host cells, as well as muteins, analogs and fragments thereof.
  • the term also includes antibodies and anti-idiotypic antibodies.
  • these proteins or polypeptides are a phophatonin-related factor which modulates phosphatonin activity.
  • Such polypeptides can be isolated or produced using the methods identified below.
  • proteins and polypeptides described herein are obtainable by a number of processes well known to those of skill in the art, which include purification, chemical synthesis and recombinant methods.
  • Full length proteins can be purified from a neoplastic cell or a tumor biopsy as identified above.
  • Sources for purifying the protein can also be serum or urine samples from an individual, such as a patient with oncogenic osteomalacia.
  • Proteins can be purified by methods such as immunoprecipitation with antibody, and standard techniques such as gel filtration, ion-exchange, reversed-phase, and affinity chromatography using a fusion protein as shown herein. For such methodology, see for example Deutscher et al.
  • this invention also provides the processes for obtaining these proteins and polypeptides as well as the products obtainable and obtained by these processes.
  • the proteins and polypeptides also can be obtained by chemical synthesis using a commercially available automated peptide synthesizer such as those manufactured by Perkin Elmer/ Applied Biosystems, Inc., Model 430A or 431 A, Foster City, CA, USA.
  • the synthesized protein or polypeptide can be precipitated and further purified, for example by high performance liquid chromatography (HPLC).
  • HPLC high performance liquid chromatography
  • this invention also provides a process for chemically synthesizing the proteins of this invention by providing the sequence of the protein and reagents, such as amino acids and enzymes and linking together the amino acids in the proper orientation and linear sequence.
  • proteins and polypeptides can be obtained by well-known recombinant methods as described, for example, in Sambrook, et al., (1989), supra, using the host cell and vector systems described above.
  • detectably labeled proteins and polypeptides can be bound to a column and used for the detection and purification of antibodies. They also are useful as immunogens for the production of antibodies as described below.
  • the proteins and fragments of this invention are useful in an in vitro assay system to screen for agents or drugs, which modulate cellular processes.
  • the FGF 23 proteins also can be combined with various liquid phase carriers, such as sterile or aqueous solutions, pharmaceutically acceptable earners, suspensions and emulsions.
  • liquid phase carriers such as sterile or aqueous solutions, pharmaceutically acceptable earners, suspensions and emulsions.
  • non-aqueous solvents include propyl ethylene glycol, polyethylene glycol and vegetable oils.
  • the carriers When used to prepare antibodies, the carriers also can include an adjuvant that is useful to non-specifically augment a specific immune response. A skilled artisan can easily determine whether an adjuvant is required and select one. However, for the pu ⁇ ose of illustration only, suitable adjuvants include, but are not limited to Freund's Complete and Incomplete, mineral salts and polynucleotides.
  • This invention also provides methods of using a pharmaceutical composition
  • a pharmaceutical composition comprising any of a protein, analog, mutein, polypeptide fragment, antibody, antibody fragment or anti-idiotypic antibody of this invention, alone or in combination with each other or other agents, and an acceptable carrier. These compositions are useful for various diagnostic and therapeutic methods as described herein.
  • Antibodies are useful for various diagnostic and therapeutic methods as described herein.
  • the present invention also envisions utilizing an antibody capable of specifically forming a complex with FGF 23 proteins or polypeptides as described above.
  • antibody includes polyclonal antibodies and monoclonal antibodies.
  • the antibodies include, but are not limited to mouse, rat, and rabbit or human antibodies.
  • the monoclonal antibodies of this invention can be biologically produced by introducing protein or a fragment thereof into an animal, e.g., a mouse or a rabbit.
  • the antibody producing cells in the animal are isolated and fused with myeloma cells or hetero- myeloma cells to produce hybrid cells or hybridomas. Accordingly, the hybridoma cells producing the monoclonal antibodies of this invention also are provided.
  • a monoclonal antibody being tested binds with the protein or polypeptide, then the antibody being tested and the antibodies provided by the hybridomas of this invention are equivalent. It also is possible to determine without undue experimentation, whether an antibody has the same specificity as the monoclonal antibody of this invention by determining whether the antibody being tested prevents a monoclonal antibody of this invention from binding the protein or polypeptide with which the monoclonal antibody is normally reactive. If the antibody being tested competes with the monoclonal antibody of the invention as shown by a decrease in binding by the monoclonal antibody of this invention, then it is likely that the two antibodies bind to the same or a closely related epitope.
  • antibody also is intended to include antibodies of all isotypes. Particular isotypes of a monoclonal antibody can be prepared either directly by selecting from the initial fusion, or prepared secondarily, from a parental hybridoma secreting a monoclonal antibody of different isotype by using the sib selection technique to isolate class switch variants using the procedure described in Steplewski et al. (1985) Proc. Natl. Acad. Sci. 82:8653 or Spira et al. (1984) J. Immunol. Methods 74:307.
  • antibody fragments retain some ability to selectively bind with its antigen or immunogen.
  • antibody fragments can include, but are not limited to: Fab; Fab'; F(ab') 2 , Fv; and SCA.
  • a specific example of "a biologically active antibody fragment” is a CDR region of the antibody. Methods of making these fragments are known in the art, see for example, Harlow and Lane, (1988) supra.
  • the antibodies utilized in practicing this invention also can be modified to create chimeric antibodies and humanized antibodies (Oi, et al. (1986) BioTechniques 4(3):214). Chimeric antibodies are those in which the various domains of the antibodies' heavy and light chains are coded for by DNA from more than one species.
  • the isolation of other hybridomas secreting monoclonal antibodies with the specificity of the monoclonal antibodies of the invention can also be accomplished by one of ordinary skill in the art by producing anti-idiotypic antibodies (Herlyn, et al. (1986) Science 232:100).
  • An anti-idiotypic antibody is an antibody which recognizes unique determinants present on the monoclonal antibody produced by the hybridoma of interest. Idiotypic identity between monoclonal antibodies of two hybridomas demonstrates that the two monoclonal antibodies are the same with respect to their recognition of the same epitopic determinant. Thus, by using antibodies to the epitopic determinants on a monoclonal antibody it is possible to identify other hybridomas expressing monoclonal antibodies of the same epitopic specificity.
  • an anti-idiotypic monoclonal antibody made to a first monoclonal antibody will have a binding domain in the hypervariable region which is the mirror image of the epitope bound by the first monoclonal antibody.
  • the anti-idiotypic monoclonal antibody could be used for immunization for production of these antibodies.
  • epitopic determinants are meant to include any determinant having specific affinity for the monoclonal antibodies of the invention.
  • Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics.
  • the antibodies utilized in this invention can be linked to a detectable agent or label.
  • a detectable agent or label There are many different labels and methods of labeling known to those of ordinary skill in the art.
  • the antibody-label complex is useful to detect the protein or fragments in a sample, using standard immunochemical techniques such as immunohistochemistry as described by Harlow and Lane (1988) supra.
  • Competitive and non-competitive immunoassays in either a direct or indirect format are examples of such assays, e.g., enzyme linked immunoassay (ELISA) radioimmunoassay (RIA) and the sandwich (immunometric) assay.
  • ELISA enzyme linked immunoassay
  • RIA radioimmunoassay
  • sandwich immunometric
  • haptens such as biotin, which reacts avidin, or dinitropherryl, pyridoxal, and fluorescein, which can react with specific anti-hapten antibodies. See Harlow and Lane (1988) supra.
  • Monoclonal antibodies also can be bound to many different carriers.
  • this invention also envisions employing compositions containing the antibodies and another substance, active or inert.
  • examples of well-known carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, agaroses and magnetite.
  • the nature of the carrier can be either soluble or insoluble for pu ⁇ oses of the invention.
  • suitable carriers for binding monoclonal antibodies or will be able to ascertain such, using routine experimentation.
  • compositions containing the antibodies, fragments thereof or cell lines which produce the antibodies are encompassed by this invention.
  • compositions are to be used pharmaceutically, they are combined with a pharmaceutically acceptable carrier.
  • the invention provides methods for identification, characterization and modulation of a selected phenotype of a tumor mass isolated from a patient with oncogenic osteomalacia. Manipulation of selected cells is useful for practicing these methods of the invention.
  • Tumors from which sample cells can be obtained for use in the present invention are tumors originated from patients with symptoms of oncogenic osteomalacia. These include, but are not limited to, hemangiopericytomas and other tumors of mesenchymal origin, carcinoma of prostate and lung, fibrous dysplasia of bone, linear sebaceous naevus syndrome, neurofibromatosis, and oat cell carcinoma.
  • Tumor cells are typically obtained from a cancer patient by resection, biopsy, or endoscopic sampling; the cells may be used directly, stored frozen, or maintained or expanded in culture. Samples of both the tumor and the patient's blood or blood fraction should be thoroughly tested to ensure sterility before co-culturing of the cells. Standard sterility tests are known to those of skill in the art and are not described in detail herein.
  • the tumor cells can be cultured in vitro to generate a cell line.
  • tumor cells can be dispersed from, for example, a biopsy sample, by standard mechanical means before use.
  • One aspect of the invention involves the comparison of transcript expression pattern between a sample cell and a control cell.
  • the selection of the control cell is determined by the sample cell initially selected and the phenotype of interest.
  • the control cell can be any of a counte ⁇ art normal cell type, a counte ⁇ art benign cell type, a counte ⁇ art non-neoplastic cell type and a non-neoplastic precursor of the neoplastic cell.
  • the sample cell can be a hemangiopericytoma cell isolated from a patient with oncogenic osteomalacia;
  • the counte ⁇ art control cell can be a hemangiopericytoma cell isolated from a patient who does not have oncogenic osteomalacia.
  • tumor cells can be dispersed from, for example, a biopsy sample, by standard mechanical means before use.
  • One aspect of the invention involves the comparison of transcript expression pattern between a sample cell and a control cell.
  • the selection of the control cell is determined by the sample cell initially selected and the phenotype of interest.
  • the control cell can be any of a counte ⁇ art normal cell type, a counte ⁇ art benign cell type, a counte ⁇ art non-neoplastic cell type and a non-neoplastic precursor of the neoplastic cell.
  • the sample cell can be a hemangiopericytoma cell isolated from a patient with oncogenic osteomalacia;
  • the counte ⁇ art control cell can be a hemangiopericytoma cell isolated from a patient who does not have oncogenic osteomalacia.
  • the isolated FGF 23 gene can be used to search for and identify single nucleotide polymo ⁇ hisms (SNP's), which are mutant variants of the gene in the human population. Identification of such polymo ⁇ hisms is useful to define human diseases to which mutations in the FGF 23 gene contribute and to perfect therapies for disease processes in which the protein encoded by the FGF 23 gene participates. Mutant variants of the gene identified in this manner can then be employed in the development, screening, and analysis of pharmaceutical agents to treat these diseases. Methods to detect such SNP's can be formatted to create diagnostic tests.
  • SNP's single nucleotide polymo ⁇ hisms
  • SNP's identified in the FGF 23 gene can provide useful sequence markers for genetic tests to analyze other genes and mutations in the region of the genome where the FGF 23 gene is located. Thus it is useful to inco ⁇ orate these SNP's into polymo ⁇ hism databases.
  • a full length cDNAs encoding the FGF23 protein is inserted into an expression vector and the protein is expressed in mammalian culture cells using standard cloning strategies. Stable cell lines that can be rapidly scaled for production ae also established. In vitro expressed FGF23 is then produced as a secreted molecule in conditioned culture medium and prepared for functional assays measuring phosphate re-abso ⁇ tion.
  • Similar conditioned media are also prepared from mammalian cells that are transfected with a control vector that does not contain any recombinant gene.
  • the phosphate transport modulating activity of FGF23 protein is analyzed using methods and techniques known in the art. Specifically, sodium-dependent phosphate uptake is measured in opossum kidney (OK) cells according to the methods described in Cai et al. (1994) New Engl. J. Med. 330: 1645-1649. Briefly, OK cells are cultured until becoming confluent, harvested and then re-seeded at a density of 1 X 10 5 cells per 24 well dish. The cells are then re-grown for several days past the time they become confluent and then re-fed with medium containing FGF23 protein as well as control conditioned medium from cells that are transfected with an the expression vector with no cloned insert.
  • OK cells are cultured until becoming confluent, harvested and then re-seeded at a density of 1 X 10 5 cells per 24 well dish. The cells are then re-grown for several days past the time they become confluent and then re-fed with medium containing FGF23 protein as well as
  • the medium is removed and the plated cells are re-fed with transport medium containing 32 P-labeled dibasic potassium phosphate and incubated at 37°C for 5 minutes. The cells are then washed, harvested and radioactivity measured via a scintillation counter to monitor uptake of 32 P.

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Abstract

Cette invention porte sur des procédés de modulation de l'homéostase des phosphates et du transport rénal des phosphates par l'administration d'agents qui modifient l'expression du gène FGF 23 ou modifient l'activité de la protéine FGF 23. Les procédés de cette invention sont utiles pour moduler le transport rénal des phosphates, soulager les symptômes associés à l'ostéomalacie oncogène et traiter la maladie relative à l'homéostase des phosphates. L'invention porte également sur des procédés de réduction de la réabsorption des phosphates qui consiste à administrer à un sujet la protéine FGF 23 ou des polynucléotides codant cette protéine. L'invention porte, en outre, sur des procédés de détection et de surveillance de l'expression du gène FGF 23, de modulation de l'activité des cellules associées au transport du calcium ou des phosphates et de modulation du phénotype d'une cellule néoplasique associée à l'ostéomalacie oncogène. L'invention porte enfin sur des procédés de criblage d'agents candidats visant à identifier des compositions qui modifient l'activité du gène et de la protéine FGF 23.
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EP1303536A1 (fr) * 2000-07-19 2003-04-23 Advanced Research And Technology Institute, Inc. Nouveau facteur de croissance fibroblastique (fgf23) et ses methodes d'utilisation
WO2003057733A1 (fr) * 2001-12-28 2003-07-17 Kirin Beer Kabushiki Kaisha Anticorps diriges contre le facteur de croissance 23 des fibroblastes
EP1664075A2 (fr) * 2003-08-20 2006-06-07 Curagen Corporation Methodes permettant de diagnostiquer et de traiter des affections qui modifient le transport du phosphate chez les mammiferes
WO2009091556A2 (fr) * 2008-01-17 2009-07-23 The General Hospital Corporation Méthodes et nécessaires de diagnostic utilisant le facteur de croissance des fibroblastes 23
US7883705B2 (en) 2007-02-14 2011-02-08 Kyowa Hakko Kirin Co., Ltd. Anti FGF23 antibody and a pharmaceutical composition comprising the same
US7981419B2 (en) 2000-08-11 2011-07-19 Kyowa Hakko Kirin Co., Ltd. Method for treating hypophosphatemic bone diseases using FGF-23 antibody

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FUKUMOTO S. ET AL.: "Fibroblast growth factor-23 is the phosphaturic factor in tumor-induced osteomalacia and may be phosphatonin", CURRENT OPINION IN NEPHROLOGY AND HYPERTENSION, vol. 11, no. 4, 1 July 2002 (2002-07-01), pages 385 - 389, XP002955219 *
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Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1303536A1 (fr) * 2000-07-19 2003-04-23 Advanced Research And Technology Institute, Inc. Nouveau facteur de croissance fibroblastique (fgf23) et ses methodes d'utilisation
EP1303536A4 (fr) * 2000-07-19 2005-01-05 Advanced Res & Tech Inst Nouveau facteur de croissance fibroblastique (fgf23) et ses methodes d'utilisation
US7981419B2 (en) 2000-08-11 2011-07-19 Kyowa Hakko Kirin Co., Ltd. Method for treating hypophosphatemic bone diseases using FGF-23 antibody
WO2003057733A1 (fr) * 2001-12-28 2003-07-17 Kirin Beer Kabushiki Kaisha Anticorps diriges contre le facteur de croissance 23 des fibroblastes
US7923012B2 (en) 2001-12-28 2011-04-12 Kyowa Hakko Kirin Co., Ltd. Antibody against fibroblast growth factor-23
EP1664075A2 (fr) * 2003-08-20 2006-06-07 Curagen Corporation Methodes permettant de diagnostiquer et de traiter des affections qui modifient le transport du phosphate chez les mammiferes
EP1664075A4 (fr) * 2003-08-20 2009-03-04 Curagen Corp Methodes permettant de diagnostiquer et de traiter des affections qui modifient le transport du phosphate chez les mammiferes
US7883705B2 (en) 2007-02-14 2011-02-08 Kyowa Hakko Kirin Co., Ltd. Anti FGF23 antibody and a pharmaceutical composition comprising the same
US9290569B2 (en) 2007-02-14 2016-03-22 Kyowa Hakko Kirin Co., Ltd. Anti FGF23 antibody and a pharmaceutical composition comprising the same
US10202446B2 (en) 2007-02-14 2019-02-12 Kyowa Hakko Kirin Co., Ltd. Anti FGF23 antibody and a pharmaceutical composition comprising the same
WO2009091556A2 (fr) * 2008-01-17 2009-07-23 The General Hospital Corporation Méthodes et nécessaires de diagnostic utilisant le facteur de croissance des fibroblastes 23
WO2009091556A3 (fr) * 2008-01-17 2009-10-22 The General Hospital Corporation Méthodes et nécessaires de diagnostic utilisant le facteur de croissance des fibroblastes 23

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