WO2001041805A1 - Novel cdna 22360 associated with renal disease and other disease states - Google Patents

Novel cdna 22360 associated with renal disease and other disease states Download PDF

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Publication number
WO2001041805A1
WO2001041805A1 PCT/US2000/033658 US0033658W WO0141805A1 WO 2001041805 A1 WO2001041805 A1 WO 2001041805A1 US 0033658 W US0033658 W US 0033658W WO 0141805 A1 WO0141805 A1 WO 0141805A1
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tke
nucleic acid
protein
acid molecule
seq
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PCT/US2000/033658
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French (fr)
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Hong-Wei Sun
William E. Munger
Ronald J. Falk
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Gene Logic, Inc.
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Priority to AU24298/01A priority Critical patent/AU2429801A/en
Publication of WO2001041805A1 publication Critical patent/WO2001041805A1/en

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    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • AHUMAN NECESSITIES
    • A01AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01KANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00Genetically modified animals
    • A01K2217/05Animals comprising random inserted nucleic acids (transgenic)
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00Medicinal preparations containing peptides

Definitions

  • the invention relates generally to changes in gene expression associated with renal disorders, with inflammatory diseases, and with cell cycles, and to methods of using such genes or gene products as research or diagnostic tools, or as targets in therapeutic intervention.
  • this invention relates to a novel human gene which has been identified in kidney tissue from a patient diagnosed with immunoglobulm A nepkropathy (IgAN).
  • This gene serves as a marker for renal diseases such as glomerulonephritis, including mesangial proliferation; necrotizing crescentic glomerulonephritis (NCGN); minimal change disease and sclerosis, 2) serves as a marker for inflammatory diseases including noninfectious systemic inflammatory responses (SIRS), and 3) provides a means of identifying agents that modulate its expression in renal mesangial cells and other tissues.
  • renal diseases such as glomerulonephritis, including mesangial proliferation; necrotizing crescentic glomerulonephritis (NCGN); minimal change disease and sclerosis
  • NCGN necrotizing crescentic glomerulonephritis
  • SIRS noninfectious systemic inflammatory responses
  • 3) provides a means of identifying agents that modulate its expression in renal mesangial cells and other tissues.
  • IgA nepkropathy is the most common type of immunologically mediated glomerulonephritis (GN) and is characterized by deposition in tbe glomerular mesangium of IgA together witk C3, C5b-9, and properdin. In patients, tke co-deposition of IgA together with IgG and/or IgM can lead to a more progressive course of disease. Fifteen to forty percent of primary glomerulonephritis in parts of Europe, Asia and Japan has been linked to IgAN and it is well accepted that IgAN can lead to ESRD.
  • IgAN often presents either as asymptomatic microscopic hematuria and/or proteinuria (most common in adults), or episodic gross hematuria following upper respiratory and other infections or exercise.
  • the course of IgAN is variable, with some patients showing no decline in glomerular filtration rate (GFR) over decades and others developing the nephrotic syndrome, hypertension and renal failure.
  • GFR glomerular filtration rate
  • Diabetic nephropathy is also a common cause of end-stage renal disease (ESRD) and accounts for 35% of the ESRD population in the United States. It results in considerable morbidity, mortality and expense. The average cost of managing one diabetic patient with ESRD is approximately $50,000 a year (Kobrin SM. Kidney International Supplement, Vol. 63 (Dec. 1997) pp. S144-150). Approximately 5.8 million people in the United States have been diagnosed by a physician as being diabetic, and an additional four to five million people have undiagnosed diabetes.
  • Tkis compensation meckanism is a pkysiological response in wkick tke cells of tke kidney increase in size and protein content witkout syntkesizing DNA or dividing (Kujubu et al. American Journal of Physiology, Vol. 260, no. 6, pt. 2 (1991) pp. F823-827).
  • Tbe compensatory growtk of tke kidney is a kigkly regulated process (Wolf et al. Kidney International, Vol. 39, no. 3 (1991) pp. 401-420). Early studies demonstrated significant increases in ribosome syntbesis, including increased rDNA transcription rates (Ouellette et al. American Journal of Physiology, Vol. 253, no. 4, pt. 1 (1987) pp. C506-513).
  • Renal kypertropky involves a gradual and progressive increase in mRNA levels resulting in tke coordinated expression of positive and negative growtk control elements.
  • Tke present invention is based on tke discovery of a new gene family tkat is differentially expressed in various forms of renal disease, inflammation, or in various stages of cell growtk, compared witk normal cells and tissues.
  • Tke invention includes isolated nucleic acid molecules selected from tke group consisting of an isolated nucleic acid molecule tkat encodes tke amino acid sequence of SEQ ID NO: 2, an isolated nucleic acid molecule tkat encodes a fragment of at least 6 amino acids of SEQ ID NO: 2, an isolated nucleic acid molecule tkat kybridizes to a nucleic acid molecule comprising SEQ ID NO: 1 under conditions of sufficient stringency to produce a clear signal, an isolated nucleic acid molecule tkat kybridizes to a nucleic acid molecule tkat encodes tke amino acid sequence of SEQ ID NO: 2 under conditions of sufficient stringency to produce a clear signal, and an isolated nucleic acid molecule tkat encodes a protein tkat
  • Tke present invention furtker includes tke nucleic acid molecules operably linked to one or more expression control elements, including vectors comprising tke isolated nucleic acid molecules.
  • Tke invention furtker includes kost cells transformed to contain tke nucleic acid molecules of tke invention and metkods for producing a protein comprising tke step of culturing a kost cell transformed witk a nucleic acid molecule of tke invention under conditions in wkick tke protein is expressed.
  • Tke invention furtker provides an isolated polypeptide selected from tke group consisting of an isolated polypeptide comprising tke amino acid sequence of SEQ ID NO: 2, an isolated polypeptide comprising a functional or antigenic fragment of at least 6 amino acids of SEQ ID NO: 2, an isolated polypeptide comprising conservative amino acid substitutions of SEQ ID NO: 2, an isolated polypeptide comprising naturally occurring amino acid sequence variants of SEQ ID NO: 2, and an isolated polypeptide tkat exkibits at least about 35% amino acid sequence identity to SEQ ID NO: 2.
  • Tke invention furtker provides an isolated antibody, including a monoclonal or polyclonal antibody or antigen-binding antibody fragment, tkat specifically binds to a polypeptide of tke invention.
  • Tbe invention furtker provides metkods of identifying an agent wkick modulates tke expression of a nucleic acid encoding tke protein kaving tke sequence of SEQ ID NO: 2 or tke related proteins described berein, comprising: exposing cells wkick express tke nucleic acid to tke agent; and determining wketker tke agent modulates expression of said nucleic acid, tkereby identifying an agent wkick modulates tke expression of a nucleic acid encoding tke protein.
  • Tke invention furtker provides metkods of identifying an agent wkick modulates tke level of or at least one activity of a protein comprising tke sequence of SEQ ID NO: 2 or tke related proteins described kerein, comprising: exposing cells wkick express tke protein to tke agent; and determining wketker tke agent modulates tke level of or at least one activity of said protein, tkereby identifying an agent wkick modulates tke level of or at least one activity of tke protein.
  • Tke invention furtker provides metkods of identifying binding partners for a protein comprising tke sequence of SEQ ID NO: 2 or a related protein described kerein, comprising: exposing said protein to a potential binding partner; and determining if tke potential binding partner binds to said protein, tkereby identifying binding partners for tke protein.
  • Tke present invention furtker provides metkods of modulating tke expression of a nucleic acid encoding tke protein kaving tke sequence of SEQ ID NO: 2 or tke related proteins described kerein, comprising: administering an effective amount of an agent wkick modulates tke expression of a nucleic acid encoding tke protein.
  • Tke invention also provides metkods of modulating at least one activity of a protein comprising tke sequence of SEQ ID NO: 2 or tke related proteins described kerein, comprising: administering an effective amount of an agent wkick modulates at least one activity of tke protein.
  • Tke present invention furtker includes non-kuman transgenic animals modified to contain tke nucleic acid molecules of tke invention or mutated nucleic acid molecules suck tkat expression of tke encoded polypeptides of tke invention is prevented.
  • Tke invention furtker provides metkods of diagnosing renal or otker disease states, comprising: acquiring a tissue or blood or other sample from a subject; and determining tke level of expression of nucleic acid molecules of tke invention or polypeptides of tke invention.
  • Figure 1 Figure 1 skows tke expression levels of mRNA encoding tke protein of
  • SEQ ID NO: 2 as determined by quantitative PCR analysis in diseased and control kidney biopsy tissues, relative to tbe expression of GAPDH.
  • Min. Ck refers to minimum ckange
  • IgAN refers to IgA nepkropatky
  • NCGN refers to necrotizing crescentic glomerular nepkropatky.
  • Figure 2 Figure 2 skows tke tissue distribution of RNA encoding tke protein of
  • SEQ ID NO: 2 as analyzed by Nortkern blot in kuman keart (lane 1), brain (2), placenta (3), lung (4), liver (5), muscle (6), kidney (7) and pancreas (lane 8).
  • M RNA marker.
  • Figure 3 Figure 3 skows tke expression levels of mRNA encoding tke protein of
  • SEQ ID NO: 2 as determined by quantitative PCR analysis in various tissues relative to tbe expression of GAPDH.
  • Fi ure 4 Figure 4 skows tke expression levels of mRNA encoding tke protein of SEQ ID NO: 2 in kuman peripkeral blood leukocytes (PBL) as determined by quantitative
  • PBL samples were obtained from normal kuman controls or patients witk IgAN or witk anti-neutropkil cytoplasmic autoantibody (ANCA) - associated renal disease.
  • ANCA cytoplasmic autoantibody
  • Figure 5 Figure 5 skows tke expression levels of mRNA encoding tke protein of SEQ ID NO: 2 in kuman umbilical vein endotkelial cells (HUVEC) and kuman umbilical artery endotkelial cells (HUAEC) as determined by quantitative PCR, relative to tke expression of GAPDH. Cells were treated witk TNF ⁇ at time 0 in a time course study.
  • Figure 6 Figure 6 skows tke expression levels of mRNA encoding tke protein of
  • SEQ ID NO: 2 in tke endotkelial cell line EA an immortalized kybrid between primary kuman endotkelial cells and tke tumor cell line A549) as determined by quantitative PCR relative to tbe expression of GAPDH.
  • Growtk arrest was induced by two separate metkods: 1) culture in serum free conditions for various lengtks of time; 2) kigk density contact inhibition.
  • Figure 7 shows the expression levels of mRNA encoding the protein of
  • SEQ ID NO: 2 as determined by quantitative PCR relative to the expression of GAPDH.
  • Samples are primary kuman renal epitkelial cells under log, semi-kigk or high density culture conditions leading to growth arrest, and primary human renal mesangial cells under similar conditions of growth arrest as well as cytokine treatment (IL-1 and IL-6).
  • Figure 8 Figure 8 skows plots predicting various structural features and tke antigenic and kydropkilic regions of tke amino acid sequence of SEQ ID NO: 2.
  • Tke present invention is based in part on a new gene family tkat is differentially expressed in renal disease, inflammation, and in various stages of cell growtk, compared witk normal cells or tissues.
  • Tke kuman gene encodes a protein of 275 amino acids.
  • Tke proteins of tke invention can serve as a diagnostic agent or marker or as a target for agents tkat modulate tke expression or activity of tke proteins.
  • agents may be identified tkat modulate biological processes associated witk renal disease, kidney transplantation and kidney regeneration.
  • Tke present invention is furtker based on tbe development of metkods for isolating binding partners that bind a protein of the invention. Additionally, tkese proteins provide novel targets for screening syntketic small molecules and combinatorial or naturally occurring compound libraries to discover novel tkerapeutics to regulate kidney function.
  • Tke present invention provides isolated proteins, allelic variants of tke proteins, and conservative amino acid substitutions of tke proteins.
  • tke "protein” or “polypeptide” refers, in part, to a protein tkat kas tke kuman amino acid sequence depicted in SEQ ID NO: 2.
  • Tke invention includes naturally occurring allelic variants and proteins tkat kave a slightly different amino acid sequence tkan tkat specifically recited above. Allelic variants, tkougk possessing a skgktly different amino acid sequence tkan tkose recited above, will still kave tke same or similar biological functions associated witk tke 275 amino acid protein.
  • tke family of proteins related to tke kuman amino acid sequence of SEQ ID NO: 2 refers, in part, to proteins tkat kave been isolated from organisms in addition to humans. The methods used to identify and isolate other members of the family of proteins related to the 275 amino acid protein are described below.
  • the proteins of the present invention are preferably in isolated form.
  • a protein is said to be isolated wben pkysical, meckanical or ckemical metkods are employed to remove tke protein from cellular constituents tkat are normally associated witk tke protein.
  • Tbe proteins of tke present invention furtker include insertion, deletion or conservative amino acid substitution variants of SEQ ID NO: 2.
  • a conservative variant refers to at least one alteration in tke amino acid sequence tkat does not adversely affect tke biological functions of the protein.
  • a substitution, insertion or deletion is said to adversely affect the protein when the altered sequence prevents or disrupts a biological function associated witk tke protein.
  • tke overall ckarge, structure or kydropkobic/kydropkilic properties of tke protein can be altered witbout adversely affecting a biological activity.
  • tke amino acid sequence can be altered, for example to render tbe peptide more kydropkobic or kydropkilic, witkout adversely affecting tke biological activities of tke protein.
  • tke allelic variants will kave an amino acid sequence kaving at least about 35%, 40%, 50%, 60%, 65%, 70% or 75% amino acid sequence identity witk tke sequence set fortk in SEQ ID NO: 2, more preferably at least about 80%, even more preferably at least about 90%, and most preferably at least about 95% sequence identity.
  • Identity or komology witk respect to suck sequences is defined kerein as tke percentage of amino acid residues in tke candidate sequence tkat are identical witk tke known peptides, after aligning tke sequences and introducing gaps, if necessary, to ackieve tke maximum percent komology, and not considering any conservative substitutions as part of the sequence identity (see section B for the relevant paramenters). Fusion proteins, or N-terminal, C-terminal or internal extensions, deletions, or insertions into the peptide sequence shall not be construed as affecting homology.
  • Contemplated variants further include those containing predetermined mutations by, e.g., homologous recombination, site-directed or PCR mutagenesis, and the corresponding proteins of other animal species, including but not limited to rabbit, mouse, rat, porcine, bovine, ovine, equine and non-human primate species, and the alleles or other naturally occurring variants of the family of proteins; and derivatives wherein the protein has been covalently modified by substitution, chemical, enzymatic, or other appropriate means witk a moiety otker tkan a naturally occurring amino acid (for example a detectable moiety such as an enzyme or radioisotope).
  • the proteins of the present invention also include molecules having a portion of tke amino acid sequence disclosed in SEQ ID NO: 2 suck as fragments kaving a consecutive sequence of at least about 3, 4, 5, 6, 10, 15, 20, 25, 30, 35 or more amino acid residues of the protein.
  • Such fragments also referred to as peptides or polypeptides, may contain antigenic regions, functional regions of the protein identified as regions of the amino acid sequence which correspond to known protein domains, as well as regions of pronounced hydrophilicity. Tke regions are all easily identifiable by using commonly available protein sequence analysis software such as Mac VectorTM (Oxford Molecular). See also Figure 8.
  • members of the family of proteins can be used: 1) to identify agents which modulate at least one activity of the protein; 2) to identify binding partners for the protein, 3) as an antigen to raise polyclonal or monoclonal antibodies, 4) as a therapeutic agent or target, and 5) as a diagnostic agent or marker.
  • nucleic acid is defined as RNA or DNA that encodes a protein or peptide as defined above, is complementary to a nucleic acid sequence encoding such peptides, hybridizes to the complement of a nucleic acid that encodes the amino acid sequence of SEQ ID NO: 2 and remains stably bound to it under appropriate stringency conditions, encodes a polypeptide skaring at least about 35%, 40%, 50%, 60%, 65%, 70% or 75%, preferably at least about 80%, more preferably at least about 90%, and most preferably at least about 95% or more amino acid sequence identity with the peptide sequences, or exhibits at least about 35%, 40%, 50%, 60%, 65%, 70% or 75%, preferably at least about 80%, more preferably at least about 90%, and most preferably at least about 95% or more nucleotide sequence identity
  • genomic DNA e.g., genomic DNA, cDNA, mRNA and antisense molecules, as well as nucleic acids based on alternative backbones or including alternative bases whether derived from natural sources or synthesized.
  • hybridizing or complementary nucleic acids are defined further as being novel and non-obvious over any prior art nucleic acid including that which encodes, hybridizes under appropriate stringency conditions, or is complementary to nucleic acid encoding a protein according to the present invention.
  • Homology or identity at tke nucleotide or amino acid sequence level is determined by BLAST (Basic Local Alignment Searck Tool) analysis using tke algoritkm employed by tke programs blastp, blastn, blastx, tblastn and tblastx (Altsckul, SF et al. (1997) Nucleic Acid Res, 25:3389-3402, and Karlin et al. (1990) PNAS 87:2264-2268, fully incorporated by reference) which are tailored for sequence similarity searching.
  • BLAST Basic Local Alignment Searck Tool
  • the approach used by the BLAST program is to first consider similar segments, with and without gaps, between a query sequence and a database sequence, then to evaluate the statistical significance of all matches that are identified and finally to summarize only those matckes wkick satisfy a preselected tkreskold of significance.
  • wkick is fully incorporated by reference.
  • the search parameters for histogram, descriptions, alignments, expect i.e., the statistical significance threshold for reporting matches against database sequences
  • cutoff matrix and filter
  • the default scoring matrix used by blastp, blastx, tblastn, and tblastx is the BLOSUM62 matrix (recommended for query sequences over 85 nucleotide bases or amino acids in length (Henikoff et al. Proceedings of the National Academy of Science USA, Vol. 89 (1992) pp. 10915-10919, fully incorporated by reference).
  • tke scoring matrix is set by tke ratios of M (i.e., tke reward score for a pair of matcking residues) to N (i.e., tke penalty score for mismatcking residues), wkerein tke default values for M and N are 5 and -4, respectively.
  • "Stringent conditions" are tkose tkat (1) employ low ionic strengtk and kigk temperature for wasking, for example, 0.015 M NaCl/0.0015 M sodium citrate/0.1% SDS at 50°C, or (2) employ during kybridization a denaturing agent suck as formamide, for example, 50% (vol/vol) formamide witk 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium pkospkate buffer at pH 6.5 witk 750 mM NaCl, 75 mM sodium citrate at 42°C.
  • Anotker example is kybridization in 50% formamide, 5x SSC (0.75M NaCl, 0.075 M sodium citrate), 50 mM sodium pkospkate (pH 6.8), 0.1% sodium pyropkospkate, 5x Denkardt's solution, sonicated salmon sperm DNA (50 ⁇ g/ml), 0.1% SDS, and 10% dextran sulfate at 42°C, with washes at 42°C. in 0.2x SSC and 0.1% SDS.
  • a skilled artisan can readily determine and vary the stringency conditions appropriately to obtain a clear and detectable hybridization signal.
  • Preferred molecules are those that hybridize under the above conditions to the complement of SEQ ID NO: 1 and which encode a functional protein. Even more preferred hybridizing molecules are those that hybridize under the above conditions to tbe complement strand of tke open reading frame of SEQ ID NO:l.
  • nucleic acid molecule is said to be "isolated" wken tke nucleic acid molecule is substantially separated from contaminant nucleic acid molecules encoding other polypeptides.
  • the present invention further provides fragments of the encoding nucleic acid molecule.
  • a fragment of an encoding nucleic acid molecule refers to a small portion of the entire protein coding sequence. Tke size of tke fragment will be determined by tke intended use. For example, if tke fragment is ckosen so as to encode an active portion of tke protein, tke fragment will need to be large enougk to encode tke functional region(s) of tke protein. For instance, fragments which encode peptides corresponding to predicted antigenic regions may be prepared (see Figure 8).
  • tken tke fragment length is chosen so as to obtain a relatively small number of false positives during probing/priming (see tke discussion in section H).
  • Fragments of tke encoding nucleic acid molecules of tke present invention i.e., syntketic oligonucleotides
  • tkat are used as probes or specific primers for the polymerase chain reaction (PCR), or to synthesize gene sequences encoding proteins of the invention can easily be synthesized by chemical techniques, for example, the phospkotriester metkod ofMatteucci et al. (Journal of the American Chemical Society, Vol. 103 (1981) pp.
  • DNA segments can readily be prepared by well known metkods, suck as syntkesis of a group of oligonucleotides that define various modular segments of the gene, followed by ligation of oligonucleotides to build tke complete modified gene.
  • Tke encoding nucleic acid molecules of tke present invention may furtker be modified so as to contain a detectable label for diagnostic and probe purposes.
  • a variety of suck labels are known in the art and can readily be employed with the encoding molecules herein described. Suitable labels include, but are not limited to, biotin, radiolabeled nucleotides and the like. A skilled artisan can readily employ any such label to obtain labeled variants of the nucleic acid molecules of the invention.
  • tke kuman nucleic acid molecule having SEQ ID NO: 1 allows a skilled artisan to isolate nucleic acid molecules tkat encode otker members of tke protein family in addition to tke sequences kerein described.
  • a skilled artisan can readily use tke amino acid sequence of SEQ ID NO: 2 to generate antibody probes to screen expression libraries prepared from appropriate cells.
  • polyclonal antiserum from mammals such as rabbits immunized with the purified protein (as described below) or monoclonal antibodies can be used to probe a mammakan cDNA or genomic expression kbrary, such as lambda gtll library, to obtain the appropriate coding sequence for other members of the protein family.
  • the cloned cDNA sequence can be expressed as a fusion protein, expressed directly using its own control sequences, or expressed by constructions using control sequences appropriate to the particular host used for expression of tke enzyme.
  • tke coding sequence kerein described can be synthesized and used as a probe to retrieve DNA encoding a member of the protein family from any mammalian organism. Oligomers containing approximately 18-20 nucleotides (encoding about a 6-7 amino acid stretch) are prepared and used to screen genomic DNA or cDNA libraries to obtain hybridization under stringent conditions or conditions of sufficient stringency to eliminate an undue level of false positives.
  • pairs of oUgonucleotide primers can be prepared for use in a polymerase chain reaction (PCR) to selectively clone an encoding nucleic acid molecule.
  • PCR polymerase chain reaction
  • a PCR denature- anneal-extend cycle for using such PCR primers is well known in tke art and can readily be adapted for use in isolating otker encoding nucleic acid molecules.
  • Nucleic acid molecules encoding otker members of tbe protein family may also be identified in existing genomic or otker sequence information using any available computational metkod, including but not limited to: PSI-BLAST (Ascbul SF et al. (1997) Nucliec Acids Res. 26:3389-3402); PHI-BLAST (Zkang, et al. (1998) Nucliec Acids Res. 26:3986-3990); 3D-PSSM (Kelly et al. (2000) J. Mol. Biol. 299:499-520); and otker computational analysis metkods (Shi et al. (1999) Biochem. Biophys. Res. Commun. 262:132-138 and Matsunami et al. (2000) Nature 404:601-604).
  • PSI-BLAST Alpha-BLAST
  • PHI-BLAST Zakang, et al. (1998) Nucliec Acids Res. 26:3986-3990
  • Tke present invention furtker provides recombinant DNA molecules (rDNAs) tkat contain a coding sequence.
  • a rDNA molecule is a DNA molecule tkat kas been subjected to molecular manipulation in situ. Methods for generating rDNA molecules are well known in the art, for example, see Sambrook et al. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, NY. Cold Spring Harbor Laboratory Press, 1989.
  • a coding DNA sequence is operably linked to expression control sequences and/or vector sequences.
  • Tke ckoice of vector and/or expression control sequences to wkick one of tke protein family encoding sequences of tke present invention is operably linked depends directly, as is well known in tke art, on tke functional properties desired, e.g., protein expression, and tke kost cell to be transformed.
  • a vector contemplated by tke present invention is at least capable of directing the replication or insertion into tke kost chromosome, and preferably also expression, of tke structural gene included in tke rDNA molecule.
  • Expression control elements tkat are used for regulating tke expression of an operably linked protein encoding sequence are known in the art and include, but are not limited to, inducible promoters, constitutive promoters, secretion signals, and other regulatory elements.
  • the inducible promoter is readily controlled, such as being responsive to a nutrient in the host cell's medium.
  • the vector containing a coding nucleic acid molecule will include a prokaryotic repkcon, i.e., a DNA sequence having tke ability to direct autonomous replication and maintenance of the recombinant DNA molecule extra-chromosomally in a prokaryotic host cell, such as a bacterial host cell, transformed therewith.
  • a prokaryotic repkcon i.e., a DNA sequence having tke ability to direct autonomous replication and maintenance of the recombinant DNA molecule extra-chromosomally in a prokaryotic host cell, such as a bacterial host cell, transformed therewith.
  • a prokaryotic host cell such as a bacterial host cell, transformed therewith.
  • vectors tkat include a prokaryotic replicon may also include a gene wkose expression confers a detectable marker such as a drug resistance. Typical bacterial drug resistance genes are tkose tkat confer resistance to ampicillin or tetracycline.
  • Vectors tkat include a prokaryotic replicon can furtker include a prokaryotic or bacteriopbage promoter capable of directing the expression (transcription and translation) of tke coding gene sequences in a bacterial kost cell, suck as E. coli.
  • a promoter is an expression control element formed by a DNA sequence tkat permits binding of RNA polymerase and transcription to occur.
  • Promoter sequences compatible witk bacterial hosts are typically provided in plasmid vectors containing convenient restriction sites for insertion of a DNA segment of tke present invention.
  • Typical of suck vector plasmids are pUC8, pUC9, pBR322 and pBR329 available from BioRad Laboratories, (Richmond, CA), pPL and pKK223 available from Pharmacia (Piscataway, NJ).
  • Expression vectors compatible with eukaryotic cells can also be used to form rDNA molecules that contain a coding sequence.
  • Eukaryotic cell expression vectors are well known in the art and are available from several commercial sources. Typically, such vectors are provided containing convenient restriction sites for insertion of tke desired DNA segment. Typical of suck vectors are pSVL and pKSV-10 (Pkarmacia), pBPV-l/pML2d (International Biotechnologies, Inc.), pTDTl (ATCC, #31255), the vector pCDM8 described herein, and the like eukaryotic expression vectors. Vectors may be modified to include kidney cell specific promoters if needed.
  • Eukaryotic cell expression vectors used to construct the rDNA molecules of tke present invention may furtker include a selectable marker that is effective in an eukaryotic cell, preferably a drug resistance selection marker.
  • a preferred drug resistance marker is the gene whose expression results in neomycin resistance, i.e., the neomycin phosphotransferase (neo) gene. (Southern et al. Journal of Molecular and Applied Genetics, Vol. 1, no. 4 (1982) pp.
  • the selectable marker can be present on a separate plasmid, and the two vectors are introduced by co- transfection of tke kost cell, and selected by culturing in tke appropriate.drug for tke selectable marker.
  • the present invention furtker provides kost cells transformed witk a nucleic acid molecule tkat encodes a protein of tke present invention.
  • Tke kost cell can be either prokaryotic or eukaryotic.
  • Eukaryotic cells useful for expression of a protein of tke invention are not limited, so long as tke cell kne is compatible witk cell culture metkods and compatible witk tke propagation of tke expression vector and expression of tke gene product.
  • Preferred eukaryotic kost cells include, but are not limited to, yeast, insect and mammalian cells, preferably vertebrate cells such as those from a mouse, rat, monkey or human cell line.
  • Preferred eukaryotic host cells include Chinese hamster ovary (CHO) cells available from the ATCC as CCL61, NIH Swiss mouse embryo cells (NIH/3T3) available from the ATCC as CRL 1658, baby hamster kidney cells (BHK), and the like eukaryotic tissue culture cell lines. Any prokaryotic host can be used to express a rDNA molecule encoding a protein of the invention.
  • the preferred prokaryotic host is E. coli.
  • Transformation of appropriate cell hosts with a rDNA molecule of the present invention is accomplished by well known methods tkat typically depend on tke type of vector used and kost system employed. Witk regard to transformation of prokaryotic kost cells, electroporation and salt treatment metkods are typically employed, see, for example, Coken et al. Proceedings of the National Academy of Science USA, Vol. 69, no. 8 (1972) pp. 2110-2114; and Maniatis et al. Molecular Cloning: A Laboratory Mammal. Cold Spring Harbor, NY. Cold Spring Harbor Laboratory Press, 1982).
  • Witk regard to transformation of vertebrate cells witk vectors containing rDNAs, electroporation, cationic lipid or salt treatment metkods are typically employed, see, for example, Graham et al. Virology, Vol. 52, no. 2 (1973) pp. 456-467; and Wigler et al. Proceedings of the National Academy of Science USA, Vol. 76 (1979) pp. 1373-1376.
  • Successfully transformed cells i.e., cells that contain a rDNA molecule of tke present invention
  • cells resulting from the introduction of an rDNA of the present invention can be cloned to produce single colonies. Cells from those colonies can be harvested, lysed and their DNA content examined for the presence of the rDNA using a method such as that described by Southern, Journal of Molecular Biology, Vol. 98, no. 3 (1975) pp. 503-517; or Berent et al. Biotechnic and Histochemistry, Vol. 3 (1985) pp. 208; or the proteins produced from the cell assayed via an immunological method.
  • the present invention further provides methods for producing a protein of the invention using nucleic acid molecules herein described.
  • the production of a recombinant form of a protein typically involves the following steps: First, a nucleic acid molecule is obtained tkat encodes a protein of tke invention, suck as tke nucleic acid molecule comprising, consisting essentially of, or consisting of SEQ ID NO: 1, or nucleotides 121-948 of SEQ ID NO: 1, or nucleotides 121-945 of SEQ ID NO: 1. If the encoding sequence is uninterrupted by introns, as is this open reading frame, it is directly suitable for expression in any host.
  • Tke nucleic acid molecule is tken preferably placed in operable linkage with suitable control sequences, as described above, to form an expression unit containing tke protein open reading frame.
  • Tke expression unit is used to transform a suitable kost and tke transformed kost is cultured under conditions tkat allow tke production of tke recombinant protein.
  • tke recombinant protein is isolated from tke medium or from tke cells; recovery and purification of tke protein may not be necessary in some instances wkere some impurities may be tolerated.
  • Eack of tke foregoing steps can be done in a variety of ways.
  • the desired coding sequences may be obtained from genomic fragments and used directly in appropriate hosts.
  • the construction of expression vectors tkat are operable in a variety of kosts is accomplisked using appropriate replicons and control sequences, as set forth above.
  • Tke control sequences, expression vectors, and transformation metkods are dependent on tke type of kost cell used to express tke gene and were discussed in detail earker.
  • Suitable restriction sites can, if not normally available, be added to tke ends of tke coding sequence so as to provide an excisable gene to insert into these vectors.
  • a skilled artisan can readily adapt any host/expression system known in tke art for use witk tke nucleic acid molecules of tke invention to produce recombinant protein.
  • Another embodiment of the present invention provides methods for use in isolating and identifying binding partners of proteins of the invention
  • a protein of the invention is mixed with a potential binding partner or an extract or fraction of a cell under conditions that allow the association of potential binding partners with the protein of the invention.
  • peptides, polypeptides, proteins or other molecules that have become associated with a protein of the invention are separated from the mixture.
  • the binding partner that bound to the protein of the invention can then be removed and further analyzed.
  • tbe entire protein for instance a protein comprising tke entire amino acid sequence of SEQ ID NO: 2 can be used.
  • a fragment of tke protein can be used.
  • a cellular extract refers to a preparation or fraction wkick is made from a lysed or disrupted cell.
  • Tke preferred source of cellular extracts will be cells derived from kuman kidney tissue or cells, for instance, renal biopsy tissue or a tissue culture cell model of renal disease.
  • cellular extracts may be prepared from normal kuman kidney tissue or available cell lines, particularly kidney derived cell lines.
  • metkods can be used to obtain an extract of a cell.
  • Cells can be disrupted using eitker pkysical or ckemical disruption metkods.
  • pkysical disruption metkods include, but are not limited to, sonication and meckanical skearing.
  • ckemical lysis metkods include, but are not limited to, detergent lysis and enzyme lysis.
  • a skilled artisan can readily adapt metkods for preparing cellular extracts in order to obtain extracts for use in tke present metkods.
  • tke extract is mixed witk tke protein of tke invention under conditions in wkick association of tke protein witk tke binding partner can occur.
  • conditions can be used, the most preferred being conditions that closely resemble conditions found in the cytoplasm of a human cell.
  • Features such as osmolarity, pH, temperature, and the concentration of cellular extract used, can be varied to optimize the association of the protein with the binding partner.
  • the bound complex is separated from the mixture.
  • a variety of techniques can be utilized to separate the mixture. For example, antibodies specific to a protein of the invention can be used to immunoprecipitate the binding partner complex.
  • the binding partner can be dissociated from the complex using conventional methods. For example, dissociation can be accomplished by altering the salt concentration or pH of the mixture.
  • the protein of the invention can be immobilized on a solid support.
  • the protein can be attached to a nitrocellulose matrix or acrylic beads. Attachment of the protein to a solid support aids in separating peptide binding partner pairs from other constituents found in the extract.
  • the identified binding partners can be either a single protein or a complex made up of two or more proteins. Alternatively, binding partners may be identified using a Far- Western assay according to the procedures of Takayama et al. Methods in Molecular Biology, Vol. 69 (1997) pp. 171-184 or identified through the use of epitope tagged proteins or GST fusion proteins.
  • the nucleic acid molecules of the invention can be used in a yeast two- hybrid system.
  • the yeast two-hybrid system has been used to identify other protein partner pairs and can readily be adapted to employ the nucleic acid molecules herein described.
  • Another embodiment of the present invention provides methods for identifying agents that modulate the expression of a nucleic acid encoding a protein of the invention such as a protein having the amino acid sequence of SEQ ID NO: 2. Such assays may utilize any available means of monitoring for changes in the expression level of the nucleic acids of the invention.
  • an agent is said to modulate the expression of a nucleic acid of the invention, for instance a nucleic acid encoding the protein having the sequence of SEQ ID NO: 2, if it is capable of up- or down-regulating expression of the nucleic acid in a cell compared to a control.
  • cell lines that contain reporter gene fusions between the open reading frame defined by nucleotides 121-948 or nucleotide 121-945 of SEQ ID NO: 1, and/or the 5' and/or the 3' regulatory elements, and any assayable fusion partner may be prepared.
  • Numerous assayable fusion partners are known and readily available including the firefly luciferase gene and the gene encoding chloramphenicol acetyltransferase (Alam et al.
  • RNA or mRNA is isolated by standard procedures such those disclosed in Sambrook et al. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, NY. Cold Spring Harbor Laboratory Press, 1989.
  • Probes to detect differences in RNA expression levels between cells exposed to the agent and control cells may be prepared from the nucleic acids of the invention. It is preferable, but not necessary, to design probes which hybridize only with target nucleic acids under conditions of high stringency. Only highly complementary nucleic acid hybrids form under conditions of high stringency. Accordingly, the stringency of the assay conditions determines the amount of complementarity which should exist between two nucleic acid strands in order to form a hybrid. Stringency should be chosen to maximize the difference in stability between tke probe:target kybrid and potential probe:non-target kybrids.
  • Probes may be designed from tke nucleic acids of tke invention through methods known in the art. For instance, the G+C content of the probe and the probe length can affect probe binding to its target sequence. Methods to optimize probe specificity are commonly available in Sambrook et al. (Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, NY. Cold Spring Harbor Laboratory Press, 1989); or Ausubel et al. (Current Protocols in Molecular Biology. NY, Greene Publishing Company, 1995).
  • Hybridization conditions are modified using known methods, such as those described by Sambrook et al. and Ausubel et al. as required for each probe.
  • Hybridization of total cellular RNA or RNA enriched for polyA RNA can be accomplished in any available format.
  • total cellular RNA or RNA enriched for polyA RNA can be affixed to a solid support and the solid support exposed to at least one probe comprising at least one, or part of one of the sequences of the invention under conditions in which the probe will specifically hybridize.
  • nucleic acid fragments comprising at least one, or part of one of the sequences of the invention can be affixed to a solid support, such as a silicon chip or a porous glass wafer.
  • the solid support can then be exposed to total cellular RNA or polyA RNA from a sample under conditions in which the affixed sequences will specifically hybridize.
  • Such solid supports and hybridization methods are widely available, for example, those disclosed by Beattie (1995) (WO 95/11755).
  • Hybridization for qualitative and quantitative analysis of mRNAs may also be carried out by using a RNase Protection Assay (i.e., RPA, see Ma et al. Methods, Vol. 10, no. 3 (1996) pp. 273-238).
  • RPA RNase Protection Assay
  • an expression vehicle comprising cDNA encoding the gene product and a phage specific DNA dependent RNA polymerase promoter (e.g., T7, T3 or SP6 RNA polymerase) is linearized at the 3' end of the cDNA molecule, downstream from the phage promoter, wherein such a linearized molecule is subsequently used as a template for synthesis of a labeled antisense transcript of the cDNA by in vitro transcription.
  • a phage specific DNA dependent RNA polymerase promoter e.g., T7, T3 or SP6 RNA polymerase
  • the labeled transcript is then hybridized to a mixture of isolated RNA (i.e., total or fractionated mRNA) by incubation at 45 °C overnight in a buffer comprising 80% formamide, 40 mM Pipes, pH 6.4, 0.4 M NaCl and 1 mM EDTA.
  • the resulting hybrids are then digested in a buffer comprising 40 ⁇ g/ml ribonuclease A and 2 ⁇ g/ml ribonuclease. After deactivation and extraction of extraneous proteins, the samples are loaded onto urea/polyacrylamide gels for analysis.
  • cells or cell lines are first identified that express the gene products of the invention physiologically (e.g., see for example, Figure 1 for tissue distribution via Northern blot, however, RPAs may serve the identical purpose of expression selection).
  • Cell and/or cell lines so identified would be expected to comprise the necessary cellular machinery such that the fidelity of modulation of the transcriptional apparatus is maintained with regard to exogenous contact of agent with appropriate surface transduction mechanisms and/or the cytosolic cascades.
  • such cells or cell lines would be transduced or transfected with an expression vehicle (e.g., a plasmid or viral vector) construct comprising an operable non-translated 5 '-promoter containing end of the structural gene encoding the instant gene products fused to one or more antigenic fragments, which are peculiar to tke instant gene products, wherein said fragments are under the transcriptional control of said promoter and are expressed as polypeptides whose molecular weight can be distinguished from the naturally occurring polypeptides or may further comprise an immunologically distinct tag or other detectable marker.
  • an expression vehicle e.g., a plasmid or viral vector
  • Cells or cell lines transduced or transfected as outlined above are then contacted with agents under appropriate conditions; for example, the agent in a pharmaceutically acceptable excipient is contacted with cells in an aqueous physiological buffer such as phosphate buffered saline (PBS) at physiological pH, Eagles balanced salt solution (BSS) at physiological pH, PBS or BSS comprising serum or conditioned media comprising PBS or BSS and/or serum incubated at 37 °C .
  • PBS phosphate buffered saline
  • BSS Eagles balanced salt solution
  • Said conditions may be modulated as deemed necessary by one of skill in the art.
  • the cells will be disrupted and the polypeptides of the lysate are fractionated such that a polypeptide fraction is pooled and contacted with an antibody to be further processed by immuno logical assay (e.g., ELISA, immunoprecipitation or Western blot).
  • immuno logical assay e.g., ELISA, immunoprecipitation or Western blot.
  • the pool of proteins isolated from the "agent-contacted” sample will be compared with a control sample where only the excipient is contacted with the cells and an increase or decrease in the immunologically generated signal from the "agent- contacted" sample compared to the control will be used to distinguish the effectiveness of the agent.
  • Another embodiment of the present invention provides methods for identifying agents that modulate the level of or at least one activity of a protein of the invention such as the protein having the amino acid sequence of SEQ ID NO: 2. Such methods or assays may utilize any means of monitoring or detecting the desired activity.
  • the relative amounts of a protein of the invention between a cell population that has been exposed to the agent to be tested compared to an un-exposed control cell population may be assayed.
  • probes such as specific antibodies are used to monitor the differential expression of the protein in the different cell populations.
  • Cell lines or populations are exposed to the agent to be tested under appropriate conditions and time.
  • Cellular lysates may be prepared from the exposed cell line or population and a control, unexposed cell line or population. The cellular lysates are then analyzed with the probe.
  • Antibody probes are prepared by immunizing suitable mammalian hosts in appropriate immunization protocols using the peptides, polypeptides or proteins of the invention if they are of sufficient length, or, if desired, or if required to enhance immunogenicity, conjugated to suitable carriers. Methods for preparing immunogenic conjugates with carriers such as BSA, KLH, or other carrier proteins are well known in the art. In some circumstances, direct conjugation using, for example, carbodiimide reagents may be effective; in other instances linking reagents such as those supplied by Pierce Chemical Co. (Rockford, IL), may be desirable to provide accessibility to the hapten.
  • the hapten peptides can be extended at either the amino or carboxy terminus with a cysteine residue or interspersed with cysteine residues, for example, to facilitate linking to a carrier.
  • Administration of the immunogens is conducted generally by injection over a suitable time period and with use of suitable adjuvants, as is generally understood in the art.
  • titers of antibodies are taken to determine adequacy of antibody formation.
  • Wkile tke polyclonal antisera produced in tkis way may be satisfactory for some applications, for pkarmaceutical compositions, use of monoclonal preparations is preferred.
  • Immortalized cell lines wkick secrete tke desired monoclonal antibodies may be prepared using the standard method of Kohler and Milstein (Nature, Vol. 256, no. 5517 (Aug. 1975) pp. 495-497) or modifications which effect immortalization of lymphocytes or spleen cells, as is generally known.
  • the immortalized cell lines secreting tke desired antibodies are screened by immunoassay in which the antigen is the peptide hapten, polypeptide or protein.
  • the cells can be cultured either in vitro or by production in ascites fluid.
  • the desired monoclonal antibodies are then recovered from the culture supernatant or from the ascites supernatant. Fragments of the monoclonal antibodies or the polyclonal antisera which contain the immunologically significant portion can be used as antagonists, as well as the intact antibodies. Use of immunologically reactive antibody fragments, such as the Fab, Fab', of F(ab') 2 fragments is often preferable, especially in a therapeutic context, as these fragments are generally less immunogenic than the whole immunoglobulm.
  • the antibodies or fragments may also be produced, using current technology, by recombinant means. Antibody regions that bind specifically to the desired regions of the protein can also be produced in the context of chimeras with multiple species origin, such as humanized antibodies.
  • Agents that are assayed in the above method can be randomly selected or rationally selected or designed.
  • an agent is said to be randomly selected when the agent is chosen randomly without considering the specific sequences involved in the association of a protein of the invention alone or with its associated substrates, binding partners, etc.
  • An example of randomly selected agents is the use a chemical library or a peptide combinatorial library, or a growth broth of an organism.
  • an agent is said to be rationally selected or designed when the agent is chosen on a nonrandom basis which takes into account the sequence of the target site and/or its conformation in connection with the agent's action.
  • Agents can be rationally selected or rationally designed by using the peptide sequences that make up these sites.
  • a rationally selected peptide agent can be a peptide whose amino acid sequence is identical to or a derivative of any functional consensus site.
  • the agents of the present invention can be, as examples, peptides, small molecules, vitamin derivatives, as well as carbohydrates. Dominant negative proteins, DNAs encoding these proteins, antibodies to these proteins, peptide fragments of these proteins or mimics of these proteins may be introduced into cells to affect function. "Mimic” used herein refers to the modification of a region or several regions of a peptide molecule to provide a structure chemically different from the parent peptide but topographically and functionally similar to the parent peptide (see Grant GA. in: Meyers (ed.) Molecular Biology and Biotechnology (New York, VCH Publishers, 1995), pp. 659-664).
  • Tke peptide agents of tke invention can be prepared using standard solid phase (or solution phase) peptide synthesis methods, as is known in the art.
  • the DNA encoding these peptides may be synthesized using commercially available oligonucleotide synthesis instrumentation and produced recombinantly using standard recombinant production systems. The production using solid phase peptide synthesis is necessitated if non-gene- encoded amino acids are to be included.
  • Another class of agents of the present invention are antibodies immunoreactive with critical positions of proteins of the invention.
  • Antibody agents are obtained by immunization of suitable mammalian subjects with peptides, containing as antigenic regions, those portions of the protein intended to be targeted by the antibodies.
  • the proteins and nucleic acids of the invention are expressed in various types of diseased renal tissue, as well as in otker organs, tissues and cells, for example during stages of inflammation associated witk infliltrating leukocytes or during cell growtk arrest.
  • Renal disease and renal disorder refer to any disease of tke kidney, including but not limited to IgA nephropathy (IgAN), necrotizing crescentic glomerulonephritis (NCGN), and minimal change disease.
  • Agents that up- or down-regulate or modulate the expression of the protein or at least one activity of the protein may be used to up- or down- regulate or modulate biological and pathologic processes associated with the protein's function and activity.
  • a subject can be any mammal, so long as the mammal is in need of modulation of a pathological or biological process mediated by a protein of tke invention.
  • Tke term "mammal" is defined as an individual belonging to the class Mammalia. The invention is particularly useful in the treatment of human subjects.
  • Pathological processes refer to a category of biological processes which produce a deleterious effect.
  • expression of a protein of the invention may be associated with kidney cell growth regeneration and/or recovery from kidney disease.
  • an agent is said to modulate a pathological process when the agent reduces the degree or severity of the process.
  • kidney damage or ESRD may be prevented or disease progression modulated by the administration of agents that up- or down-regulate or modulate in some way the expression or at least one activity of a protein of the invention.
  • the agents of the present invention can be provided alone, or in combination with other agents that modulate a particular pathological process.
  • an agent of the present invention can be administered in combination with other known drugs or may be combined with dialysis or anti-rejection drugs used during transplantation.
  • two agents are said to be administered in combination when the two agents are administered simultaneously or are administered independently in a fashion such that the agents will act at the same time.
  • the agents of the present invention can be administered via parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal, or buccal routes. Alternatively, or concurrently, administration may be by the oral route.
  • the dosage administered will be dependent upon the age, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired.
  • the present invention further provides compositions containing one or more agents which modulate expression or at least one activity of a protein of the invention. While individual needs vary, determination of optimal ranges of effective amounts of each component is within tke skill of tke art.
  • Typical dosages comprise 0.1 to 100 ⁇ g/kg body wt.
  • the preferred dosages comprise 0.1 to 10 ⁇ g/kg body weight.
  • the most preferred dosages comprise 0.1 to 1 ⁇ g/kg body weight.
  • compositions of the present invention may contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically for delivery to the site of action.
  • suitable formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form, for example, water-soluble salts.
  • suspensions of the active compounds as appropriate oily injection suspensions may be administered.
  • Suitable lipophilic solvents or vehicles include fatty oils, for example, sesame oil, or syntketic fatty acid esters, for example, etkyl oleate or triglycerides.
  • Aqueous injection suspensions may contain substances wkick increase tke viscosity of tke suspension include, for example, sodium carboxymetbyl cellulose, sorbitol, and/or dextran.
  • the suspension may also contain stabilizers. Liposomes can also be used to encapsulate the agent for delivery into tke cell.
  • Tke pkarmaceutical formulation for systemic administration according to tke invention may be formulated for enteral, parenteral or topical administration. Indeed, all three types of formulations may be used simultaneously to achieve systemic administration of the active ingredient. Suitable formulations for oral administration include hard or soft gelatin capsules, pills, tablets, including coated tablets, elixirs, suspensions, syrups or inkalations and controlled release forms tkereof.
  • tke compounds of tkis invention may be used alone or in combination, or in combination with other therapeutic or diagnostic agents.
  • the compounds of this invention may be coadministered along with other compounds typically prescribed for these conditions according to generally accepted medical practice.
  • the compounds of this invention can be utilized in vivo, ordinarily in mammals, such as humans, sheep, horses, cattle, pigs, dogs, cats, rats and mice, or in vitro.
  • Transgenic animals containing mutant, knock-out or modified genes corresponding to the cDNA sequence of SEQ ID NO: 1, or the open reading frame encoding the polypeptide sequence of SEQ ID NO: 2, or fragments tkerof kaving a consecutive sequence of at least about 3, 4, 5, 6, 10, 15, 20, 25, 30, 35 or more amno acid residues, are also included in the invention.
  • Transgenic animals are genetically modified animals into whick recombinant, exogenous or cloned genetic material has been experimentally transferred. Such genetic material is often referred to as a "transgene".
  • the nucleic acid sequence of the transgene in this case a form of SEQ ID NO: 1 may be integrated either at a locus of a genome where that particular nucleic acid sequence is not otherwise normally found or at the normal locus for the transgene.
  • the transgene may consist of nucleic acid sequences derived from the genome of the same species or of a different species than the species of the target animal.
  • germ cell line transgenic animal refers to a transgenic animal in which the genetic alteration or genetic information was introduced into a germ line cell, thereby conferring the ability of the transgenic animal to transfer the genetic information to offspring. If such offspring in fact possess some or all of that alteration or genetic information, then they too are transgenic animals.
  • the alteration or genetic information may be foreign to the species of animal to which the recipient belongs, foreign only to the particular individual recipient, or may be genetic information already possessed by the recipient. In the last case, the altered or introduced gene may be expressed differently than the native gene.
  • Transgenic animals can be produced by a variety of different methods including transfection, electroporation, microinjection, gene targeting in embryonic stem cells and recombinant viral and retroviral infection (see, e.g., U.S. Patent No. 4,736,866; U.S. Patent No. 5,602,307; Mullins et al. Hypertension (1993) 22(4):630-633; Brenin et al. Surgical Oncology, Vol. 6, no. 2 (1997) pp. 99-110; "Recombinant Gene Expression Protocols” in: Tuan (ed.), Methods in Molecular Biology, No. 62 (Humana Press, 1997)).
  • mice kave A number of recombinant or transgenic mice kave been produced, including tkose wkick express an activated oncogene sequence (U.S. Patent No. 4,736,866); express simian SV40 T-antigen (U.S. Patent No. 5,728,915); lack tke expression of interferon regulatory factor 1 (IRF-1) (U.S. Patent No. 5,731,490); exkibit dopaminergic dysfunction (U.S. Patent No. 5,723,719); express at least one human gene which participates in blood pressure control (U.S. Patent No. 5,731,489); display greater similarity to the conditions existing in naturally occurring Alzheimer's disease (U.S. Patent No.
  • mice and rats remain the animals of choice for most transgenic experimentation, in some instances it is preferable or even necessary to use alternative animal species.
  • Transgenic procedures have been successfully utilized in a variety of non-murine animals, including sheep, goats, pigs, dogs, cats, monkeys, chimpanzees, hamsters, rabbits, cows and guinea pigs (see, e.g., Kim et al. Molecular Reproduction and Development, Vol. 46, no. 4 (1997) pp. 515-526; Houdebine. Reproduction, Nutrition, Development, Vol. 35, no. 6 (1995) pp. 609-617; Petters Reproduction, Fertility and Development, Vol. 6, no. 5 (1994) pp. 643- 645; Schnieke et al. Science Vol. 278, no. 5346 (1997) pp. 2130-2133; and Amoah, Journal of Animal Science, Vol. 75, no. 2 (1997) pp. 578-585.
  • the method of introduction of nucleic acid fragments into recombination competent mammalian cells can be by any method which favors co-transformation of multiple nucleic acid molecules.
  • Detailed procedures for producing transgenic animals are readily available to one skilled in the art, including the disclosures in U.S. Patent No. 5,489,743 and U.S. Patent No. 5,602,307.
  • the genes and proteins of the invention are differentially expressed in the tissue of patients with IgAN compared with normal renal tissue, they maybe used to diagnose or monitor IgAN, reanl function, or to track disease progression.
  • One means of diagnosing a renal disease using the nucleic acid molecules or proteins of the invention involves obtaining kidney tissue from living subjects. Obtaining tissue samples from living sources is problematic for tissues such as kidney. However, due to the nature of the treatment paradigms for kidney patients, biopsy may be necessary. If possible, renal biopsy tissue may be obtained percutaneously.
  • Another means of diagnosing renal disease using the nucleic acid molecules or proteins of tke invention involves obtaining blood samples from living subjects.
  • nucleic acid probes comprising all or at least part of tke sequence of SEQ ID NO: 1 may be used to determine the expression of a nucleic acid molecule in forensic/pathology specimens.
  • nucleic acid assays may be carried out by any means of conducting a transcriptional profiling analysis.
  • forensic methods of the invention may target the proteins of the invention, particularly a protein comprising the amino acid sequence of SEQ ID NO: 2, to determine up- or down- regulation of the genes (Shiverick et al, Biochim Biophys Acta (1975) 393:124-33).
  • Methods of the invention may involve treatment of tissues with collagenases or other proteases to make the tissue amenable to cell lysis (Semenov DE et al, Biull Eksp BiolMed (1987) 104:113-6). Further, it is possible to obtain biopsy samples from different regions of the kidney for analysis.
  • Assays to detect nucleic acid or protein molecules of the invention may be in any available format.
  • Typical assays for nucleic acid molecules include hybridization or PCR based formats.
  • Typical assays for the detection of proteins, polypeptides or peptides of the invention include the use of antibody probes in any available format such as in situ binding assays, etc. See Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1988. In preferred embodiments, assays are carried-out with appropriate controls.
  • the above methods may also be used in other diagnostic protocols, including protocols and methods to detect disease states in other tissues or organs, for example the colon, pancreas, and spleen, particularly when inflammation associated with infiltrating leukocytes is involved.
  • Kidney tissue was obtained from patients with various forms and stages of diagnosed renal disease.
  • Total cellular RNA was prepared from the kidney tissue described above as well as from control kidney tissue using the procedure of Newburger et al. Journal of Biological Chemistry, Vol. 266, no. 24 (1991) pp. 16171-16177 and Newburger et al. Proceedings of National Academy of Science USA, Vol. 85 (1988) pp. 5215-5219. Synthesis of cDNA was performed as previously described by Prashar et al. in WO
  • cDNA was syntkesized according to tke protocol described in tbe GIBCO/BRL kit for cDNA syntkesis.
  • Tke reaction mixture for first-strand syntkesis included 6 ⁇ g of total RNA, and 200 ng of a mixture of 1-base anchored oligo(dT) primers with all three possible anchored bases
  • reaction mixture may include 10 ⁇ g of total RNA, and 2 pmol of one of the 2-base anchored oligo(dT) primers a keel suck as RP5.0 (CTCTCAAGGATCTTACCGCTT 18 AT (SEQ ID NO: 4)), or
  • RP6.0 TAATACCGCGCCACATAGCAT 18 CG (SEQ ID NO: 5)
  • RP9.2 CAGGGTAGACGACGCTACGCT lg GA (SEQ ID NO: 6)
  • Tkis mixture was tken layered witk mineral oil and incubated at 65 °C for seven minutes followed by 50°C for anotber seven minutes.
  • 2 ⁇ l of Superscript® reverse transcriptase 200 units/ ⁇ l; GIBCO/BRL was added quickly and mixed, and tke reaction continued for one kour at 45-50 °C.
  • Second- strand syntkesis was performed at 16 °C for two kours. At tke end of tke reaction, tke cDNAs were precipitated witk etkanol and tke yield of cDNA was calculated. In our experiments, -200 ng of cDNA was obtained from 10 ⁇ g of total RNA.
  • the adapter oligonucleotide sequences were Al (TAGCGTCCGGCGCAGCGACGGCCAG (SEQ ID NO: 7)) and A2 (GATCCTGGCCGTCGGCTGTCTGTCGGCGC (SEQ ID NO: 8)).
  • oligonucleotide A2 was first phosphorylated at the 5 ' end using T4 polynucleotide kinase (PNK). After phosphorylation, PNK was heat-denatured, and 1 ⁇ g of the oligonucleotide Al was added along with lOx annealing buffer (1 M NaCl/100 mM Tris- HC1, pH8.0/10 mM EDTA, pH8.0) in a final volume of 20 ⁇ l. This mixture was then heated at 65 °C for ten minutes followed by slow cooling to room temperature for thirty minutes, resulting in formation of the Y adapter at a final concentration of 100 ng/ ⁇ l.
  • PNK polynucleotide kinase
  • reaction mixture was diluted with water to a final volume of 80 ⁇ l (adapter ligated cDNA concentration, -50 pg/ ⁇ l) and heated at 65 °C for ten minutes to denature T4 DNA ligase, and 2 ⁇ l aliquots (witk ⁇ 100 pg of cDNA) were used for PCR.
  • Tke following sets of primers were used for PCR amplification of tke adapter ligated 3 '-end cDNAs:
  • oligonucleotide Al or Al.l was 5'- end-labeled using 15 ⁇ l of [ ⁇ - 32 P]ATP (Amersham; 3000 Ci/mmol) and PNK in a final volume of 20 ⁇ l for thirty minutes at 37°C. After heat denaturing PNK at 65 °C for twenty minutes, the labeled oligonucleotide was diluted to a final concentration of 2 ⁇ M in 80 ⁇ l witk unlabeled oligonucleotide Al.l.
  • Tke PCR mixture (20 ⁇ l) consisted of 2 ⁇ l (- 100 pg) of tke template, 2 ⁇ l of lOx PCR buffer (100 mM Tris-HCl, pH 8.3/500 mM KC1), 2 ⁇ l of 15 mM MgCl 2 to yield 1.5 mM final Mg 2+ concentration optimum in tke reaction mixture, 200 ⁇ M dNTPs, 200 nM each 5' and 3' PCR primers, and one unit of Am litaq Gold®. Primers and dNTPs were added after prekeating tke reaction mixture containing tke rest of tke components at 85 °C.
  • Tkis "kot start" PCR was done to avoid artifactual amplification arising out of arbitrary annealing of PCR primers at lower temperature during transition from room temperature to 94 °C in the first PCR cycle.
  • PCR consisted of five cycles of 94 °C for thirty seconds, 55 °C for two minutes, and 72 °C for sixty seconds followed by 25 cycles of 94 °C for thirty seconds, 60°C for two minutes, and 72°C for sixty seconds. A higher number of cycles resulted in smeary gel patterns.
  • PCR products (2.5 ⁇ l) were analyzed on 6% polyacrylamide sequencing gel.
  • tke ligated cDNA sample was digested witk a secondary restriction enzyme(s) in a final vol of 20 ⁇ l. From tkis solution, 3 ⁇ l was used as template for PCR. Tkis template vol of 3 ⁇ l carried ⁇ 100 pg of tke cDNA and 10 mM MgCl 2 (from tke lOx enzyme buffer), wkick diluted to tke optimum of 1.5 mM in tke final PCR volume of 20 ⁇ l. Since Mg 2+ comes from tke restriction enzyme buffer, it was not included in tke reaction mixture wken amplifying secondarily cut cDNA.
  • Example 2 Cloning of a Full Length human cDNA Corresponding to the EST fragment
  • the full length cDNA corresponding to the EST fragment in Example 1 was obtained by the oligo-pulling method. Briefly, a gene-specific oligo was designed based on the sequence of the EST identified in Example 1. The oligo was labeled witk biotin and used to bybridize witk 2 ⁇ g of single strand plasmid DNA (cDNA recombinants) from a kuman kidney cDNA library following tke procedures of Sambrook et al. (Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, NY. Cold Spring Harbor Laboratory Press, 1985).
  • Tke kybridized cDNAs were separated by streptavidin-conjugated beads and eluted by heating.
  • the eluted cDNA was converted to double strand plasmid DNA and used to transform E. coli cells (DH10B) and the longest cDNA was screened. After confirmation by PCR using gene-specific primers, the cDNA clone was subjected to DNA sequencing.
  • the nucleotide sequence of the full-length cDNA corresponding to the differentially regulated EST band is set forth in SEQ ID NO: 1.
  • the cDNA comprises about 2839 base pairs with an open reading frame from nucleotide 121 to 945 (to 948, including the stop codon) encoding a 275 amino acid protein, as is set forth in SEQ ID NO: 2.
  • the predicted structural characteristics, hydrophilicity and antigenicity of peptide fragments within SEQ ID NO: 2 are shown in Figure 8.
  • a cAMP- and cGMP-dependent protein kinase phosphorylation site is present at amino acid position 194-197; seven casein kinase II phosphorylation sites are present starting at amino acids 8, 53, 75, 204, 234, 244 and 249; and Protein Kinase C phosphorylation sites are present at amino acid 13, 131 an 193.
  • Six amino acid N-myristoylation motifs occur througkout tke protein starting at amino acids 9, 18, 36, 49, 161 and 238.
  • FIG. 3 to 7 skow tke results of tke quantitative PCR analysis of expression levels of mRNA corresponding to SEQ ID NO: 1 in various kuman tissue or blood samples.
  • Real time PCR detection was accomplisbed by the use of the ABI PRISM 7700 Sequence Detection System.
  • the 7700 measures the fluorescence intensity of the sample each cycle and is able to detect the presence of specific amplicons within the PCR reaction.
  • Each sample was assayed for the level of GAPDH and mRNA corresponding to SEQ ID NO: 1.
  • GAPDH detection was performed using Perkin Elmer part #402869 according to the manufacturer's directions.
  • Primers were designed from SEQ ID NO: 1 using Primer Express, a program developed by PE to efficiently find primers and probes for specific sequences. These primers were used in conjunction with SYBR green (Molecular Probes), a nonspecific double stranded DNA dye, to measure the expression level mRNA corresponding to SEQ ID NO: 1, which was then normalized to the GAPDH level in each sample.
  • SYBR green Molecular Probes
  • Tissue distribution analysis shows prominent expression in the glomeruli, medulla and cortex of the kidney, and additionally in the pancreas, colon and spleen, indicating that the gene and proteins of the invention could be involved in the pathogenesis of diseases afftecting these organs or other tissues, particularly when inflammation associated witk infiltrating leukocytes is involved.

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Abstract

The invention relates generally to changes in gene expression associated with renal disease, with inflammation, and with cell growth arrest, and to methods of using differentially expressed genes or gene products as drug discovery agents, diagnostic tools or as targets in therapeutic intervention. Specifically, the invention relates to a novel human gene that corresponds to an mRNA which is differentially-regulated in kidney tissue from patients diagnosed with immunoglobulin A nephropathy (IgAN).

Description

NOVEL cDNA 22360 ASSOCIATED WITH RENAL DISEASE AND OTHER DISEASE STATES
INVENTORS: Hong- Wei Sun, William E. Munger and Ronald J. Falk
CONTINUING APPLICATION INFORMATION
This application claims priority to U.S. Provisional Application No. 60/170,348 filed December 13, 1999, which is herein incorporated by reference in its entirity.
FIELD OF THE INVENTION
The invention relates generally to changes in gene expression associated with renal disorders, with inflammatory diseases, and with cell cycles, and to methods of using such genes or gene products as research or diagnostic tools, or as targets in therapeutic intervention. Specifically, this invention relates to a novel human gene which has been identified in kidney tissue from a patient diagnosed with immunoglobulm A nepkropathy (IgAN). This gene: 1) serves as a marker for renal diseases such as glomerulonephritis, including mesangial proliferation; necrotizing crescentic glomerulonephritis (NCGN); minimal change disease and sclerosis, 2) serves as a marker for inflammatory diseases including noninfectious systemic inflammatory responses (SIRS), and 3) provides a means of identifying agents that modulate its expression in renal mesangial cells and other tissues.
BACKGROUND OF THE INVENTION
Abnormal renal function is among the most common ailments requiring intensive medical care. In addition, the incidence and prevalence rates of end-stage renal disease (ESRD) in the United States continue to increase. In 1995, the incidence rate was 262 per million population, with a point prevalence rate of 975 per million population. The exact number of individuals with abnormal renal function who have not yet progressed to ESRD is difficult to assess. The incidence rate per million population of treated end-stage renal disease (ESRD) has been increasing at similar rates in most countries that record counts of new ESRD patients per year. Data from the United States Renal Data System (USRDS) suggest an exponential growth for both incidence rates and prevalence rates (Port FK. Disease- A-Month, Vol. 44, no. 5 (May 1998) pp. 214-234).
IgA nepkropathy (IgAN) is the most common type of immunologically mediated glomerulonephritis (GN) and is characterized by deposition in tbe glomerular mesangium of IgA together witk C3, C5b-9, and properdin. In patients, tke co-deposition of IgA together with IgG and/or IgM can lead to a more progressive course of disease. Fifteen to forty percent of primary glomerulonephritis in parts of Europe, Asia and Japan has been linked to IgAN and it is well accepted that IgAN can lead to ESRD.
IgAN often presents either as asymptomatic microscopic hematuria and/or proteinuria (most common in adults), or episodic gross hematuria following upper respiratory and other infections or exercise. The course of IgAN is variable, with some patients showing no decline in glomerular filtration rate (GFR) over decades and others developing the nephrotic syndrome, hypertension and renal failure.
Diabetic nephropathy is also a common cause of end-stage renal disease (ESRD) and accounts for 35% of the ESRD population in the United States. It results in considerable morbidity, mortality and expense. The average cost of managing one diabetic patient with ESRD is approximately $50,000 a year (Kobrin SM. Kidney International Supplement, Vol. 63 (Dec. 1997) pp. S144-150). Approximately 5.8 million people in the United States have been diagnosed by a physician as being diabetic, and an additional four to five million people have undiagnosed diabetes. Althougk tke incidence of new cases of diabetes appears to be declining from a peak of 300 per 100,000 population in 1973, to 230 per 100,000 in 1981, its prevalence continues to rise due to a 19 percent decline since 1970 in deatks caused by diabetes. In 1982, 34,583 deatks were attributed to diabetes, resulting in diabetes being ranked as tbe seventk leading underlying cause of deatk. Medical and surgical complications of diabetes due to macro- and microvascular disease result in 5,800 new cases of blindness, 4,500 perinatal deatks, 40,000 lower extremity amputations and 3,000 deatks due to diabetic coma (ketotic and byperosmolar) and at least 4,000 new cases of end-stage renal disease per year (Verk K. Verhandelingen - Koninklijke Academie Voor Geneeskunde Van Belgie, Vol. 51, no. 2 (1989) pp. 81-151). A ckaracteristic and early manifestation of diabetes in kumans is renal kypertropky (Bakris GL et al. Disease- A-Month, Vol. 39, no. 8 (1993) pp. 573-611). Tkis compensation meckanism is a pkysiological response in wkick tke cells of tke kidney increase in size and protein content witkout syntkesizing DNA or dividing (Kujubu et al. American Journal of Physiology, Vol. 260, no. 6, pt. 2 (1991) pp. F823-827). Tbe compensatory growtk of tke kidney is a kigkly regulated process (Wolf et al. Kidney International, Vol. 39, no. 3 (1991) pp. 401-420). Early studies demonstrated significant increases in ribosome syntbesis, including increased rDNA transcription rates (Ouellette et al. American Journal of Physiology, Vol. 253, no. 4, pt. 1 (1987) pp. C506-513). Subsequent studies bave demonstrated tke induction of early response genes suck as tke 7YS family; proto-oncogenes ras,fos, myc; structural protein genes suck as vimentin and β-actin; and transport protein genes (Na+K+- ATPase, ADP-ATP translocase and calcyclin (Norman et al. Proceedings of the National Academy of Science USA, Vol. 85, no. 18 (1988) pp. 6768-6772; Moskowitz et al. Journal of Urology, Vol. 154, no. 4 (1995) pp. 1560-1565; Sawczuk et al. Journal of Urology, Vol. 140, no. 5, pt. 2 (1988) pp. 1145-1148; Kujubu et al. American Journal of Physiology, Vol. 260, no. 6, pt. 2 (1991) pp. F823-827; Beer et al. Journal of Cellular Physiology, Vol. 131, no. 1 (1987) pp. 29-35). Renal kypertropky involves a gradual and progressive increase in mRNA levels resulting in tke coordinated expression of positive and negative growtk control elements. Suck sustained message amplification observed during renal compensation represents a distinct cellular response wkick is distinguiskable from tke responses tkat ckaracterize several patkways of cell differentiation (e.g., proto-oncogene expression patterns in kyperplasia in liver).
If intervention is expected to be successful in baiting or slowing down renal disease progression, means of accurately assessing tke early manifestations of renal disease need to be establisbed. One way to accurately assess tke early manifestations of renal disease is to identify markers wkick are uniquely associated witk disease progression. Likewise, tke development of tkerapeutics to prevent or repair kidney damage relies on tke identification of kidney genes responsible for kidney cell growtk and function. SUMMARY OF THE INVENTION
Tke present invention is based on tke discovery of a new gene family tkat is differentially expressed in various forms of renal disease, inflammation, or in various stages of cell growtk, compared witk normal cells and tissues. Tke invention includes isolated nucleic acid molecules selected from tke group consisting of an isolated nucleic acid molecule tkat encodes tke amino acid sequence of SEQ ID NO: 2, an isolated nucleic acid molecule tkat encodes a fragment of at least 6 amino acids of SEQ ID NO: 2, an isolated nucleic acid molecule tkat kybridizes to a nucleic acid molecule comprising SEQ ID NO: 1 under conditions of sufficient stringency to produce a clear signal, an isolated nucleic acid molecule tkat kybridizes to a nucleic acid molecule tkat encodes tke amino acid sequence of SEQ ID NO: 2 under conditions of sufficient stringency to produce a clear signal, and an isolated nucleic acid molecule tkat encodes a protein tkat exkibits at least about 35% amino acid sequence identity to SEQ ID NO: 2.
Tke present invention furtker includes tke nucleic acid molecules operably linked to one or more expression control elements, including vectors comprising tke isolated nucleic acid molecules. Tke invention furtker includes kost cells transformed to contain tke nucleic acid molecules of tke invention and metkods for producing a protein comprising tke step of culturing a kost cell transformed witk a nucleic acid molecule of tke invention under conditions in wkick tke protein is expressed. Tke invention furtker provides an isolated polypeptide selected from tke group consisting of an isolated polypeptide comprising tke amino acid sequence of SEQ ID NO: 2, an isolated polypeptide comprising a functional or antigenic fragment of at least 6 amino acids of SEQ ID NO: 2, an isolated polypeptide comprising conservative amino acid substitutions of SEQ ID NO: 2, an isolated polypeptide comprising naturally occurring amino acid sequence variants of SEQ ID NO: 2, and an isolated polypeptide tkat exkibits at least about 35% amino acid sequence identity to SEQ ID NO: 2.
Tke invention furtker provides an isolated antibody, including a monoclonal or polyclonal antibody or antigen-binding antibody fragment, tkat specifically binds to a polypeptide of tke invention. Tbe invention furtker provides metkods of identifying an agent wkick modulates tke expression of a nucleic acid encoding tke protein kaving tke sequence of SEQ ID NO: 2 or tke related proteins described berein, comprising: exposing cells wkick express tke nucleic acid to tke agent; and determining wketker tke agent modulates expression of said nucleic acid, tkereby identifying an agent wkick modulates tke expression of a nucleic acid encoding tke protein.
Tke invention furtker provides metkods of identifying an agent wkick modulates tke level of or at least one activity of a protein comprising tke sequence of SEQ ID NO: 2 or tke related proteins described kerein, comprising: exposing cells wkick express tke protein to tke agent; and determining wketker tke agent modulates tke level of or at least one activity of said protein, tkereby identifying an agent wkick modulates tke level of or at least one activity of tke protein.
Tke invention furtker provides metkods of identifying binding partners for a protein comprising tke sequence of SEQ ID NO: 2 or a related protein described kerein, comprising: exposing said protein to a potential binding partner; and determining if tke potential binding partner binds to said protein, tkereby identifying binding partners for tke protein.
Tke present invention furtker provides metkods of modulating tke expression of a nucleic acid encoding tke protein kaving tke sequence of SEQ ID NO: 2 or tke related proteins described kerein, comprising: administering an effective amount of an agent wkick modulates tke expression of a nucleic acid encoding tke protein. Tke invention also provides metkods of modulating at least one activity of a protein comprising tke sequence of SEQ ID NO: 2 or tke related proteins described kerein, comprising: administering an effective amount of an agent wkick modulates at least one activity of tke protein.
Tke present invention furtker includes non-kuman transgenic animals modified to contain tke nucleic acid molecules of tke invention or mutated nucleic acid molecules suck tkat expression of tke encoded polypeptides of tke invention is prevented.
Tke invention furtker provides metkods of diagnosing renal or otker disease states, comprising: acquiring a tissue or blood or other sample from a subject; and determining tke level of expression of nucleic acid molecules of tke invention or polypeptides of tke invention. BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 Figure 1 skows tke expression levels of mRNA encoding tke protein of
SEQ ID NO: 2 as determined by quantitative PCR analysis in diseased and control kidney biopsy tissues, relative to tbe expression of GAPDH. "Min. Ck." refers to minimum ckange; "IgAN" refers to IgA nepkropatky; "NCGN" refers to necrotizing crescentic glomerular nepkropatky.
Figure 2 Figure 2 skows tke tissue distribution of RNA encoding tke protein of
SEQ ID NO: 2 as analyzed by Nortkern blot in kuman keart (lane 1), brain (2), placenta (3), lung (4), liver (5), muscle (6), kidney (7) and pancreas (lane 8). M=RNA marker.
Figure 3 Figure 3 skows tke expression levels of mRNA encoding tke protein of
SEQ ID NO: 2 as determined by quantitative PCR analysis in various tissues relative to tbe expression of GAPDH.
Fi ure 4 Figure 4 skows tke expression levels of mRNA encoding tke protein of SEQ ID NO: 2 in kuman peripkeral blood leukocytes (PBL) as determined by quantitative
PCR, relative to tke expression of GAPDH. PBL samples were obtained from normal kuman controls or patients witk IgAN or witk anti-neutropkil cytoplasmic autoantibody (ANCA) - associated renal disease.
Figure 5 Figure 5 skows tke expression levels of mRNA encoding tke protein of SEQ ID NO: 2 in kuman umbilical vein endotkelial cells (HUVEC) and kuman umbilical artery endotkelial cells (HUAEC) as determined by quantitative PCR, relative to tke expression of GAPDH. Cells were treated witk TNFα at time 0 in a time course study.
Figure 6 Figure 6 skows tke expression levels of mRNA encoding tke protein of
SEQ ID NO: 2 in tke endotkelial cell line EA (an immortalized kybrid between primary kuman endotkelial cells and tke tumor cell line A549) as determined by quantitative PCR relative to tbe expression of GAPDH. Growtk arrest was induced by two separate metkods: 1) culture in serum free conditions for various lengtks of time; 2) kigk density contact inhibition.
Figure 7 Figure 7 shows the expression levels of mRNA encoding the protein of
SEQ ID NO: 2 as determined by quantitative PCR relative to the expression of GAPDH. Samples are primary kuman renal epitkelial cells under log, semi-kigk or high density culture conditions leading to growth arrest, and primary human renal mesangial cells under similar conditions of growth arrest as well as cytokine treatment (IL-1 and IL-6).
Figure 8 Figure 8 skows plots predicting various structural features and tke antigenic and kydropkilic regions of tke amino acid sequence of SEQ ID NO: 2.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
I. General Description
Tke present invention is based in part on a new gene family tkat is differentially expressed in renal disease, inflammation, and in various stages of cell growtk, compared witk normal cells or tissues. Tke kuman gene encodes a protein of 275 amino acids.
Tke proteins of tke invention can serve as a diagnostic agent or marker or as a target for agents tkat modulate tke expression or activity of tke proteins. For example, agents may be identified tkat modulate biological processes associated witk renal disease, kidney transplantation and kidney regeneration.
Tke present invention is furtker based on tbe development of metkods for isolating binding partners that bind a protein of the invention. Additionally, tkese proteins provide novel targets for screening syntketic small molecules and combinatorial or naturally occurring compound libraries to discover novel tkerapeutics to regulate kidney function.
II. Specific Embodiments A. Protein Molecules
Tke present invention provides isolated proteins, allelic variants of tke proteins, and conservative amino acid substitutions of tke proteins. As used kerein, tke "protein" or "polypeptide" refers, in part, to a protein tkat kas tke kuman amino acid sequence depicted in SEQ ID NO: 2. Tke invention includes naturally occurring allelic variants and proteins tkat kave a slightly different amino acid sequence tkan tkat specifically recited above. Allelic variants, tkougk possessing a skgktly different amino acid sequence tkan tkose recited above, will still kave tke same or similar biological functions associated witk tke 275 amino acid protein.
As used kerein, tke family of proteins related to tke kuman amino acid sequence of SEQ ID NO: 2 refers, in part, to proteins tkat kave been isolated from organisms in addition to humans. The methods used to identify and isolate other members of the family of proteins related to the 275 amino acid protein are described below.
The proteins of the present invention are preferably in isolated form. As used herein, a protein is said to be isolated wben pkysical, meckanical or ckemical metkods are employed to remove tke protein from cellular constituents tkat are normally associated witk tke protein. A skilled artisan can readily employ standard purification metkods to obtain an isolated protein. Tbe proteins of tke present invention furtker include insertion, deletion or conservative amino acid substitution variants of SEQ ID NO: 2. As used kerein, a conservative variant refers to at least one alteration in tke amino acid sequence tkat does not adversely affect tke biological functions of the protein. A substitution, insertion or deletion is said to adversely affect the protein when the altered sequence prevents or disrupts a biological function associated witk tke protein. For example, tke overall ckarge, structure or kydropkobic/kydropkilic properties of tke protein can be altered witbout adversely affecting a biological activity. Accordingly, tke amino acid sequence can be altered, for example to render tbe peptide more kydropkobic or kydropkilic, witkout adversely affecting tke biological activities of tke protein. Ordinarily, tke allelic variants, tke conservative substitution variants, and tke members of tke protein family, will kave an amino acid sequence kaving at least about 35%, 40%, 50%, 60%, 65%, 70% or 75% amino acid sequence identity witk tke sequence set fortk in SEQ ID NO: 2, more preferably at least about 80%, even more preferably at least about 90%, and most preferably at least about 95% sequence identity. Identity or komology witk respect to suck sequences is defined kerein as tke percentage of amino acid residues in tke candidate sequence tkat are identical witk tke known peptides, after aligning tke sequences and introducing gaps, if necessary, to ackieve tke maximum percent komology, and not considering any conservative substitutions as part of the sequence identity (see section B for the relevant paramenters). Fusion proteins, or N-terminal, C-terminal or internal extensions, deletions, or insertions into the peptide sequence shall not be construed as affecting homology.
Contemplated variants further include those containing predetermined mutations by, e.g., homologous recombination, site-directed or PCR mutagenesis, and the corresponding proteins of other animal species, including but not limited to rabbit, mouse, rat, porcine, bovine, ovine, equine and non-human primate species, and the alleles or other naturally occurring variants of the family of proteins; and derivatives wherein the protein has been covalently modified by substitution, chemical, enzymatic, or other appropriate means witk a moiety otker tkan a naturally occurring amino acid (for example a detectable moiety such as an enzyme or radioisotope). The proteins of the present invention also include molecules having a portion of tke amino acid sequence disclosed in SEQ ID NO: 2 suck as fragments kaving a consecutive sequence of at least about 3, 4, 5, 6, 10, 15, 20, 25, 30, 35 or more amino acid residues of the protein. Such fragments, also referred to as peptides or polypeptides, may contain antigenic regions, functional regions of the protein identified as regions of the amino acid sequence which correspond to known protein domains, as well as regions of pronounced hydrophilicity. Tke regions are all easily identifiable by using commonly available protein sequence analysis software such as Mac Vector™ (Oxford Molecular). See also Figure 8.
As described below, members of the family of proteins can be used: 1) to identify agents which modulate at least one activity of the protein; 2) to identify binding partners for the protein, 3) as an antigen to raise polyclonal or monoclonal antibodies, 4) as a therapeutic agent or target, and 5) as a diagnostic agent or marker.
B. Nucleic Acid Molecules
The present invention further provides nucleic acid molecules that encode the protein having SEQ ID NO: 2 and the related proteins herein described, preferably in isolated form. As used herein, "nucleic acid" is defined as RNA or DNA that encodes a protein or peptide as defined above, is complementary to a nucleic acid sequence encoding such peptides, hybridizes to the complement of a nucleic acid that encodes the amino acid sequence of SEQ ID NO: 2 and remains stably bound to it under appropriate stringency conditions, encodes a polypeptide skaring at least about 35%, 40%, 50%, 60%, 65%, 70% or 75%, preferably at least about 80%, more preferably at least about 90%, and most preferably at least about 95% or more amino acid sequence identity with the peptide sequences, or exhibits at least about 35%, 40%, 50%, 60%, 65%, 70% or 75%, preferably at least about 80%, more preferably at least about 90%, and most preferably at least about 95% or more nucleotide sequence identity over the open reading frame of SEQ ID NO: 1.
Specifically contemplated are genomic DNA, cDNA, mRNA and antisense molecules, as well as nucleic acids based on alternative backbones or including alternative bases whether derived from natural sources or synthesized. Such hybridizing or complementary nucleic acids, however, are defined further as being novel and non-obvious over any prior art nucleic acid including that which encodes, hybridizes under appropriate stringency conditions, or is complementary to nucleic acid encoding a protein according to the present invention.
Homology or identity at tke nucleotide or amino acid sequence level is determined by BLAST (Basic Local Alignment Searck Tool) analysis using tke algoritkm employed by tke programs blastp, blastn, blastx, tblastn and tblastx (Altsckul, SF et al. (1997) Nucleic Acid Res, 25:3389-3402, and Karlin et al. (1990) PNAS 87:2264-2268, fully incorporated by reference) which are tailored for sequence similarity searching. The approach used by the BLAST program is to first consider similar segments, with and without gaps, between a query sequence and a database sequence, then to evaluate the statistical significance of all matches that are identified and finally to summarize only those matckes wkick satisfy a preselected tkreskold of significance. For a discussion of basic issues in similarity searcking of sequence databases, see Altsckul et al. (Nature Genetics, Vol. 6 (1994) pp. 119-129) wkick is fully incorporated by reference. The search parameters for histogram, descriptions, alignments, expect (i.e., the statistical significance threshold for reporting matches against database sequences), cutoff, matrix and filter (low complexity) are at the default settings. The default scoring matrix used by blastp, blastx, tblastn, and tblastx is the BLOSUM62 matrix (recommended for query sequences over 85 nucleotide bases or amino acids in length (Henikoff et al. Proceedings of the National Academy of Science USA, Vol. 89 (1992) pp. 10915-10919, fully incorporated by reference). For blastn, tke scoring matrix is set by tke ratios of M (i.e., tke reward score for a pair of matcking residues) to N (i.e., tke penalty score for mismatcking residues), wkerein tke default values for M and N are 5 and -4, respectively. Four blastn parameters were adjusted as follows: Q=10 (gap creation penalty); R=10 (gap extension penalty); wink=l (generates word bits at every winkth position along tke query); and gapw=16 (sets tke window widtk witkin wkick gapped alignments are generated). Tke equivalent Blastp parameter settings were Q=9; R=2; wink=l; and gapw=32. A Bestfit comparison between sequences, available in tke GCG package version 10.0, uses DNA parameters GAP=50 (gap creation penalty) and LEN=3 (gap extension penalty) and tke equivalent settings in protein comparisons are GAP=8 and LEN=2. "Stringent conditions" are tkose tkat (1) employ low ionic strengtk and kigk temperature for wasking, for example, 0.015 M NaCl/0.0015 M sodium citrate/0.1% SDS at 50°C, or (2) employ during kybridization a denaturing agent suck as formamide, for example, 50% (vol/vol) formamide witk 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium pkospkate buffer at pH 6.5 witk 750 mM NaCl, 75 mM sodium citrate at 42°C. Anotker example is kybridization in 50% formamide, 5x SSC (0.75M NaCl, 0.075 M sodium citrate), 50 mM sodium pkospkate (pH 6.8), 0.1% sodium pyropkospkate, 5x Denkardt's solution, sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS, and 10% dextran sulfate at 42°C, with washes at 42°C. in 0.2x SSC and 0.1% SDS. A skilled artisan can readily determine and vary the stringency conditions appropriately to obtain a clear and detectable hybridization signal. Preferred molecules are those that hybridize under the above conditions to the complement of SEQ ID NO: 1 and which encode a functional protein. Even more preferred hybridizing molecules are those that hybridize under the above conditions to tbe complement strand of tke open reading frame of SEQ ID NO:l.
As used kerein, a nucleic acid molecule is said to be "isolated" wken tke nucleic acid molecule is substantially separated from contaminant nucleic acid molecules encoding other polypeptides.
The present invention further provides fragments of the encoding nucleic acid molecule. As used herein, a fragment of an encoding nucleic acid molecule refers to a small portion of the entire protein coding sequence. Tke size of tke fragment will be determined by tke intended use. For example, if tke fragment is ckosen so as to encode an active portion of tke protein, tke fragment will need to be large enougk to encode tke functional region(s) of tke protein. For instance, fragments which encode peptides corresponding to predicted antigenic regions may be prepared (see Figure 8). If the fragment is to be used as a nucleic acid probe or PCR primer, tken tke fragment length is chosen so as to obtain a relatively small number of false positives during probing/priming (see tke discussion in section H). Fragments of tke encoding nucleic acid molecules of tke present invention (i.e., syntketic oligonucleotides) tkat are used as probes or specific primers for the polymerase chain reaction (PCR), or to synthesize gene sequences encoding proteins of the invention can easily be synthesized by chemical techniques, for example, the phospkotriester metkod ofMatteucci et al. (Journal of the American Chemical Society, Vol. 103 (1981) pp. 3185- 3191) or using automated syntkesis metkods. In addition, larger DNA segments can readily be prepared by well known metkods, suck as syntkesis of a group of oligonucleotides that define various modular segments of the gene, followed by ligation of oligonucleotides to build tke complete modified gene.
Tke encoding nucleic acid molecules of tke present invention may furtker be modified so as to contain a detectable label for diagnostic and probe purposes. A variety of suck labels are known in the art and can readily be employed with the encoding molecules herein described. Suitable labels include, but are not limited to, biotin, radiolabeled nucleotides and the like. A skilled artisan can readily employ any such label to obtain labeled variants of the nucleic acid molecules of the invention.
Modifications to the primary structure itself by deletion, addition, or alteration of tke amino acids incorporated into tke protein sequence during translation can be made witkout destroying tke activity of tke protein. Suck substitutions or otker alterations result in proteins kaving an amino acid sequence encoded by a nucleic acid falling within the contemplated scope of the present invention.
C. Isolation of Other Related Nucleic Acid Molecules
As described above, the identification and characterization of tke kuman nucleic acid molecule having SEQ ID NO: 1 allows a skilled artisan to isolate nucleic acid molecules tkat encode otker members of tke protein family in addition to tke sequences kerein described.
Briefly, a skilled artisan can readily use tke amino acid sequence of SEQ ID NO: 2 to generate antibody probes to screen expression libraries prepared from appropriate cells. Typically, polyclonal antiserum from mammals such as rabbits immunized with the purified protein (as described below) or monoclonal antibodies can be used to probe a mammakan cDNA or genomic expression kbrary, such as lambda gtll library, to obtain the appropriate coding sequence for other members of the protein family. The cloned cDNA sequence can be expressed as a fusion protein, expressed directly using its own control sequences, or expressed by constructions using control sequences appropriate to the particular host used for expression of tke enzyme.
Alternatively, a portion of tke coding sequence kerein described can be synthesized and used as a probe to retrieve DNA encoding a member of the protein family from any mammalian organism. Oligomers containing approximately 18-20 nucleotides (encoding about a 6-7 amino acid stretch) are prepared and used to screen genomic DNA or cDNA libraries to obtain hybridization under stringent conditions or conditions of sufficient stringency to eliminate an undue level of false positives.
Additionally, pairs of oUgonucleotide primers can be prepared for use in a polymerase chain reaction (PCR) to selectively clone an encoding nucleic acid molecule. A PCR denature- anneal-extend cycle for using such PCR primers is well known in tke art and can readily be adapted for use in isolating otker encoding nucleic acid molecules.
Nucleic acid molecules encoding otker members of tbe protein family may also be identified in existing genomic or otker sequence information using any available computational metkod, including but not limited to: PSI-BLAST (Ascbul SF et al. (1997) Nucliec Acids Res. 26:3389-3402); PHI-BLAST (Zkang, et al. (1998) Nucliec Acids Res. 26:3986-3990); 3D-PSSM (Kelly et al. (2000) J. Mol. Biol. 299:499-520); and otker computational analysis metkods (Shi et al. (1999) Biochem. Biophys. Res. Commun. 262:132-138 and Matsunami et al. (2000) Nature 404:601-604). D. rDNA molecules Containing a Nucleic Acid Molecule
Tke present invention furtker provides recombinant DNA molecules (rDNAs) tkat contain a coding sequence. As used kerein, a rDNA molecule is a DNA molecule tkat kas been subjected to molecular manipulation in situ. Methods for generating rDNA molecules are well known in the art, for example, see Sambrook et al. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, NY. Cold Spring Harbor Laboratory Press, 1989. In tke preferred rDNA molecules, a coding DNA sequence is operably linked to expression control sequences and/or vector sequences.
Tke ckoice of vector and/or expression control sequences to wkick one of tke protein family encoding sequences of tke present invention is operably linked depends directly, as is well known in tke art, on tke functional properties desired, e.g., protein expression, and tke kost cell to be transformed. A vector contemplated by tke present invention is at least capable of directing the replication or insertion into tke kost chromosome, and preferably also expression, of tke structural gene included in tke rDNA molecule.
Expression control elements tkat are used for regulating tke expression of an operably linked protein encoding sequence are known in the art and include, but are not limited to, inducible promoters, constitutive promoters, secretion signals, and other regulatory elements. Preferably, the inducible promoter is readily controlled, such as being responsive to a nutrient in the host cell's medium.
In one embodiment, the vector containing a coding nucleic acid molecule will include a prokaryotic repkcon, i.e., a DNA sequence having tke ability to direct autonomous replication and maintenance of the recombinant DNA molecule extra-chromosomally in a prokaryotic host cell, such as a bacterial host cell, transformed therewith. Such replicons are well known in the art. In addition, vectors tkat include a prokaryotic replicon may also include a gene wkose expression confers a detectable marker such as a drug resistance. Typical bacterial drug resistance genes are tkose tkat confer resistance to ampicillin or tetracycline.
Vectors tkat include a prokaryotic replicon can furtker include a prokaryotic or bacteriopbage promoter capable of directing the expression (transcription and translation) of tke coding gene sequences in a bacterial kost cell, suck as E. coli. A promoter is an expression control element formed by a DNA sequence tkat permits binding of RNA polymerase and transcription to occur. Promoter sequences compatible witk bacterial hosts are typically provided in plasmid vectors containing convenient restriction sites for insertion of a DNA segment of tke present invention. Typical of suck vector plasmids are pUC8, pUC9, pBR322 and pBR329 available from BioRad Laboratories, (Richmond, CA), pPL and pKK223 available from Pharmacia (Piscataway, NJ).
Expression vectors compatible with eukaryotic cells, including viral vectors, preferably those compatible with vertebrate cells such as kidney cells, can also be used to form rDNA molecules that contain a coding sequence. Eukaryotic cell expression vectors are well known in the art and are available from several commercial sources. Typically, such vectors are provided containing convenient restriction sites for insertion of tke desired DNA segment. Typical of suck vectors are pSVL and pKSV-10 (Pkarmacia), pBPV-l/pML2d (International Biotechnologies, Inc.), pTDTl (ATCC, #31255), the vector pCDM8 described herein, and the like eukaryotic expression vectors. Vectors may be modified to include kidney cell specific promoters if needed.
Eukaryotic cell expression vectors used to construct the rDNA molecules of tke present invention may furtker include a selectable marker that is effective in an eukaryotic cell, preferably a drug resistance selection marker. A preferred drug resistance marker is the gene whose expression results in neomycin resistance, i.e., the neomycin phosphotransferase (neo) gene. (Southern et al. Journal of Molecular and Applied Genetics, Vol. 1, no. 4 (1982) pp. 327-341) Alternatively, the selectable marker can be present on a separate plasmid, and the two vectors are introduced by co- transfection of tke kost cell, and selected by culturing in tke appropriate.drug for tke selectable marker.
E. Host Cells Containing an Exogenously Supplied Coding Nucleic Acid Molecule
The present invention furtker provides kost cells transformed witk a nucleic acid molecule tkat encodes a protein of tke present invention. Tke kost cell can be either prokaryotic or eukaryotic. Eukaryotic cells useful for expression of a protein of tke invention are not limited, so long as tke cell kne is compatible witk cell culture metkods and compatible witk tke propagation of tke expression vector and expression of tke gene product. Preferred eukaryotic kost cells include, but are not limited to, yeast, insect and mammalian cells, preferably vertebrate cells such as those from a mouse, rat, monkey or human cell line. Preferred eukaryotic host cells include Chinese hamster ovary (CHO) cells available from the ATCC as CCL61, NIH Swiss mouse embryo cells (NIH/3T3) available from the ATCC as CRL 1658, baby hamster kidney cells (BHK), and the like eukaryotic tissue culture cell lines. Any prokaryotic host can be used to express a rDNA molecule encoding a protein of the invention. The preferred prokaryotic host is E. coli.
Transformation of appropriate cell hosts with a rDNA molecule of the present invention is accomplished by well known methods tkat typically depend on tke type of vector used and kost system employed. Witk regard to transformation of prokaryotic kost cells, electroporation and salt treatment metkods are typically employed, see, for example, Coken et al. Proceedings of the National Academy of Science USA, Vol. 69, no. 8 (1972) pp. 2110-2114; and Maniatis et al. Molecular Cloning: A Laboratory Mammal. Cold Spring Harbor, NY. Cold Spring Harbor Laboratory Press, 1982). Witk regard to transformation of vertebrate cells witk vectors containing rDNAs, electroporation, cationic lipid or salt treatment metkods are typically employed, see, for example, Graham et al. Virology, Vol. 52, no. 2 (1973) pp. 456-467; and Wigler et al. Proceedings of the National Academy of Science USA, Vol. 76 (1979) pp. 1373-1376.
Successfully transformed cells, i.e., cells that contain a rDNA molecule of tke present invention, can be identified by well known techniques including the selection for a selectable marker. For example, cells resulting from the introduction of an rDNA of the present invention can be cloned to produce single colonies. Cells from those colonies can be harvested, lysed and their DNA content examined for the presence of the rDNA using a method such as that described by Southern, Journal of Molecular Biology, Vol. 98, no. 3 (1975) pp. 503-517; or Berent et al. Biotechnic and Histochemistry, Vol. 3 (1985) pp. 208; or the proteins produced from the cell assayed via an immunological method.
F. Production of Recombinant Proteins using a rDNA Molecule
The present invention further provides methods for producing a protein of the invention using nucleic acid molecules herein described. In general terms, the production of a recombinant form of a protein typically involves the following steps: First, a nucleic acid molecule is obtained tkat encodes a protein of tke invention, suck as tke nucleic acid molecule comprising, consisting essentially of, or consisting of SEQ ID NO: 1, or nucleotides 121-948 of SEQ ID NO: 1, or nucleotides 121-945 of SEQ ID NO: 1. If the encoding sequence is uninterrupted by introns, as is this open reading frame, it is directly suitable for expression in any host.
Tke nucleic acid molecule is tken preferably placed in operable linkage with suitable control sequences, as described above, to form an expression unit containing tke protein open reading frame. Tke expression unit is used to transform a suitable kost and tke transformed kost is cultured under conditions tkat allow tke production of tke recombinant protein. Optionally tke recombinant protein is isolated from tke medium or from tke cells; recovery and purification of tke protein may not be necessary in some instances wkere some impurities may be tolerated.
Eack of tke foregoing steps can be done in a variety of ways. For example, the desired coding sequences may be obtained from genomic fragments and used directly in appropriate hosts. The construction of expression vectors tkat are operable in a variety of kosts is accomplisked using appropriate replicons and control sequences, as set forth above. Tke control sequences, expression vectors, and transformation metkods are dependent on tke type of kost cell used to express tke gene and were discussed in detail earker. Suitable restriction sites can, if not normally available, be added to tke ends of tke coding sequence so as to provide an excisable gene to insert into these vectors. A skilled artisan can readily adapt any host/expression system known in tke art for use witk tke nucleic acid molecules of tke invention to produce recombinant protein.
G. Methods to Identify Binding Partners
Another embodiment of the present invention provides methods for use in isolating and identifying binding partners of proteins of the invention In detail, a protein of the invention is mixed with a potential binding partner or an extract or fraction of a cell under conditions that allow the association of potential binding partners with the protein of the invention. After mixing, peptides, polypeptides, proteins or other molecules that have become associated with a protein of the invention are separated from the mixture. The binding partner that bound to the protein of the invention can then be removed and further analyzed. To identify and isolate a binding partner, tbe entire protein, for instance a protein comprising tke entire amino acid sequence of SEQ ID NO: 2 can be used. Alternatively, a fragment of tke protein can be used. As used kerein, a cellular extract refers to a preparation or fraction wkick is made from a lysed or disrupted cell. Tke preferred source of cellular extracts will be cells derived from kuman kidney tissue or cells, for instance, renal biopsy tissue or a tissue culture cell model of renal disease. Alternatively, cellular extracts may be prepared from normal kuman kidney tissue or available cell lines, particularly kidney derived cell lines.
A variety of metkods can be used to obtain an extract of a cell. Cells can be disrupted using eitker pkysical or ckemical disruption metkods. Examples of pkysical disruption metkods include, but are not limited to, sonication and meckanical skearing. Examples of ckemical lysis metkods include, but are not limited to, detergent lysis and enzyme lysis. A skilled artisan can readily adapt metkods for preparing cellular extracts in order to obtain extracts for use in tke present metkods.
Once an extract of a cell is prepared, tke extract is mixed witk tke protein of tke invention under conditions in wkick association of tke protein witk tke binding partner can occur. A variety of conditions can be used, the most preferred being conditions that closely resemble conditions found in the cytoplasm of a human cell. Features such as osmolarity, pH, temperature, and the concentration of cellular extract used, can be varied to optimize the association of the protein with the binding partner. After mixing under appropriate conditions, the bound complex is separated from the mixture. A variety of techniques can be utilized to separate the mixture. For example, antibodies specific to a protein of the invention can be used to immunoprecipitate the binding partner complex. Alternatively, standard chemical separation techniques such as chromatography and density/sediment centrifugation can be used. After removal of non-associated cellular constituents found in the extract, the binding partner can be dissociated from the complex using conventional methods. For example, dissociation can be accomplished by altering the salt concentration or pH of the mixture.
To aid in separating associated binding partner pairs from the mixed extract, the protein of the invention can be immobilized on a solid support. For example, the protein can be attached to a nitrocellulose matrix or acrylic beads. Attachment of the protein to a solid support aids in separating peptide binding partner pairs from other constituents found in the extract. The identified binding partners can be either a single protein or a complex made up of two or more proteins. Alternatively, binding partners may be identified using a Far- Western assay according to the procedures of Takayama et al. Methods in Molecular Biology, Vol. 69 (1997) pp. 171-184 or identified through the use of epitope tagged proteins or GST fusion proteins.
Alternatively, the nucleic acid molecules of the invention can be used in a yeast two- hybrid system. The yeast two-hybrid system has been used to identify other protein partner pairs and can readily be adapted to employ the nucleic acid molecules herein described.
H. Methods to Identify Agents that Modulate the Expression of the Nucleic Acids.
Another embodiment of the present invention provides methods for identifying agents that modulate the expression of a nucleic acid encoding a protein of the invention such as a protein having the amino acid sequence of SEQ ID NO: 2. Such assays may utilize any available means of monitoring for changes in the expression level of the nucleic acids of the invention. As used herein, an agent is said to modulate the expression of a nucleic acid of the invention, for instance a nucleic acid encoding the protein having the sequence of SEQ ID NO: 2, if it is capable of up- or down-regulating expression of the nucleic acid in a cell compared to a control.
In one assay format, cell lines that contain reporter gene fusions between the open reading frame defined by nucleotides 121-948 or nucleotide 121-945 of SEQ ID NO: 1, and/or the 5' and/or the 3' regulatory elements, and any assayable fusion partner may be prepared. Numerous assayable fusion partners are known and readily available including the firefly luciferase gene and the gene encoding chloramphenicol acetyltransferase (Alam et al.
Analytical Biochemistry, Vol. 188 (1990) pp. 245-254). Cell lines containing the reporter gene fusions are then exposed to the agent to be tested under appropriate conditions and time. Differential expression of the reporter gene between samples exposed to the agent and control samples identifies agents which modulate the expression of a nucleic acid of the invention. Additional assay formats may be used to monitor the ability of tke agent to modulate tke expression of a nucleic acid encoding a protein of tke invention suck as tke protein kaving SEQ ID NO: 2. For instance, mRNA expression may be monitored directly by kybridization to tke nucleic acids of tke invention. Cell lines are exposed to tke agent to be tested under appropriate conditions and time and total RNA or mRNA is isolated by standard procedures such those disclosed in Sambrook et al. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, NY. Cold Spring Harbor Laboratory Press, 1989.
Probes to detect differences in RNA expression levels between cells exposed to the agent and control cells may be prepared from the nucleic acids of the invention. It is preferable, but not necessary, to design probes which hybridize only with target nucleic acids under conditions of high stringency. Only highly complementary nucleic acid hybrids form under conditions of high stringency. Accordingly, the stringency of the assay conditions determines the amount of complementarity which should exist between two nucleic acid strands in order to form a hybrid. Stringency should be chosen to maximize the difference in stability between tke probe:target kybrid and potential probe:non-target kybrids.
Probes may be designed from tke nucleic acids of tke invention through methods known in the art. For instance, the G+C content of the probe and the probe length can affect probe binding to its target sequence. Methods to optimize probe specificity are commonly available in Sambrook et al. (Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, NY. Cold Spring Harbor Laboratory Press, 1989); or Ausubel et al. (Current Protocols in Molecular Biology. NY, Greene Publishing Company, 1995).
Hybridization conditions are modified using known methods, such as those described by Sambrook et al. and Ausubel et al. as required for each probe. Hybridization of total cellular RNA or RNA enriched for polyA RNA can be accomplished in any available format. For instance, total cellular RNA or RNA enriched for polyA RNA can be affixed to a solid support and the solid support exposed to at least one probe comprising at least one, or part of one of the sequences of the invention under conditions in which the probe will specifically hybridize. Alternatively, nucleic acid fragments comprising at least one, or part of one of the sequences of the invention can be affixed to a solid support, such as a silicon chip or a porous glass wafer. The solid support can then be exposed to total cellular RNA or polyA RNA from a sample under conditions in which the affixed sequences will specifically hybridize. Such solid supports and hybridization methods are widely available, for example, those disclosed by Beattie (1995) (WO 95/11755). By examining for the ability of a given probe to specifically hybridize to an RNA sample from an untreated cell population and from a cell population exposed to the agent, agents which up or down regulate the expression of a nucleic acid encoding the protein having the sequence of SEQ ID NO: 2 are identified.
Hybridization for qualitative and quantitative analysis of mRNAs may also be carried out by using a RNase Protection Assay (i.e., RPA, see Ma et al. Methods, Vol. 10, no. 3 (1996) pp. 273-238). Briefly, an expression vehicle comprising cDNA encoding the gene product and a phage specific DNA dependent RNA polymerase promoter (e.g., T7, T3 or SP6 RNA polymerase) is linearized at the 3' end of the cDNA molecule, downstream from the phage promoter, wherein such a linearized molecule is subsequently used as a template for synthesis of a labeled antisense transcript of the cDNA by in vitro transcription. The labeled transcript is then hybridized to a mixture of isolated RNA (i.e., total or fractionated mRNA) by incubation at 45 °C overnight in a buffer comprising 80% formamide, 40 mM Pipes, pH 6.4, 0.4 M NaCl and 1 mM EDTA. The resulting hybrids are then digested in a buffer comprising 40 μg/ml ribonuclease A and 2 μg/ml ribonuclease. After deactivation and extraction of extraneous proteins, the samples are loaded onto urea/polyacrylamide gels for analysis. In another assay format, cells or cell lines are first identified that express the gene products of the invention physiologically (e.g., see for example, Figure 1 for tissue distribution via Northern blot, however, RPAs may serve the identical purpose of expression selection). Cell and/or cell lines so identified would be expected to comprise the necessary cellular machinery such that the fidelity of modulation of the transcriptional apparatus is maintained with regard to exogenous contact of agent with appropriate surface transduction mechanisms and/or the cytosolic cascades. Further, such cells or cell lines would be transduced or transfected with an expression vehicle (e.g., a plasmid or viral vector) construct comprising an operable non-translated 5 '-promoter containing end of the structural gene encoding the instant gene products fused to one or more antigenic fragments, which are peculiar to tke instant gene products, wherein said fragments are under the transcriptional control of said promoter and are expressed as polypeptides whose molecular weight can be distinguished from the naturally occurring polypeptides or may further comprise an immunologically distinct tag or other detectable marker. Such a process is well known in the art (see Sambrook et al. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, NY. Cold Spring Harbor Laboratory Press, 1989).
Cells or cell lines transduced or transfected as outlined above are then contacted with agents under appropriate conditions; for example, the agent in a pharmaceutically acceptable excipient is contacted with cells in an aqueous physiological buffer such as phosphate buffered saline (PBS) at physiological pH, Eagles balanced salt solution (BSS) at physiological pH, PBS or BSS comprising serum or conditioned media comprising PBS or BSS and/or serum incubated at 37 °C . Said conditions may be modulated as deemed necessary by one of skill in the art. Subsequent to contacting the cells with the agent, said cells will be disrupted and the polypeptides of the lysate are fractionated such that a polypeptide fraction is pooled and contacted with an antibody to be further processed by immuno logical assay (e.g., ELISA, immunoprecipitation or Western blot). The pool of proteins isolated from the "agent-contacted" sample will be compared with a control sample where only the excipient is contacted with the cells and an increase or decrease in the immunologically generated signal from the "agent- contacted" sample compared to the control will be used to distinguish the effectiveness of the agent.
I. Methods to Identify Agents that Modulate the Level of or at Least One Activity of the Proteins.
Another embodiment of the present invention provides methods for identifying agents that modulate the level of or at least one activity of a protein of the invention such as the protein having the amino acid sequence of SEQ ID NO: 2. Such methods or assays may utilize any means of monitoring or detecting the desired activity.
In one format, the relative amounts of a protein of the invention between a cell population that has been exposed to the agent to be tested compared to an un-exposed control cell population may be assayed. In this format, probes such as specific antibodies are used to monitor the differential expression of the protein in the different cell populations. Cell lines or populations are exposed to the agent to be tested under appropriate conditions and time. Cellular lysates may be prepared from the exposed cell line or population and a control, unexposed cell line or population. The cellular lysates are then analyzed with the probe.
Antibody probes are prepared by immunizing suitable mammalian hosts in appropriate immunization protocols using the peptides, polypeptides or proteins of the invention if they are of sufficient length, or, if desired, or if required to enhance immunogenicity, conjugated to suitable carriers. Methods for preparing immunogenic conjugates with carriers such as BSA, KLH, or other carrier proteins are well known in the art. In some circumstances, direct conjugation using, for example, carbodiimide reagents may be effective; in other instances linking reagents such as those supplied by Pierce Chemical Co. (Rockford, IL), may be desirable to provide accessibility to the hapten. The hapten peptides can be extended at either the amino or carboxy terminus with a cysteine residue or interspersed with cysteine residues, for example, to facilitate linking to a carrier. Administration of the immunogens is conducted generally by injection over a suitable time period and with use of suitable adjuvants, as is generally understood in the art. During the immunization schedule, titers of antibodies are taken to determine adequacy of antibody formation.
Wkile tke polyclonal antisera produced in tkis way may be satisfactory for some applications, for pkarmaceutical compositions, use of monoclonal preparations is preferred.
Immortalized cell lines wkick secrete tke desired monoclonal antibodies may be prepared using the standard method of Kohler and Milstein (Nature, Vol. 256, no. 5517 (Aug. 1975) pp. 495-497) or modifications which effect immortalization of lymphocytes or spleen cells, as is generally known. The immortalized cell lines secreting tke desired antibodies are screened by immunoassay in which the antigen is the peptide hapten, polypeptide or protein. When the appropriate immortalized cell culture secreting the desired antibody is identified, the cells can be cultured either in vitro or by production in ascites fluid.
The desired monoclonal antibodies are then recovered from the culture supernatant or from the ascites supernatant. Fragments of the monoclonal antibodies or the polyclonal antisera which contain the immunologically significant portion can be used as antagonists, as well as the intact antibodies. Use of immunologically reactive antibody fragments, such as the Fab, Fab', of F(ab')2 fragments is often preferable, especially in a therapeutic context, as these fragments are generally less immunogenic than the whole immunoglobulm. The antibodies or fragments may also be produced, using current technology, by recombinant means. Antibody regions that bind specifically to the desired regions of the protein can also be produced in the context of chimeras with multiple species origin, such as humanized antibodies.
Agents that are assayed in the above method can be randomly selected or rationally selected or designed. As used herein, an agent is said to be randomly selected when the agent is chosen randomly without considering the specific sequences involved in the association of a protein of the invention alone or with its associated substrates, binding partners, etc. An example of randomly selected agents is the use a chemical library or a peptide combinatorial library, or a growth broth of an organism. As used herein, an agent is said to be rationally selected or designed when the agent is chosen on a nonrandom basis which takes into account the sequence of the target site and/or its conformation in connection with the agent's action. Agents can be rationally selected or rationally designed by using the peptide sequences that make up these sites. For example, a rationally selected peptide agent can be a peptide whose amino acid sequence is identical to or a derivative of any functional consensus site.
The agents of the present invention can be, as examples, peptides, small molecules, vitamin derivatives, as well as carbohydrates. Dominant negative proteins, DNAs encoding these proteins, antibodies to these proteins, peptide fragments of these proteins or mimics of these proteins may be introduced into cells to affect function. "Mimic" used herein refers to the modification of a region or several regions of a peptide molecule to provide a structure chemically different from the parent peptide but topographically and functionally similar to the parent peptide (see Grant GA. in: Meyers (ed.) Molecular Biology and Biotechnology (New York, VCH Publishers, 1995), pp. 659-664). A skilled artisan can readily recognize that there is no limit as to the structural nature of tke agents of tke present invention. Tke peptide agents of tke invention can be prepared using standard solid phase (or solution phase) peptide synthesis methods, as is known in the art. In addition, the DNA encoding these peptides may be synthesized using commercially available oligonucleotide synthesis instrumentation and produced recombinantly using standard recombinant production systems. The production using solid phase peptide synthesis is necessitated if non-gene- encoded amino acids are to be included.
Another class of agents of the present invention are antibodies immunoreactive with critical positions of proteins of the invention. Antibody agents are obtained by immunization of suitable mammalian subjects with peptides, containing as antigenic regions, those portions of the protein intended to be targeted by the antibodies.
J. Uses for Agents that Modulate at Least One Activity of the Proteins.
As provided in the Examples, the proteins and nucleic acids of the invention, such as the protein having tke amino acid sequence of SEQ ID NO: 2, are expressed in various types of diseased renal tissue, as well as in otker organs, tissues and cells, for example during stages of inflammation associated witk infliltrating leukocytes or during cell growtk arrest. Renal disease and renal disorder refer to any disease of tke kidney, including but not limited to IgA nephropathy (IgAN), necrotizing crescentic glomerulonephritis (NCGN), and minimal change disease. Agents that up- or down-regulate or modulate the expression of the protein or at least one activity of the protein, such as agonists or antagonists, may be used to up- or down- regulate or modulate biological and pathologic processes associated with the protein's function and activity.
As used herein, a subject can be any mammal, so long as the mammal is in need of modulation of a pathological or biological process mediated by a protein of tke invention. Tke term "mammal" is defined as an individual belonging to the class Mammalia. The invention is particularly useful in the treatment of human subjects.
Pathological processes refer to a category of biological processes which produce a deleterious effect. For example, expression of a protein of the invention may be associated with kidney cell growth regeneration and/or recovery from kidney disease. As used herein, an agent is said to modulate a pathological process when the agent reduces the degree or severity of the process. For instance, kidney damage or ESRD may be prevented or disease progression modulated by the administration of agents that up- or down-regulate or modulate in some way the expression or at least one activity of a protein of the invention. The agents of the present invention can be provided alone, or in combination with other agents that modulate a particular pathological process. For example, an agent of the present invention can be administered in combination with other known drugs or may be combined with dialysis or anti-rejection drugs used during transplantation. As used herein, two agents are said to be administered in combination when the two agents are administered simultaneously or are administered independently in a fashion such that the agents will act at the same time.
The agents of the present invention can be administered via parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal, or buccal routes. Alternatively, or concurrently, administration may be by the oral route. The dosage administered will be dependent upon the age, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired.
The present invention further provides compositions containing one or more agents which modulate expression or at least one activity of a protein of the invention. While individual needs vary, determination of optimal ranges of effective amounts of each component is within tke skill of tke art. Typical dosages comprise 0.1 to 100 μg/kg body wt. The preferred dosages comprise 0.1 to 10 μg/kg body weight. The most preferred dosages comprise 0.1 to 1 μg/kg body weight.
In addition to the pharmacologically active agent, the compositions of the present invention may contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically for delivery to the site of action. Suitable formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form, for example, water-soluble salts. In addition, suspensions of the active compounds as appropriate oily injection suspensions may be administered. Suitable lipophilic solvents or vehicles include fatty oils, for example, sesame oil, or syntketic fatty acid esters, for example, etkyl oleate or triglycerides. Aqueous injection suspensions may contain substances wkick increase tke viscosity of tke suspension include, for example, sodium carboxymetbyl cellulose, sorbitol, and/or dextran. Optionally, the suspension may also contain stabilizers. Liposomes can also be used to encapsulate the agent for delivery into tke cell.
Tke pkarmaceutical formulation for systemic administration according to tke invention may be formulated for enteral, parenteral or topical administration. Indeed, all three types of formulations may be used simultaneously to achieve systemic administration of the active ingredient. Suitable formulations for oral administration include hard or soft gelatin capsules, pills, tablets, including coated tablets, elixirs, suspensions, syrups or inkalations and controlled release forms tkereof.
In practicing tke metkods of tkis invention, tke compounds of tkis invention may be used alone or in combination, or in combination with other therapeutic or diagnostic agents. In certain preferred embodiments, the compounds of this invention may be coadministered along with other compounds typically prescribed for these conditions according to generally accepted medical practice. The compounds of this invention can be utilized in vivo, ordinarily in mammals, such as humans, sheep, horses, cattle, pigs, dogs, cats, rats and mice, or in vitro.
K. Transgenic Animals
Transgenic animals containing mutant, knock-out or modified genes corresponding to the cDNA sequence of SEQ ID NO: 1, or the open reading frame encoding the polypeptide sequence of SEQ ID NO: 2, or fragments tkerof kaving a consecutive sequence of at least about 3, 4, 5, 6, 10, 15, 20, 25, 30, 35 or more amno acid residues, are also included in the invention. Transgenic animals are genetically modified animals into whick recombinant, exogenous or cloned genetic material has been experimentally transferred. Such genetic material is often referred to as a "transgene". The nucleic acid sequence of the transgene, in this case a form of SEQ ID NO: 1 may be integrated either at a locus of a genome where that particular nucleic acid sequence is not otherwise normally found or at the normal locus for the transgene. The transgene may consist of nucleic acid sequences derived from the genome of the same species or of a different species than the species of the target animal.
The term "germ cell line transgenic animal" refers to a transgenic animal in which the genetic alteration or genetic information was introduced into a germ line cell, thereby conferring the ability of the transgenic animal to transfer the genetic information to offspring. If such offspring in fact possess some or all of that alteration or genetic information, then they too are transgenic animals.
The alteration or genetic information may be foreign to the species of animal to which the recipient belongs, foreign only to the particular individual recipient, or may be genetic information already possessed by the recipient. In the last case, the altered or introduced gene may be expressed differently than the native gene.
Transgenic animals can be produced by a variety of different methods including transfection, electroporation, microinjection, gene targeting in embryonic stem cells and recombinant viral and retroviral infection (see, e.g., U.S. Patent No. 4,736,866; U.S. Patent No. 5,602,307; Mullins et al. Hypertension (1993) 22(4):630-633; Brenin et al. Surgical Oncology, Vol. 6, no. 2 (1997) pp. 99-110; "Recombinant Gene Expression Protocols" in: Tuan (ed.), Methods in Molecular Biology, No. 62 (Humana Press, 1997)).
A number of recombinant or transgenic mice kave been produced, including tkose wkick express an activated oncogene sequence (U.S. Patent No. 4,736,866); express simian SV40 T-antigen (U.S. Patent No. 5,728,915); lack tke expression of interferon regulatory factor 1 (IRF-1) (U.S. Patent No. 5,731,490); exkibit dopaminergic dysfunction (U.S. Patent No. 5,723,719); express at least one human gene which participates in blood pressure control (U.S. Patent No. 5,731,489); display greater similarity to the conditions existing in naturally occurring Alzheimer's disease (U.S. Patent No. 5,720,936); have a reduced capacity to mediate cellular adhesion (U.S. Patent No. 5,602,307); possess a bovine growth hormone gene (Clutter et al. Genetics, Vol. 143, no. 4 (1996) pp. 1753-1760); or, are capable of generating a fully human antibody response (McCarthy. The Lancet, Vol. 349, no. 9049 (1997) pp. 405).
While mice and rats remain the animals of choice for most transgenic experimentation, in some instances it is preferable or even necessary to use alternative animal species. Transgenic procedures have been successfully utilized in a variety of non-murine animals, including sheep, goats, pigs, dogs, cats, monkeys, chimpanzees, hamsters, rabbits, cows and guinea pigs (see, e.g., Kim et al. Molecular Reproduction and Development, Vol. 46, no. 4 (1997) pp. 515-526; Houdebine. Reproduction, Nutrition, Development, Vol. 35, no. 6 (1995) pp. 609-617; Petters Reproduction, Fertility and Development, Vol. 6, no. 5 (1994) pp. 643- 645; Schnieke et al. Science Vol. 278, no. 5346 (1997) pp. 2130-2133; and Amoah, Journal of Animal Science, Vol. 75, no. 2 (1997) pp. 578-585.
The method of introduction of nucleic acid fragments into recombination competent mammalian cells can be by any method which favors co-transformation of multiple nucleic acid molecules. Detailed procedures for producing transgenic animals are readily available to one skilled in the art, including the disclosures in U.S. Patent No. 5,489,743 and U.S. Patent No. 5,602,307.
L. Diagnostic Methods As the genes and proteins of the invention are differentially expressed in the tissue of patients with IgAN compared with normal renal tissue, they maybe used to diagnose or monitor IgAN, reanl function, or to track disease progression. One means of diagnosing a renal disease using the nucleic acid molecules or proteins of the invention involves obtaining kidney tissue from living subjects. Obtaining tissue samples from living sources is problematic for tissues such as kidney. However, due to the nature of the treatment paradigms for kidney patients, biopsy may be necessary. If possible, renal biopsy tissue may be obtained percutaneously. Another means of diagnosing renal disease using the nucleic acid molecules or proteins of tke invention involves obtaining blood samples from living subjects.
Tke use of molecular biological tools bas become routine in forensic tecknology. For example, nucleic acid probes comprising all or at least part of tke sequence of SEQ ID NO: 1 may be used to determine the expression of a nucleic acid molecule in forensic/pathology specimens. Further, nucleic acid assays may be carried out by any means of conducting a transcriptional profiling analysis. In addition to nucleic acid analysis, forensic methods of the invention may target the proteins of the invention, particularly a protein comprising the amino acid sequence of SEQ ID NO: 2, to determine up- or down- regulation of the genes (Shiverick et al, Biochim Biophys Acta (1975) 393:124-33).
Methods of the invention may involve treatment of tissues with collagenases or other proteases to make the tissue amenable to cell lysis (Semenov DE et al, Biull Eksp BiolMed (1987) 104:113-6). Further, it is possible to obtain biopsy samples from different regions of the kidney for analysis.
Assays to detect nucleic acid or protein molecules of the invention may be in any available format. Typical assays for nucleic acid molecules include hybridization or PCR based formats. Typical assays for the detection of proteins, polypeptides or peptides of the invention include the use of antibody probes in any available format such as in situ binding assays, etc. See Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1988. In preferred embodiments, assays are carried-out with appropriate controls.
The above methods may also be used in other diagnostic protocols, including protocols and methods to detect disease states in other tissues or organs, for example the colon, pancreas, and spleen, particularly when inflammation associated with infiltrating leukocytes is involved.
Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the compounds of the present invention and practice the claimed methods. The following working examples therefore, specifically point out preferred embodiments of the present invention, and are not to be construed as limiting in any way the remainder of the disclosure.
EXAMPLES
Example 1 Identification of Differentially Expressed Kidney mRNA Kidney tissue was obtained from patients with various forms and stages of diagnosed renal disease.
Total cellular RNA was prepared from the kidney tissue described above as well as from control kidney tissue using the procedure of Newburger et al. Journal of Biological Chemistry, Vol. 266, no. 24 (1991) pp. 16171-16177 and Newburger et al. Proceedings of National Academy of Science USA, Vol. 85 (1988) pp. 5215-5219. Synthesis of cDNA was performed as previously described by Prashar et al. in WO
97/05286 and in Praskar et al Proceedings of the National Academy of Science USA, Vol. 93 (1996) pp. 659-663. Briefly, cDNA was syntkesized according to tke protocol described in tbe GIBCO/BRL kit for cDNA syntkesis. Tke reaction mixture for first-strand syntkesis included 6 μg of total RNA, and 200 ng of a mixture of 1-base anchored oligo(dT) primers with all three possible anchored bases
(ACGTAATACGACTCACTATAGGGCGAATTGGGTCGACTTTTTTTTTTTTTTTTT nl (SEQ ID NO: 3) wherein nl=A C or G) along with otker components for first-strand syntkesis reaction except reverse transcriptase. Tkis mixture was incubated at 65 °C for 5 minutes, chilled on ice and the process repeated. Alternatively, the reaction mixture may include 10 μg of total RNA, and 2 pmol of one of the 2-base anchored oligo(dT) primers a keel suck as RP5.0 (CTCTCAAGGATCTTACCGCTT 18AT (SEQ ID NO: 4)), or
RP6.0 (TAATACCGCGCCACATAGCAT 18CG (SEQ ID NO: 5)), or RP9.2 (CAGGGTAGACGACGCTACGCTlgGA (SEQ ID NO: 6)) along witk otker components for first-strand syntkesis reaction except reverse transcriptase. Tkis mixture was tken layered witk mineral oil and incubated at 65 °C for seven minutes followed by 50°C for anotber seven minutes. At tkis stage, 2 μl of Superscript® reverse transcriptase (200 units/μl; GIBCO/BRL) was added quickly and mixed, and tke reaction continued for one kour at 45-50 °C. Second- strand syntkesis was performed at 16 °C for two kours. At tke end of tke reaction, tke cDNAs were precipitated witk etkanol and tke yield of cDNA was calculated. In our experiments, -200 ng of cDNA was obtained from 10 μg of total RNA. The adapter oligonucleotide sequences were Al (TAGCGTCCGGCGCAGCGACGGCCAG (SEQ ID NO: 7)) and A2 (GATCCTGGCCGTCGGCTGTCTGTCGGCGC (SEQ ID NO: 8)). One microgram of oligonucleotide A2 was first phosphorylated at the 5 ' end using T4 polynucleotide kinase (PNK). After phosphorylation, PNK was heat-denatured, and 1 μg of the oligonucleotide Al was added along with lOx annealing buffer (1 M NaCl/100 mM Tris- HC1, pH8.0/10 mM EDTA, pH8.0) in a final volume of 20 μl. This mixture was then heated at 65 °C for ten minutes followed by slow cooling to room temperature for thirty minutes, resulting in formation of the Y adapter at a final concentration of 100 ng/μl. About 20 ng of the cDNA was digested with 4 units oϊBgl II in a final volume of 10 μl for thirty minutes at 37°C. Two microliters (~4 ng of digested cDNA) of this reaction mixture was then used for ligation to 100 ng (~ 50-fold) of the Y-shaped adapter in a final volume of 5 μl for sixteen hours at 15 °C. After ligation, the reaction mixture was diluted with water to a final volume of 80 μl (adapter ligated cDNA concentration, -50 pg/μl) and heated at 65 °C for ten minutes to denature T4 DNA ligase, and 2 μl aliquots (witk ~ 100 pg of cDNA) were used for PCR. Tke following sets of primers were used for PCR amplification of tke adapter ligated 3 '-end cDNAs:
TGAAGCCGAGACGTCGGTCG(T)18 nl, n2 (SEQ ID NO: 9) (wkerein nl, n2 = AA, AC, AG AT CA CC CG CT GA GC GG and GT) as tke 3' primer witk Al as tke 5' primer or alternatively
RP 5.0, RP 6.0, or RP 9.2 used as 3' primers witk primer Al.l serving as tke 5' primer. To detect tke PCR products on tke display gel, 24 pmol of oligonucleotide Al or Al.l was 5'- end-labeled using 15 μl of [γ-32 P]ATP (Amersham; 3000 Ci/mmol) and PNK in a final volume of 20 μl for thirty minutes at 37°C. After heat denaturing PNK at 65 °C for twenty minutes, the labeled oligonucleotide was diluted to a final concentration of 2 μM in 80 μl witk unlabeled oligonucleotide Al.l. Tke PCR mixture (20 μl) consisted of 2 μl (- 100 pg) of tke template, 2 μl of lOx PCR buffer (100 mM Tris-HCl, pH 8.3/500 mM KC1), 2 μl of 15 mM MgCl2 to yield 1.5 mM final Mg2+ concentration optimum in tke reaction mixture, 200 μM dNTPs, 200 nM each 5' and 3' PCR primers, and one unit of Am litaq Gold®. Primers and dNTPs were added after prekeating tke reaction mixture containing tke rest of tke components at 85 °C. Tkis "kot start" PCR was done to avoid artifactual amplification arising out of arbitrary annealing of PCR primers at lower temperature during transition from room temperature to 94 °C in the first PCR cycle. PCR consisted of five cycles of 94 °C for thirty seconds, 55 °C for two minutes, and 72 °C for sixty seconds followed by 25 cycles of 94 °C for thirty seconds, 60°C for two minutes, and 72°C for sixty seconds. A higher number of cycles resulted in smeary gel patterns. PCR products (2.5 μl) were analyzed on 6% polyacrylamide sequencing gel. For double or multiple digestion following adapter ligation, 13.2 μl of tke ligated cDNA sample was digested witk a secondary restriction enzyme(s) in a final vol of 20 μl. From tkis solution, 3 μl was used as template for PCR. Tkis template vol of 3 μl carried ~ 100 pg of tke cDNA and 10 mM MgCl2 (from tke lOx enzyme buffer), wkick diluted to tke optimum of 1.5 mM in tke final PCR volume of 20 μl. Since Mg2+ comes from tke restriction enzyme buffer, it was not included in tke reaction mixture wken amplifying secondarily cut cDNA. Individual cDNA fragments corresponding to mRNA species were separated by denaturing polyacrylamide gel electrophoresis and visualized by autoradiography. Bands were extracted from the display gels as described by Liang et al. (Current Opinions in Immunology, Vol. 7 (1995) pp. 274-280), reamplified using the 5' and 3' primers, and subcloned into pCR- Script with high efficiency using the PCR-Script cloning kit from Stratagene. Plasmids were sequenced by cycle sequencing on an automated sequencer. Alternatively, bands were extracted (cored) from the display gels, PCR amplified and sequenced directly without subcloning. The expression of a band corresponding to SEQ ID NO: 1 varied depending on the form of renal disease and on the stage of disease in the kidney tissue: up-regulation was observed in mild and moderate IgAN, but decreased expression was observed in necrotizing crescentic glomerulonephritis (NCGN). See Figure 1.
Example 2 Cloning of a Full Length human cDNA Corresponding to the EST fragment The full length cDNA corresponding to the EST fragment in Example 1 was obtained by the oligo-pulling method. Briefly, a gene-specific oligo was designed based on the sequence of the EST identified in Example 1. The oligo was labeled witk biotin and used to bybridize witk 2 μg of single strand plasmid DNA (cDNA recombinants) from a kuman kidney cDNA library following tke procedures of Sambrook et al. (Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, NY. Cold Spring Harbor Laboratory Press, 1985). Tke kybridized cDNAs were separated by streptavidin-conjugated beads and eluted by heating. The eluted cDNA was converted to double strand plasmid DNA and used to transform E. coli cells (DH10B) and the longest cDNA was screened. After confirmation by PCR using gene-specific primers, the cDNA clone was subjected to DNA sequencing.
The nucleotide sequence of the full-length cDNA corresponding to the differentially regulated EST band is set forth in SEQ ID NO: 1. For SEQ ID NO: 1, the cDNA comprises about 2839 base pairs with an open reading frame from nucleotide 121 to 945 (to 948, including the stop codon) encoding a 275 amino acid protein, as is set forth in SEQ ID NO: 2. The predicted structural characteristics, hydrophilicity and antigenicity of peptide fragments within SEQ ID NO: 2 are shown in Figure 8. Homology searches on the amino acid sequence of SEQ ID NO: 2 found motifs within the protein that correspond to known functional domains with complete identity: a cAMP- and cGMP- dependent protein kinase phosphorylation site is present at amino acid position 194-197; seven casein kinase II phosphorylation sites are present starting at amino acids 8, 53, 75, 204, 234, 244 and 249; and Protein Kinase C phosphorylation sites are present at amino acid 13, 131 an 193. Six amino acid N-myristoylation motifs occur througkout tke protein starting at amino acids 9, 18, 36, 49, 161 and 238. Additionally, imperfect motif matckes, containing only 1 mismatck, are found at amino acid 173 (an ATP/GTP -binding site (P-loop) motif), 153 (a eukaryotic tkiol (cysteine) protease active site), 131 (a nuclear transition proteinl signature sequence), 92 (an RNA- binding region RNP-1 signature), 89-112 (a C2H2 type zinc finger domain), and at amino acids 89-111 (a C-type lectin domain signature and profile). Example 3 Northern blot and PCR Expression Analysis The tissue distribution of RNA encoding the protein of SEQ ID NO: 2 was analyzed by Northern blot as well as PCR expression analysis of RNA isolated from various tissues. RNA was isolated from various tissues such as human heart, brain, placenta, lung, liver, skeletal muscle, kidney and pancreas using standard procedures. Nortkern blots were prepared using a probe derived from SEQ ID NO: 1 witk kybridization conditions as described by Sambrook et al. (Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, NY. Cold Spring Harbor Laboratory Press, 1985). See Figure 2.
Quantitative PCR Analysis of Expression Levels Figures 3 to 7 skow tke results of tke quantitative PCR analysis of expression levels of mRNA corresponding to SEQ ID NO: 1 in various kuman tissue or blood samples. Real time PCR detection was accomplisbed by the use of the ABI PRISM 7700 Sequence Detection System. The 7700 measures the fluorescence intensity of the sample each cycle and is able to detect the presence of specific amplicons within the PCR reaction. Each sample was assayed for the level of GAPDH and mRNA corresponding to SEQ ID NO: 1. GAPDH detection was performed using Perkin Elmer part #402869 according to the manufacturer's directions. Primers were designed from SEQ ID NO: 1 using Primer Express, a program developed by PE to efficiently find primers and probes for specific sequences. These primers were used in conjunction with SYBR green (Molecular Probes), a nonspecific double stranded DNA dye, to measure the expression level mRNA corresponding to SEQ ID NO: 1, which was then normalized to the GAPDH level in each sample.
Differentially Expressed mRNA in Renal and non-Renal Tissues
Tissue distribution analysis (see Figure 3) shows prominent expression in the glomeruli, medulla and cortex of the kidney, and additionally in the pancreas, colon and spleen, indicating that the gene and proteins of the invention could be involved in the pathogenesis of diseases afftecting these organs or other tissues, particularly when inflammation associated witk infiltrating leukocytes is involved.
Differentially Expressed mRNA in Peripkeral Blood Leukocytes Quantitative PCR, as described above, furtker skows tkat a band corresponding to SEQ ID NO: 1 is upregulated in the circulating leukocytes of IgAN patients. This indicates that the gene and proteins of the invention have diagnostic and prognostic applications that use a patient's diseased renal tissue and/or blood, to monitor expression in circulating (periheral blood) leukocytes and/or in infiltrating leukocytes. See Figure 4.
Identification of Differentially Expressed mRNA in Inflammation
A role for the genes and proteins of the invention in inflammation is demonstrated in the functional analysis of mRNA expression in two studies: treatment with the inflammatory cytokine, TNFα, elicits a complex pattern of up and down regulation in human umbilical venous endothelial cells (HUVEC) and human umbilical arterial endothelial cells (HUAEC) (Figure 5), and similar results are observed in renal mesangial cells treated with the inflammatory cytokines IL-1 and IL-6 (Figure 7).
Differentially Expressed mRNA at various Stages of the Cell Cycle. A role in cell cycle events was shown by the upregulation of a band corresponding to SEQ ID NO: 1 under growth arrest conditions in endothelial cells (Figure 6) as well as renal epithelial and mesangial cells (Figure 7). This indicates that the gene and/or proteins of the invention exert important biological effects primarily under non-proliferative conditions.
Although the present invention has been described in detail with reference to examples above, it is understood that various modifications can be made without departing from the spirit of the invention. Accordingly, the invention is limited only by the following claims. All cited patents, patent applications and publications referred to in this application are herein incorporated by reference in their entirety.

Claims

WHAT IS CLAIMED:
1. An isolated nucleic acid molecule selected from the group consisting of: (a) an isolated nucleic acid molecule that encodes the amino acid sequence of SEQ ID NO: 2; (b) an isolated nucleic acid molecule that encodes a fragment of at least 6 amino acids of SEQ ID NO: 2; (c) an isolated nucleic acid molecule which hybridizes to the complement of a nucleic acid molecule comprising SEQ ID NO: 1 under conditions of sufficient stringency to produce a clear signal; (d) an isolated nucleic acid molecule which hybridizes to the complement of a nucleic acid molecule that encodes the amino acid sequence of SEQ ID NO: 2 under conditions of sufficient stringency to produce a clear signal; (e) an isolated nucleic acid molecule that encodes a protein that exhibits at least about 35% amino acid sequence identity to SEQ ID NO: 2; and (f) an isolated nucleic acid molecule which hybridizes to any of the above nucleic acid molecules.
2. The isolated nucleic acid molecule of claim 1, wherein the nucleic acid molecule comprises the sequence of SEQ ID NO: 1.
3. The isolated nucleic acid molecule of claim 2, wherein the nucleic acid molecule consists of the sequence of SEQ ID NO: 1.
4. The isolated nucleic acid molecule of claim 1, wherein the nucleic acid molecule comprises nucleotides 121-948 of SEQ ID NO: 1 or nucleotides 121-945 of SEQ ID NO: 1.
5. The isolated nucleic acid molecule of claim 1, wherein the nucleic acid molecule consists of nucleotides 121-948 of SEQ ID NO: 1 or nucleotides 121-945 of SEQ ID NO: 1.
6. The isolated nucleic acid molecule of any one of claims 1-5, wherein said nucleic acid molecule is operably linked to one or more expression control elements.
7. A vector comprising an isolated nucleic acid molecule of any one of claims 1-5.
8. A host cell transformed to contain the nucleic acid molecule of any one of claims 1-
5.
9. A host cell comprising a vector of claim 7.
10. A host cell of claim 9, wherein said host is selected from the group consisting of prokaryotic host cells and eukaryotic host cells.
11. A method for producing a polypeptide comprising culturing a host cell transformed with the nucleic acid molecule of any one of claims 1-5 under conditions in which the protein encoded by said nucleic acid molecule is expressed.
12. The method of claim 11, wherein said host cell is selected from the group consisting of prokaryotic hosts and eukaryotic hosts.
13. An isolated polypeptide produced by the method of claim 11.
14. An isolated polypeptide or protein selected from the group consisting of an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 2, an isolated polypeptide comprising a fragment of at least 6 amino acids of SEQ ID NO: 2, an isolated polypeptide comprising conservative amino acid substitutions of SEQ ID NO: 2, an isolated polypeptide comprising naturally occurring amino acid sequence variants of SEQ ID NO: 2, and an isolated peptide exhibiting at least about 35% amino acid sequence identity with SEQ ID NO: 2.
15. An isolated antibody that binds to a polypeptide of either claim 13 or 14.
16. The antibody of claim 14 wherein said antibody is a monoclonal or a polyclonal antibody, or an immunologically reactive fragment thereof.
17. A method of identifying an agent which modulates the expression of a nucleic acid encoding a protein of claim 14 comprising: exposing cells which express the nucleic acid to the agent; and determining whether the agent modulates expression of said nucleic acid, thereby identifying an agent which modulates the expression of a nucleic acid encoding the protein.
18. A method of identifying an agent which modulates the level of or at least one activity of a protein of claim 14 comprising: exposing cells which express the protein to the agent; determining whether the agent modulates the level of or at least one activity of said protein, thereby identifying an agent which modulates the level of or at least one activity of the protein.
19. The method of claim 18, wherein the agent modulates one activity of the protein.
20. A method of identifying binding partners for a protein of claim 14, comprising: exposing said protein to a potential binding partner; and determining if the potential binding partner binds to said protein, thereby identifying binding partners for the protein.
21. A method of modulating the expression of a nucleic acid encoding a protein of claim 14 comprising: administering an effective amount of an agent which modulates the expression of a nucleic acid encoding tke protein.
22. A metkod of modulating at least one activity of a protein of claim 14 comprising: administering an effective amount of an agent wkick modulates at least one activity of tke protein.
23. A non-kuman transgenic animal modified to contain a nucleic acid molecule of any of claims 1-5.
24. Tke transgenic animal of claim 23, wkerein tke nucleic acid molecule contains a mutation tkat prevents expression of tke encoded protein.
25. A metkod of diagnosing a disease state in a subject, comprising determining the level of expression of a nucleic acid molecule of any one of claims 1-5.
26. A method of diagnosing a disease state in a subject, comprising determining the level of expression of a protein of claim 14.
27. The method of claim 25, wherein the disease state is a renal disease.
28. The method of claim 25, wherein the disease state is associated with infiltrating leukocytes.
29. The method of claim 25, wherein the disease state is an inflammatory disease.
30. The method of claim 25, wherein the disease state is an inflammatory disease associated with infiltrating leukocytes.
31. The method of claim 26, wherein the disease state is a renal disease.
32. The method of claim 26, wherein the disease state is associated with infiltrating leukocytes.
33. Tke metkod of claim 26, wkerein tke disease state is an inflammatory disease.
34. Tke metkod of claim 26, wkerein tke disease state is an inflammatory disease associated witk infiltrating leukocytes.
35. Tke metkod of any one of claims 27-34 wkerein tke expression of said nucleic acid molecule or protein is determined in a blood sample.
36. Tke metkod of any one of claims 27-34 wkerein tke expression of said nucleic acid molecule or protein is determined in a tissue sample.
PCT/US2000/033658 1999-12-13 2000-12-13 Novel cdna 22360 associated with renal disease and other disease states WO2001041805A1 (en)

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Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
DATABASE MEDLINE [online] US NATIONAL LIBRARY OF MEDICINE, (BETHESDA, MD, USA); March 1998 (1998-03-01), STRAUSBERG R.: "Human cancer genome anatomy project", XP002939478, Database accession no. AA873801 *
HARLOW: "Antibodies: A laboratory manual", 1988, COLD SPRING HARBOR LABORATORY, XP002939481 *
HE ET AL.: "Collagen and colagenase mRNAs in normal and sclerotic glomeruli: Predictors of progression and response to therapy", KIDNEY INTERNATIONAL, vol. 47, June 1995 (1995-06-01), pages S39 - S43, XP002939480 *
SAMBROOK JOSEPH: "Molecular cloning: A laboratory manual", 1989, COLD SPRING HARBOR PRESS, COLD SPRING HARBOR, NY, XP002939479 *

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