WO2002077162A2 - Gene (t23490) associe a l'hyperplasie benigne de la prostate - Google Patents

Gene (t23490) associe a l'hyperplasie benigne de la prostate Download PDF

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Publication number
WO2002077162A2
WO2002077162A2 PCT/US2002/007728 US0207728W WO02077162A2 WO 2002077162 A2 WO2002077162 A2 WO 2002077162A2 US 0207728 W US0207728 W US 0207728W WO 02077162 A2 WO02077162 A2 WO 02077162A2
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nucleic acid
protein
acid molecule
seq
nos
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PCT/US2002/007728
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WO2002077162A3 (fr
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Prakash Kulkarni
Robert H. Getzenberg
William E. Munger
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Gene Logic, Inc.
Japan Tobacco, Inc.
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Priority to AU2002250315A priority Critical patent/AU2002250315A1/en
Publication of WO2002077162A2 publication Critical patent/WO2002077162A2/fr
Publication of WO2002077162A3 publication Critical patent/WO2002077162A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out

Definitions

  • the invention relates generally to the changes in gene expression in prostate tissue removed from male patients with benign prostatic hyperplasia (BPH).
  • BPH benign prostatic hyperplasia
  • the invention specifically relates to a novel human gene which is differentially expressed in BPH tissue compared to normal prostate tissue.
  • BPH is the most common benign tumor in men over the age of 60. It is estimated that one in four men living to the age of 80 will require treatment for this disease. BPH is usually noted clinically after the age of 50, the incidence increasing with age, but as many as two thirds of men between the ages of 40 and 49 demonstrate histological evidence of the disease.
  • the anatomic location of the prostate at the bladder neck enveloping the urethra plays an important role in the pathology of BPH, including bladder outlet obstruction.
  • Two prostate components are thought to play a role in bladder outlet obstruction. The first is the relative increase in prostate tissue mass. The second component is the prostatic smooth muscle tone.
  • the causative factors of BPH in man have been intensively studied (see Ziada et al, (1999) Urology 53:1-6). In general, the two most important factors appear to be aging and the presence of functional testes. Although these factors appear to be key to the development of BPH, both appear to be nonspecific.
  • the present invention is based on the discovery of a new gene family that is differentially expressed in BPH tissue compared to normal prostate tissue.
  • the invention includes isolated nucleic acid molecules selected from the group consisting of an isolated nucleic acid molecule that encodes the amino acid sequence of SEQ ID NOS: 2, 4 or 6, an isolated nucleic acid molecule that encodes a fragment of at least 10 amino acids of SEQ ID NOS: 2, 4 or 6, an isolated nucleic acid molecule which hybridizes to the complement of a nucleic acid molecule comprising SEQ ID NOS: 1, 3 or 5, and an isolated nucleic acid molecule which hybridizes to the complement of a nucleic acid molecule that encodes the amino acid sequence of SEQ ID NOS: 2, 4 or 6.
  • Nucleic acid molecules of the invention may have about 50%-60% nucleotide sequence identity to SEQ ID NOS: 1, 3 or 5, preferably about 70-75% sequence identity, more preferably about 80-85% sequence identity, and even more preferably at least about 90% sequence identity to SEQ LD NOS: 1, 3 or 5.
  • the present invention further includes the nucleic acid molecules operably linked to one or more expression control elements, including vectors comprising the isolated nucleic acid molecules.
  • the invention further includes host cells transformed to contain the nucleic acid molecules of the invention. Also provided are methods for producing a protein comprising the step of culturing a host cell transformed with a nucleic acid molecule of the invention under conditions in which the protein is expressed.
  • the invention further provides an isolated polypeptide selected from the group consisting of an isolated polypeptide comprising the amino acid sequence of SEQ ID NOS: 2, 4 or 6, an isolated polypeptide comprising a fragment of at least 10 amino acids of SEQ ID NOS: 2, 4 or 6, an isolated polypeptide comprising conservative amino acid substitutions of SEQ ID NOS: 2, 4 or 6 and an isolated polypeptide comprising naturally occurring amino acid sequence variants of SEQ ID NOS: 2, 4 or 6.
  • Polypeptides of the invention also include polypeptides with an amino acid sequence having at least about 50%, 60%, 70% or 75% amino acid sequence identity with the sequence set forth in SEQ ID NOS: 2, 4 or 6, more preferably at least about 80%, even more preferably at least about 90%, and most preferably at least about 95% sequence identity with the sequence set forth in SEQ ID NOS: 2, 4 or 6.
  • the invention further provides an isolated antibody or antigen-binding antibody fragment that specifically binds to a polypeptide of the invention, including monoclonal and polyclonal antibodies.
  • the invention further provides methods of identifying an agent which modulates the expression of a nucleic acid molecule encoding a protein of the invention, comprising the steps of exposing cells which express the nucleic acid molecule to the agent; and determining whether the agent modulates expression of said nucleic acid molecule, thereby identifying an agent which modulates the expression of a nucleic acid molecule encoding the protein.
  • the invention further provides methods of identifying an agent which modulates the level of or at least one activity of a protein of the invention, comprising the steps of exposing cells which express the protein to the agent; and 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.
  • the invention further provides methods of identifying binding partners for a protein of the invention, comprising the steps of 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.
  • the present invention further provides methods of modulating the expression of a nucleic acid molecule encoding a protein of the invention, comprising the step of administering an effective amount of an agent which modulates the expression of a nucleic acid molecule encoding the protein of the invention.
  • the invention also provides methods of modulating at least one activity of a protein of the invention, comprising the step of administering an effective amount of an agent which modulates at least one activity of the protein of the invention.
  • the present invention further includes non-human transgenic animals modified to contain the nucleic acid molecules of the invention, or non-human transgenic animals modified to contain mutated nucleic acid molecules such that expression of the encoded polypeptides of the invention is altered or prevented.
  • the present invention also includes non-human transgenic animals in which all or a portion of a gene comprising all or a portion of SEQ ID NOS : 1 , 3 or 5 has been knocked out or deleted from the genome of the animal.
  • the invention further provides methods of diagnosing BPH or other disease states, comprising the steps of acquiring a tissue, blood, urine or other sample from a subject and determining the level of expression of a nucleic acid molecule of the invention or polypeptide of the invention.
  • compositions comprising a diluent and a polypeptide or protein selected from the group consisting of an isolated polypeptide comprising the amino acid sequence of SEQ LD NOS: 2, 4 or 6, an isolated polypeptide comprising a fragment of at least 6 amino acids of SEQ ID NOS: 2, 4 or 6, an isolated polypeptide comprising conservative amino acid substitutions of SEQ ID NOS: 2, 4 or 6, naturally occurring amino acid sequence variants of SEQ ID NOS: 2, 4 or 6, and an isolated polypeptide with an amino acid sequence having at least about 50%, 60%, 70% or 75% amino acid sequence identity with the sequence set forth in SEQ ID NOS: 2, 4 or 6, preferably at least about 80%, more preferably at least about 90%, and most preferably at least about 95 % sequence identity with the sequence set forth in SEQ ID NOS: 2, 4 or 6.
  • Figure 1 shows the expression of Clone No. T23490, SEQ LD NOS: 1, 3 and 5 as analyzed using an Affymetrix GeneChip in normal human control prostate and BPH samples, including BPH samples from men without symptoms ("without"), BPH samples from men who were diagnosed with prostate cancer ("Cancer") and BPH samples from men with symptoms ("With”). In all cases, the subregion of the prostate analyzed in the normal and BPH patient samples was the transitional zone. Averages are shown with error bars representing the standard deviation. T23490 is upregulated in samples from symptomatic BPH versus normal control patients.
  • Figure 2 shows the results of semi-quantitative PCR (40 cycles) for the expression of T23490 (SEQ ID NOS: 1, 3 and 5) in prostate tissue samples from normal men and from patients with BPH, including BPH samples from men without symptoms, BPH samples from men who were diagnosed with prostate cancer and BPH samples from men with symptoms. In all cases, the subregion of the prostate analyzed in the normal and BPH patient samples was the transitional zone. Each sample was assayed at least 3 times, normalized to and internal reference (Cyclophilin A) and the mean and SDM are shown for each sample. The group average is shown at the right of each group.
  • Figure 3 shows the results of a Northern Assay in which the expression level of T23490 was measured across a panel of normal human tissues. For each tissue type, the mean +/- SDM is shown as a vertical bar for samples obtained from 3 or more normal individuals.
  • Fi ure 4 Figure 4 is a hydrophobicity/hydrophilicity plot of the protein encoded by the first open reading frame of T23490 (SEQ ID NO: 2). Analysis was done using the methods of Goldman et al. and of Kyte-Doolittle.
  • Figure 5 is a hydrophobicity plot of the protein encoded by the second open reading frame of T23490 (SEQ ID NO: 4). Analysis was done using the methods of Goldman et al. and of Kyte-Doolittle.
  • Figure 6 is a hydrophobicity plot of the protein encoded by the third open reading frame of T23490 (SEQ ID NO: 6). Analysis was done using the methods of Goldman et al. and of Kyte-Doolittle.
  • the present invention is based in part on the identification of a new gene family that is differentially expressed in human BPH tissue compared to normal human prostate tissue.
  • This gene family corresponds to the human cDNA of SEQ ID NO: 1 (the nucleotide sequences of SEQ ID NOS: 3 and 5 are identical).
  • Genes that encode the human proteins of SEQ ID NOS: 2, 4 and 6 may also be found in other animal species, particularly mammalian species.
  • genes and proteins of the invention may be used as diagnostic agents or markers to detect BPH or the progression of BPH in a sample. They can also serve as a target for agents that can be used to modulate gene expression or the activity of the protein. For example, agents may be identified that modulate biological processes associated with prostate growth, including the hyperplastic process of BPH.
  • the present invention is further based on the development of methods for isolating binding partners that bind to the proteins.
  • Probes based on the proteins are used as capture probes to isolate potential binding partners, such as other proteins.
  • 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.
  • proteins provide novel targets for the screening of synthetic small molecules and combinatorial or naturally occurring compound libraries to discover novel therapeutics to regulate prostate function.
  • the present invention provides isolated proteins, allelic variants of the proteins, and conservative amino acid substitutions of the proteins.
  • polypeptide refers, in part, to a protein that has the human amino acid sequence depicted in SEQ ID NOS: 2, 4 or 6.
  • the terms also refer to naturally occurring allelic variants and proteins that have a slightly different amino acid sequence than that specifically recited above. Allelic variants, though possessing a slightly different amino acid sequence than those recited above, will still have the same or similar biological functions associated with these proteins.
  • the family of proteins related to the human amino acid sequences of SEQ ID NOS: 2, 4 or 6 refers to proteins that have been isolated from organisms in addition to humans. The methods used to identify and isolate other members of the family of proteins related to these proteins are described below.
  • the proteins of the present invention are preferably in isolated form.
  • a protein is said to be isolated when physical, mechanical or chemical methods are employed to remove the protein from cellular constituents that are normally associated with the protein. A skilled artisan can readily employ standard purification methods to obtain an isolated protein.
  • the proteins of the present invention further include insertion, deletion or conservative amino acid substitution variants of SEQ ID NOS: 2, 4 or 6.
  • a conservative variant refers to alterations in the amino acid sequence that do not adversely affect the 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 with the protein.
  • the overall charge, structure or hydrophobic/hydrophilic properties of the protein can be altered without adversely affecting a biological activity.
  • the amino acid sequence can be altered, for example to render the peptide more hydrophobic or hydrophilic, without adversely affecting the biological activities of the protein.
  • allelic variants, the conservative substitution variants, and the members of the protein family will have an amino acid sequence having at least about 50%, 60%, 70%» or 75% amino acid sequence identity with the sequence set forth in SEQ ID NOS: 2, 4 or 6, more preferably at least about 80%, even more preferably at least about 90%, and most preferably at least about 95% sequence identity.
  • Identity or homology with respect to such sequences is defined herein as the percentage of amino acid residues in the candidate sequence that are identical with the known peptides, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent homology, and not considering any conservative substitutions as part of the sequence identity (see section B for the relevant parameters). Fusion proteins, or N-terminal, C-terminal or internal extensions, deletions, or insertions into the peptide sequence shall not be construed as affecting homology.
  • the proteins of the present invention include molecules having the amino acid sequence disclosed in SEQ ID NOS: 2, 4 or 6; fragments thereof having a consecutive sequence of at least about 3, 4, 5, 6, 10, 15, 20, 25, 30, 35 or more amino acid residues of these proteins; amino acid sequence variants wherein one or more amino acid residues has been inserted N- or C-terminal to, or within, the disclosed coding sequence; and amino acid sequence variants of the disclosed sequence, or their fragments as defined above, that have been substituted by another residue.
  • 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. The regions are all easily identifiable by using commonly available protein sequence analysis software such as MacVector (Oxford Molecular).
  • 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 with a moiety other than a naturally occurring amino acid (for example a detectable moiety such as an enzyme or radioisotope).
  • the present invention further provides compositions comprising a protein or polypeptide of the invention and a diluent.
  • Suitable diluents can be aqueous or non-aqueous solvents or a combination thereof, and can comprise additional components, for example water-soluble salts or glycerol, that contribute to the stability, solubility, activity, and/or storage of the protein or polypeptide.
  • members of the family of proteins can be used: (1) to identify agents which modulate the level of or 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 of BPH and other hyperplastic diseases.
  • 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 such a nucleic acid of SEQ ID NOS: 1, 3 or 5 and remains stably bound to it under appropriate stringency conditions, or encodes a polypeptide sharing at least about 50%), 60%, 70% or 75% sequence identity, preferably at least about 80%, more preferably at least about 85%, and even more preferably at least about 90% or 95% or more identity with the peptide sequences of SEQ ID NOS: 2, 4 or 6, or exhibits at least 50%, 60%, 70%o or 75%) preferably at least about 80%, more preferably at least about 85%, and even more preferably at least about 90% or 95% or more nucleotide sequence identity over the open
  • 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 unobvious 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.
  • BLAST Basic Local Alignment Search Tool
  • blastp, blastn, blastx, tblastn and tblastx Altschul S.F. et al. (1997) Nucleic Acids Res 25:3389-3402, and Karlin et al, (1990) Proc NatlAcad Sci USA 87:2264-2268, both fully incorporated by reference
  • 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 matches which satisfy a preselected threshold of significance.
  • the scoring matrix is set by the ratios of M ⁇ i.e., the reward score for a pair of matching residues) to N (/. e. , the penalty score for mismatching residues), wherein the default values for M and N are 5 and -4, respectively.
  • M i.e., the reward score for a pair of matching residues
  • N /. e. , the penalty score for mismatching residues
  • “Stringent conditions” are those that (1) employ low ionic strength and high temperature for washing, for example, 0.015 M NaCl/0.0015 M sodium citrate/0.1 % SDS at 50°C, or (2) employ during hybridization a denaturing agent such as formamide, for example; 50% (vol/vol) formamide with 0.1%> bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mM NaCl, 75 mM sodium citrate at 42°C.
  • a denaturing agent such as formamide, for example; 50% (vol/vol) formamide with 0.1%> bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mM NaCl, 75 mM sodium citrate at 42°C.
  • Another example is hybridization in 50% formamide, 5x SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5x Denhardt's solution, sonicated salmon sperm DNA (50 ⁇ g/ml), 0.1%>
  • 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 the complement strand of the open reading frame of SEQ ID NO:
  • nucleic acid molecule is said to be "isolated” when the 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. The size of the fragment will be determined by the intended use. For example, if the fragment is chosen so as to encode an active portion of the protein, the fragment will need to be large enough to encode the functional region(s) of the protein. For instance, fragments which encode peptides corresponding to predicted antigenic regions may be prepared (see Figures 4-6). If the fragment is to be used as a nucleic acid probe or PCR primer, then the fragment length is chosen so as to obtain a relatively small number of false positives during probing/priming (see the discussion in Section H).
  • Fragments of the encoding nucleic acid molecules of the present invention ⁇ i.e., synthetic oligonucleotides) that 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 phosphoramidite method of Matteucci et al, (1981) J Am Chem Soc 103: 3185-3191, or using automated synthesis methods.
  • larger DNA segments can readily be prepared by well known methods, such as synthesis of a group of oligonucleotides that define various modular segments of the gene, followed by ligation of oligonucleotides to build the complete modified gene.
  • the encoding nucleic acid molecules of the present invention may further be modified so as to contain a detectable label for diagnostic and probe purposes.
  • a detectable label for diagnostic and probe purposes.
  • 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.
  • polyclonal antiserum from mammals such as rabbits immunized with the purified protein (as described below) or monoclonal antibodies can be used to probe a mammalian cDNA or genomic expression library, 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 the enzyme.
  • coding sequence herein 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 oligonucleotide 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 the art and can readily be adapted for use in isolating other encoding nucleic acid molecules.
  • Nucleic acid molecules encoding other members of the protein family may also be identified in existing genomic or other sequence information using any available computational method, including but not limited to: PSI-BLAST (Altschul, et al. (1997)
  • rDNA molecules Containing a Nucleic Acid Molecule The present invention further provides recombinant DNA (rDNA) molecules that contain a coding sequence.
  • a rDNA molecule is a DNA molecule that has 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 Laboratory Press, Cold Spring Harbor, NY, 1989.
  • a coding DNA sequence is operably linked to expression control sequences and/or vector sequences.
  • a vector contemplated by the present invention is at least capable of directing the replication or insertion into the host chromosome, and preferably also expression, of the structural gene included in the rDNA molecule.
  • Expression control elements that are used for regulating the 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 replicon, i.e., a DNA sequence having the ability to direct autonomous replication and maintenance of the recombinant DNA molecule exfrachromosomally in a prokaryotic host cell, such as a bacterial host cell, transformed therewith.
  • a prokaryotic replicon i.e., a DNA sequence having the ability to direct autonomous replication and maintenance of the recombinant DNA molecule exfrachromosomally 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 that include a prokaryotic replicon may also include a gene whose expression confers a detectable marker such as a drug resistance.
  • Typical bacterial drug resistance genes are those that confer resistance to ampicillin, kanamycin, chloramphenicol, or tetracycl
  • Vectors that include a prokaryotic replicon can further include a prokaryotic or bacteriophage promoter capable of directing the expression (transcription and translation) of the coding gene sequences in a bacterial host cell, such as E. coli.
  • a promoter is an expression control element formed by a DNA sequence that permits binding of RNA polymerase and transcription to occur. Promoter sequences compatible with bacterial hosts are typically provided in plasmid vectors containing convenient restriction sites for insertion of a DNA segment of the present invention.
  • Typical of such vector plasmids are pUC8, ⁇ UC9, 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 including viral 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 the desired DNA segment. Typical of such vectors are pSNL and pKSN-10 (Pharmacia), pBPN-l/pML2d (International Biotechnologies, Inc.), pTDTl (ATCC, #31255), the vector ⁇ CDM8 described herein, and the like eukaryotic expression vectors. Vectors may be modified to include prostate cell specific promoters if needed.
  • Eukaryotic cell expression vectors used to construct the rD ⁇ A molecules of the present invention may further include a selectable marker that is effective in a 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 ⁇ ne ⁇ ) gene (Southern et al, (1982) JMol Anal Genet 1: 327-341).
  • the selectable marker can be present on a separate plasmid, and the two vectors are introduced by co- transfection of the host cell, and selected by culturing in the appropriate drug for the selectable marker.
  • the present invention further provides host cells transformed with a nucleic acid molecule that encodes a protein of the present invention.
  • the host cell can be either prokaryotic or eukaryotic.
  • Eukaryotic cells useful for expression of a protein of the invention are not limited, so long as the cell line is compatible with cell culture methods and compatible with the propagation of the expression vector and expression of the gene product.
  • Preferred eukaryotic host 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
  • NTH Swiss mouse embryo cells available from the ATCC as CRL 1658
  • BHK baby hamster kidney cells
  • eukaryotic tissue culture cell lines NTH Swiss mouse embryo cells (NIH/3T3) available from the ATCC as CRL 1658
  • BHK baby hamster kidney cells
  • 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 that typically depend on the type of vector used and host system employed. With regard to transformation of prokaryotic host cells, electroporation and salt treatment methods are typically employed, see, for example, Cohen et al, (1972) Proc Natl Acad Sci USA 69: 2110; and Sambrook et al, Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989.
  • Successfully transformed cells i.e., cells that contain a rDNA molecule of the present invention
  • cells resulting from the introduction of a 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 (1975) J. Mol. Biol. 98:503, or Berent et al, (1985) Biotech 3: 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:
  • a nucleic acid molecule that encodes a protein of the invention, such as a nucleic acid molecule comprising, consisting essentially of or consisting of S ⁇ Q LD NOS: 1, 3 or 5, nucleotides 663-1523 or 663-1526 of S ⁇ Q ID NO: 1 or nucleotides 211-621 or 211-624 of S ⁇ Q ID NO: 3 or nucleotides 63-275 or 63-278 of S ⁇ Q LD NO: 5. If the encoding sequence is uninterrupted by introns, as are these open reading frames, it is directly suitable for expression in any host.
  • the nucleic acid molecule is then preferably placed in operable linkage with suitable control sequences, as described above, to form an expression unit containing the protein open reading frame.
  • the expression unit is used to transform a suitable host and the transformed host is cultured under conditions that allow the production of the recombinant protein.
  • the recombinant protein is isolated from the medium or from the cells; recovery and purification of the protein may not be necessary in some instances where some impurities may be tolerated.
  • the desired coding sequences may be obtained from genomic fragments and used directly in appropriate hosts.
  • the construction of expression vectors that are operable in a variety of hosts is accomplished using appropriate replicons and control sequences, as set forth above.
  • the control sequences, expression vectors, and transformation methods are dependent on the type of host cell used to express the gene and were discussed in detail earlier.
  • Suitable restriction sites can, if not normally available, be added to the ends of the 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 the art for use with the nucleic acid molecules of the 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.
  • the entire protein for instance a protein comprising the entire amino acid sequence of SEQ ID NOS: 2, 4 or 6 can be used.
  • a fragment of the protein can be used.
  • a cellular extract refers to a preparation or fraction which is made from a lysed or disrupted cell.
  • the preferred source of cellular extracts will be cells derived from human prostate tissue, for instance, prostate biopsy tissue or cultured prostate cells from subjects with BPH.
  • cellular extracts may be prepared from normal human prostate tissue or available cell lines, particularly prostate derived cell lines.
  • a variety of methods can be used to obtain an extract of a cell.
  • Cells can be disrupted using either physical or chemical disruption methods.
  • physical disruption methods include, but are not limited to, sonication and mechanical shearing.
  • chemical lysis methods include, but are not limited to, detergent lysis and enzyme lysis.
  • a skilled artisan can readily adapt methods for preparing cellular extracts in order to obtain extracts for use in the present methods.
  • the extract is mixed with the protein of the invention under conditions in which association of the protein with the 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.
  • 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.
  • 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.
  • binding partners may be identified using a Far- Western assay according to the procedures of Takayama et al, (1997) Methods Mol Biol 69:171-184 or Sauder et al, ⁇ 1996) J GenVirol 77:991-996, 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 or other in vivo protein-protein detection 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 LD NOS: 2, 4 or 6. 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 if it is capable of up- or down-regulating expression of the nucleic acid in a cell.
  • cell lines that contain reporter gene fusions between nucleotides from within the open reading frames defined by nucleotides 663-1523 of SEQ ID NO: 1, nucleotides 211-621 of SEQ ID NO: 3, nucleotides 63-275 of SEQ ID NO: 5 and/or the 5 'and/or 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, (1990) AnalBiochem 188: 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 the agent to modulate the expression of a nucleic acid encoding a protein of the invention, such as the protein having SEQ ID NOS: 2, 4 or 6.
  • mRNA expression may be monitored directly by hybridization to the nucleic acids of the invention.
  • Cell lines are exposed to the 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 ⁇ supra).
  • the preferred cells will be those derived from human prostate tissue, for instance, prostate biopsy tissue or cultured prostate cells from normal or BPH patients, for example BRF-55T cells (immortalized human prostate cells obtained from an individual with BPH, see lype et al, (1998) Int J Oncol 12:257-63). Alternatively, other available cells or cell lines may be used.
  • 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 the probe:target hybrid and probe :non-target hybrids.
  • Probes may be designed from the nucleic acids of the 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, ⁇ supra) or Ausubel et al, Short Protocols in Molecular Biology. 4th Ed.. John Wiley & Sons, New York, 1999.
  • 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, porous glass wafer or membrane.
  • 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. (1996) Methods 10: 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
  • 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
  • cells or cell lines are first identified which express the gene products of the invention physiologically ⁇ e.g., using assays of tissue distribution via Northern blot, although
  • RPAs may serve the identical purpose of expression selection).
  • Cells 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 are 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 the 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
  • the agent comprises a pharmaceutically acceptable excipient and 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
  • serum 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.
  • 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, 4 or 6. 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.
  • 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.
  • Immortalized cell lines which secrete the desired monoclonal antibodies may be prepared using the standard method of Kohler and Milstein ⁇ Nature (1975) 256:495-497) or modifications which effect immortalization of lymphocytes or spleen cells, as is generally known.
  • the immortalized cell lines secreting the 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 reactive (antigen-binding) portion can be used as antagonists, as well as the intact antibodies. Use of immunologically reactive antibody fragments, such as the Fab, Fab', or F(ab') 2 fragments is often preferable, especially in a therapeutic context, as these fragments are generally less immunogenic than the whole immunoglobulin.
  • the antibodies or antibody 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 utilizing 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 G.A. Grant in: Molecular Biology and Biotechnology. Meyers, ed.. pp. 659-664, NCH Publishers Inc., New York, 1995). A skilled artisan can readily recognize that there is no limit as to the structural nature of the agents of the present invention.
  • the peptide agents of the 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 differentially expressed in BPH tissue compared to normal tissue.
  • Agents that modulate or up-or-down- regulate the expression of the protein or agents that are agonists or antagonists of at least one activity of the protein may be used to 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 the invention.
  • mammal is meant 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 prostate cell growth or hyperplasia.
  • an agent is said to modulate a pathological process when the agent reduces the degree or severity of the process.
  • BPH may be prevented or disease progression modulated by the administration of agents which 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.
  • 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 the skill of the art. Typical dosages comprise 0.1 to 100 ⁇ g/kg body wt. The preferred dosages comprise 0.1 to 10 ⁇ g/kg body wt. The most preferred dosages comprise 0.1 to 1 ⁇ g/kg body wt.
  • 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, e.g., sesame oil, or synthetic fatty acid esters, e.g., ethyl oleate or triglycerides.
  • Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, and the substances may include, for example, sodium carboxymethyl cellulose, sorbitol, and/or dextran.
  • the suspension may also contain stabilizers. Liposomes can also be used to encapsulate the agent for delivery into the cell.
  • the pharmaceutical formulation for systemic administration according to the 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 inhalations and controlled release forms thereof.
  • the compounds of this 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 NOS: 1, 3, or 5, or the open reading frame encoding the polypeptide sequence of SEQ ID NOS: 2, 4, or 6 or fragments thereof having a consecutive sequence of at least about 3, 4, 5, 6, 10, 15, 20, 25, 30, 35 or more amino acid residues, are also included in the invention.
  • Transgenic animals are genetically modified animals into which recombinant, exogenous or cloned genetic material has been experimentally transferred.
  • transgene Such genetic material is often referred to as a "transgene.”
  • the nucleic acid sequence of the transgene in some embodiments, all or a portion of SEQ ID NOS: 1, 3, or 5 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.
  • transgenic animals in which all or a portion of a gene comprising SEQ ID NOS: 1, 3, or 5 is deleted may be constructed.
  • the gene corresponding to SEQ ID NOS: 1, 3, or 5 contains one or more introns
  • the entire gene- all exons, introns and the regulatory sequences- may be deleted.
  • less than the entire gene may be deleted.
  • a single exon and/or intron may be deleted, so as to create an animal expressing a modified version of a protein of the invention.
  • 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, (1993) Hypertension 22: 630-633; Brenin et al, (1997) Surg Oncol 6: 99-110; Recombinant Gene Expression Protocols (Methods in Molecular Biology. Vol. 62). Tuan, ed., Humana Press, Totowa, NJ, 1997).
  • mice A number of recombinant or transgenic mice have been produced, including those which 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 the expression of interferon regulatory factor 1 (IRF-1) (U.S. Patent No. 5,731,490); exhibit 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, (1996) Genetics 143: 1753-1760); or are capable of generating a fully human antibody response (McCarthy (1997) Lancet 349: 405).
  • 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, (1997) Mol Reprod Dev 46: 515-526; Houdebine (1995) Reprod Nutr Dev 35: 609-617; Petters (1994) Reprod Fertil Dev 6: 643-645; Schnieke et al, (1997) Science 278: 2130-2133; and Amoah (1997) J Animal Science 75: 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 BPH tissue compared to normal prostate tissue, the genes and proteins of the invention may be used to diagnose or monitor BPH, prostate function, or to track disease progression.
  • One means of diagnosing BPH using the nucleic acid molecules or proteins of the invention involves obtaining prostate tissue from living subjects. Obtaining tissue samples from living sources is problematic for tissues such as prostate. Due to the nature of the treatment paradigms for BPH, however, biopsy may be necessary. When possible, urine, blood or peripheral lymphocyte samples may be used as the tissue sample in the assay. Commonly, in hyperplastic diseases, genes which are up- or down-regulated in the affected tissue (for example the prostate) are also up- or down-regulated in lymphocytes, which may be isolated from whole blood.
  • nucleic acid probes may be used to determine the expression level of a nucleic acid molecule comprising all or at least part of the sequence of SEQ ID NOS: 1, 3 or 5 in forensic or 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 SEQ ID NOS: 2, 4 or 6, to determine up- or down-regulation of the genes (Shiverick et al, (1975) Biochim BiophysActa 393: 124-133).
  • Methods of the invention may involve treatment of tissues with collagenases or other proteases to make the tissue amenable to cell lysis (Semenov et al, (1987) BiullEksp Biol Med 104: 113-116). Further, it is possible to obtain biopsy samples from different regions of the prostate 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 (see Harlow & Lane, Antibodies - A Laboratory Manual. Cold Spring Harbor
  • 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 in tissues in which expression of a nucleic acid molecule of the invention is detected (see Figure 3).
  • Human tissue was obtained from the transitional zone of the prostate in biopsy samples from normal individuals and from patients with BPH or prostate cancer. BPH was defined histologically in all samples. Normal tissue and asymptomatic BPH samples came from individuals who died of trauma, and did not report symptoms. Patients having BPH with symptoms were defined as those with a need for frequent urination; in these patients a radical prostatectomy had been performed. Prostate cancer patients provided age-matched tissue samples for symptomatic BPH patients, but were without symptoms and without cancer in the transitional zone under histological examination.
  • First strand cDNA synthesis was primed with a T7-(dT24) oligonucleotide.
  • the cDNA was phenol-chloroform extracted and ethanol precipitated to a final concentration of 1 g/1.
  • cRNA was fragmented (5' fragmentation buffer consisting of 200 mM Tris-acetate (pH 8.1), 500 mM KOAc, 150 mM MgOAc) for thirty-five minutes at 94C. Following the Affymetrix protocol, 55 g of fragmented cRNA was hybridized on a Human chip set and the HuGeneFL array for twenty-four hours at 60 rpm in a 45C hybridization oven.
  • the chips were washed and stained with Streptavidin Phycoerythrin (SAPE) (Molecular Probes) in Affymetrix fluidics stations.
  • SAPE Streptavidin Phycoerythrin
  • SAPE solution was added twice with an anti-streptavidin biotinylated antibody (Vector Laboratories) staining step in between.
  • Hybridization to the probe arrays was detected by fluorometric scanning (Hewlett Packard Gene Array Scanner). Data was analyzed using Affymetrix GeneChip version 3.0 and Expression Data Mining Tool (EDMT) software (version 1.0).
  • EDMT Expression Data Mining Tool
  • cDNA CT23490 Cloning of a Full Length human cDNA CT23490 Corresponding to a Differentially Expressed mRNA Species
  • the full length cDNA having SEQ ID NOS : 1 , 3 and 5 was obtained by the oligo- pulling method. Briefly, a gene-specific oligo was designed based on the sequence of the T23490 oligo. The oligo was labeled with biotin and used to hybridize with 2 ⁇ g of single strand plasmid DNA (cDNA recombinants) from a placental cDNA library following the procedures of Sambrook et al. The hybridized cDNAs were separated by streptavidin- conjugated beads and eluted by heating.
  • the eluted cDNA was converted to double-stranded plasmid DNA and used to transform E. coli cells (DH10B), and the longest cDNA was screened. After positive selection was confirmed by PCR using gene-specific primers, the cDNA clone was subjected to DNA sequencing.
  • the nucleotide sequence of the full-length human cDNA corresponding to the differentially regulated mRNA detected above is set forth in S ⁇ Q ID NOS: 1, 3 and 5
  • the cDNA comprises 2001 base pairs (1960 base pairs and a polyA tail). This sequence is similar but not identical to cDNA "FLJ20185 fis" (GenBank accession number AK000192) from human colon mucosa.
  • the AK000192 sequence lacks the first 24 bases of the sequence of the instant invention, and also contains a number of base differences. Furthermore, AK000192 was submitted to GenBank without any functional data or tissue expression results and was not recognized as being associated with BPH.
  • S ⁇ Q ID NO: 2 An open reading frame within the cDNA nucleotide sequence of S ⁇ Q ID NO: 1, at nucleotides 663-1523 (663-1526 including stop codon) encodes a protein of 287 amino acids
  • the amino acid sequence corresponding to a predicted protein encoded by S ⁇ Q ID NO: 1 is set forth in S ⁇ Q ID NO: 2.
  • This sequence is similar but not identical to an "unnamed protein product" (GenBank accession number BAA90999) from human colon mucosa.
  • the "unnamed protein product” sequence differs from the sequence set forth in S ⁇ Q ID NO: 2 at amino acid 179 of the instant invention.
  • a second open reading frame at nucleotides 211-621 encodes a predicted protein of 137 amino acids, as set forth in S ⁇ Q ID NO: 4.
  • a third open reading frame at nucleotides 63-275 encodes a predicted protein of 71 amino acids, as set forth in S ⁇ Q ID NO: 6.
  • Figure 2 shows the results of semi-quantitative PCR analysis of expression levels of mRNA corresponding to S ⁇ Q ID NO: 1 in prostate tissue samples from normal and diseased human patients. These data also show a significant (p ⁇ 0.01) increase in T23490 expression in prostate sample from patients with BPH with symptoms versus normal prostate samples.
  • Real time PCR detection was accomplished 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 Cyclophilin A (as an endogenous reference) and mRNA corresponding to SEQ ID NO: 1.
  • Cyclophilin A 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 FAM
  • a signal peptide may be present beginning at methionine 28 or 79 with a cleavage site after amino acid 91 or 103.
  • Figure 3 shows the results of a Q-PCR assay in which the expression levels of T23490 were measured across a panel of normal human tissues. For each tissue type, the mean +/-
  • SDM is shown as a vertical bar for samples obtained from experiments done in triplicate.
  • Expression is strongest in the placenta and spleen, with moderate levels detected in the kidney, liver, prostate, fetal brain, small intestine, stomach, bone marrow, testis and mammary gland. Lower levels of expression were also observed in the adrenal gland, trachea, fetal liver, colon, thyroid, spinal cord, thymus and cerebellum.
  • T23490 DNA or of protein products derived from T23490 is likely to be of diagnostic significance in inflammatory responses in tissues in which T23490 is found.
  • T23490 nucleotides or protein products derived from T23490 are, therefore, potential targets for therapeutic intervention.
  • the expression level of mRNA corresponding to SEQ ID NOS: 1, 3 or 5 is determined in prostate tissue biopsy samples, in urine, or in lymphocytes from blood samples as described in Example 1 and Figure 1, i.e., by screening mRNA samples on a GeneChip, or as described in Example 2 and Figure 2, i.e., by semi-quantitative PCR analysis using the fluorescent detection system.
  • samples from non-prostate hyperplastic tissues in malignant or non-malignant states may also be analyzed.
  • Tissue samples from patients with BPH with symptoms and from normal subjects may be used as positive and negative controls.
  • a level of expression about 1.5 times higher (or more) than that of the normal control is indicative of BPH or a likelihood of developing BPH.
  • Based on the amplitude of fluorescence measurements in semi-quantitative PCR a level of expression about 1.5-fold (or more) above the expression level in normal prostate tissue is indicative of BPH or a likelihood of developing BPH .
  • the up-regulation of T23490 in prostate could specifically identify cases in which inflammation is occurring, which would have additional diagnostic and prognostic implications and utility. More generally for inflammatory conditions, the tissue distribution results (see Figure 3) indicate that the up-regulation of T23490 is likely to be of diagnostic significance in inflammatory responses in tissues in which T23490 is found and a potential target for therapeutic intervention.

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Abstract

La présente invention se rapporte de manière générale aux modifications intervenant dans l'expression génétique de l'hyperplasie bénigne de la prostate (HBP). L'invention concerne de manière spécifique un nouveau gène humain correspondant à une espèce d'ARN messager qui est exprimé différemment dans un tissu atteint par l'HBP que dans un tissu prostatique normal.
PCT/US2002/007728 2001-03-22 2002-03-14 Gene (t23490) associe a l'hyperplasie benigne de la prostate WO2002077162A2 (fr)

Priority Applications (1)

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AU2002250315A AU2002250315A1 (en) 2001-03-22 2002-03-14 Gene (t23490) associated with benign prostatic hyperplasia

Applications Claiming Priority (2)

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US27763401P 2001-03-22 2001-03-22
US60/277,634 2001-03-22

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WO (1) WO2002077162A2 (fr)

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
DATABASE GENBANK [Online] NATIONAL CENTER FOR BIOTECHNOLOGY INFORMATION, NATIONAL LIBRARY OF MEDICINE, BETHESDA MARYLAND 26 October 2000 XP002958750 Retrieved from STN Database accession no. (BE313844) *

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