WO2005029084A1 - Agents diagnostiques et therapeutiques pour des maladies liees au transporteur slc22a12 (slc22a12) de cations organiques - Google Patents

Agents diagnostiques et therapeutiques pour des maladies liees au transporteur slc22a12 (slc22a12) de cations organiques Download PDF

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WO2005029084A1
WO2005029084A1 PCT/EP2004/010111 EP2004010111W WO2005029084A1 WO 2005029084 A1 WO2005029084 A1 WO 2005029084A1 EP 2004010111 W EP2004010111 W EP 2004010111W WO 2005029084 A1 WO2005029084 A1 WO 2005029084A1
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slc22a12
diseases
polypeptide
cancer
test compound
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PCT/EP2004/010111
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English (en)
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Stefan Golz
Ulf Brüggemeier
Andreas Geerts
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Bayer Healthcare Ag
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • 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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value

Definitions

  • the present invention is in the field of molecular biology, more particularly, the present invention relates to nucleic acid sequences and amino acid sequences of a human SLC22A12 and its regulation for the treatment of cardiovascular diseases, reproduction diseases, urological diseases and cancer in mammals.
  • OAT Organic anion transporting polypeptides play an important role in the uptake of organic anions, including bile acids, bilirubin conjugates and sulfobromophthalein, in the liver [Hsiang et al, (1999); Konig et al, (2000); Kouzuki et al, (200O)].
  • levels of organic anion transporting polypeptides can be modulated to affect the rate of drug clearance via hepatocellular uptake.
  • OATs have been found to transport eicosanoids, taurocholate, conjugated steroids, and thyroid hormones.
  • the nucleotide sequence of SLC22A12 is accessible in the databases by the accession number AB071863 and is given in SEQ ID NO:l.
  • Leabman et al. As part of a pharmacogenetics project that sought to identify genes that deternxine drug response, Leabman et al. [Leabman et al., 2002] screened for variation in the set of 24 genes encoding membrane transporters. They identified variants by screening an ethnically diverse collection of genomic DNA samples, derived from a total of 494 chromosomes. By combining population-genetic and phylogenetic analyses, tliey were able to identify amino acid residues and protein domains that may be important for human fitness. A large sample set made it possible for them also to obtain information about rare variants.
  • the 24 membrane transporters with potential roles in drug response were grouped based on transporter family: e.g., OCT1, OCT2 and OCT3 belonged to the SLC6 family; CNT1 and CNT2 belonged to the SLC28 family.
  • OAT polypeptides play an important role in the uptake of organic anions, including bile acids, bilirubin conjugates and sulfobromophthalein, in the liver [Hsiang et al. (1999), Konig et al. (2000), Kouzuki et al. (2000)]. Thus, levels of organic anion transporting polypeptides can be modulated to affect the rate of drug clearance via hepatocellular uptake. Further, OATs have been found to transport eicosanoids, taurocholate, conjugated steroids, and thyroid hormones. Polyspecific organic cation transporters in the liver, kidney, and intestine are critical for elimination of many endogenous amines as well as a wide array of drugs and environmental toxins [Gêtmann et al., (1994)]
  • the invention relates to novel disease associations of SLC22A12 polypeptides and polynucleotides.
  • the invention also relates to novel methods of screening for therapeutic agents for the treatment of cardiovascular diseases, reproduction diseases, urological diseases and cancer in a mammal.
  • the invention also relates to pharma- ceutical compositions for the treatment of cardiovascular diseases, reproduction diseases, urological diseases and cancer in a mammal comprising a SLC22A12 polypeptide, a SLC22A12 polynucleotide, or regulators of SLC22A12 or modulators of SLC22A12 activity.
  • the invention further comprises methods of diagnosing cardiovascular diseases, reproduction diseases, urological diseases and cancer in a mammal.
  • Fig. 1 shows the nucleotide sequence of a SLC22A12 polynucleotide (SEQ ID NO:l).
  • Fig. 2 shows the amino acid sequence of a SLC22A12 polypeptide (SEQ ID NO:2).
  • Fig. 3 shows the nucleotide sequence of a primer useful for the invention (SEQ ID NO:2).
  • Fig. 4 shows the nucleotide sequence of a primer useful for the invention (SEQ ID NO:4).
  • Fig. 5 shows a nucleotide sequence useful as a probe to detect proteins of the invention (SEQ ID NO:5).
  • oligonucleotide is a stretch of nucleotide residues which has a sufficient number of bases to be used as an oligomer, amplimer or probe in a polymerase chain reaction (PCR). Oligonucleotides are prepared from genomic or cDNA sequence and are used to amplify, reveal, or confirm the presence of a similar DNA or RNA in a particular cell or tissue. Oligonucleotides or oligomers comprise portions of a DNA sequence having at least about 10 nucleotides and as many as about 35 nucleotides, preferably about 25 nucleotides.
  • Probes may be derived from naturally occurring or recombinant single- or double- stranded nucleic acids or may be chemically synthesized. They are useful in detecting the presence of identical or similar sequences. Such probes may be labeled with reporter molecules using nick translation, Klenow fill-in reaction, PCR or other methods well known in the art. Nucleic acid probes may be used in southern, northern or in situ hybridizations to determine whether DNA or RNA encoding a certain protein is present in a cell type, tissue, or organ.
  • a “fragment of a polynucleotide” is a nucleic acid that comprises all or any part of a given nucleotide molecule, the fragment having fewer nucleotides than about 6 kb, preferably fewer than about 1 kb.
  • Reporter molecules are radionuclides, enzymes, fluorescent, chemiluminescent, or chromogenic agents which associate with a particular nucleotide or amino acid sequence, thereby establishing the presence of a certain sequence, or allowing for the quantification of a certain sequence.
  • Chimeric molecules may be constructed by introducing all or part ofthe nucleotide sequence of this invention into a vector containing additional nucleic acid sequence which might be expected to change any one or several of the following SLC22A12 characteristics: cellular location, distribution, ligand-binding affinities, interchain affinities, degradation/turnover rate, signaling, etc.
  • SLC22A12 polypeptide refers to those forms, fragments, or domains of a SLC22A12 polypeptide which retain the biological and/or antigenic activity of a SLC22A12 polypeptide.
  • Naturally occurring SLC22A12 polypeptide refers to a polypeptide produced by cells which have not been genetically engineered and specifically contemplates various polypeptides arising from post-translational modifications of the polypeptide including but not limited to acetylation, carboxylation, glycosylation, phosphorylation, lipidation and acylation.
  • Derivative refers to polypeptides which have been chemically modified by techniques such as ubiquitination, labeling (see above), pegylation (derivatization with polyethylene glycol), and chemical insertion or substitution of amino acids such as ornithine which do not normally occur in human proteins.
  • Constant amino acid substitutions result from replacing one amino acid with another having similar structural and/or chemical properties, such as the replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, or a threonine with a serine.
  • “Insertions” or “deletions” are typically in the range of about 1 to 5 amino acids. The variation allowed may be experimentally determined by producing the peptide synthetically while systematically making insertions, deletions, or substitutions of nucleotides in the sequence using recombinant DNA techniques.
  • a “signal sequence” or “leader sequence” can be used, when desired, to direct the polypeptide through a membrane of a cell.
  • Such a sequence may be naturally present on the polypeptides of the present invention or provided from heterologous sources by recombinant DNA techniques.
  • Oligopeptide is a short stretch of amino acid residues and may be expressed from an ohgonucleotide. Oligopeptides comprise a stretch of amino acid residues of at least 3, 5, 10 amino acids and at most 10, 15, 25 amino acids, typically of at least 9 to 13 amino acids, and of sufficient length to display biological and/or antigenic activity.
  • inhibitor is any substance which retards or prevents a chemical or physiological reaction or response. Common inhibitors include but are not limited to antisense molecules, antibodies, and antagonists.
  • Standard expression is a quantitative or qualitative measurement for comparison. It is based on a statistically appropriate number of normal samples and is created to use as a basis of comparison when performing diagnostic assays, ranning clinical trials, or following patient treatment profiles.
  • Animal as used herein may be defined to include human, domestic (e.g., cats, dogs, etc.), agricultural (e.g., cows, horses, sheep, etc.) or test species (e.g., mouse, rat, rabbit, etc.).
  • SLC22A12 polynucleotide within the meaning of the invention, shall be understood as being a nucleic acid molecule selected from a group consisting of
  • nucleic acid molecules encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 2,
  • nucleic acid molecules comprising the sequence of SEQ ID NO: 1,
  • nucleic acid molecules the complementary strand of which hybridizes under stringent conditions to a nucleic acid molecule of (i), (ii), or (iii);
  • nucleic acid molecules the sequence of which differs from the sequence of a nucleic acid molecule of (iii) due to the degeneracy ofthe genetic code;
  • polypeptide encoded by said nucleic acid molecule has SLC22A12 activity.
  • SLC22A12 polypeptide within the meaning of the invention, shall be understood as being a polypeptide selected from a group consisting of
  • polypeptides comprising the sequence of SEQ LD NO: 2
  • polypeptides encoded by SLC22A12 polynucleotides and (iv) polypeptides which show at least 99%, 98%, 95%, 90%, or 80% homology with a polypeptide of (i), (ii), or (iii);
  • polypeptide has SLC22A12 activity.
  • nucleotide sequences encoding a SLC22A12 have numerous applications in techniques known to those skilled in the art of molecular biology. These techniques include use as hybridization probes, use in the construc- tion of oligomers for PCR, use for chromosome and gene mapping, use in the recombinant production of SLC22A12, and use in generation of antisense DNA or RNA, their chemical analogs and the like. Uses of nucleotides encoding a SLC22A12 disclosed herein are exemplary of known techniques and are not intended to limit their use in any technique known to a person of ordinary skill in the art.
  • nucleotide sequences disclosed herein may be used in molecular biology techniques that have not yet been developed, provided the new techniques rely on properties of nucleotide sequences that are currently known, e.g., the triplet genetic code, specific base pair interactions, etc.
  • nucleotide sequences which encode a SLC22A12, its derivatives or its variants are preferably capable of hybridizing to the nucleotide sequence of the naturally occurring SLC22A12 polynucleotide under stringent conditions, it may be advantageous to produce nucleotide sequences encoding SLC22A12 polypeptides or its derivatives possessing a substantially different codon usage. Codons can be selected to increase the rate at which expression of the peptide occurs in a particular prokaryotic or eukaryotic expression host in accordance with the frequency with which particular codons are utilized by the host.
  • RNA transcripts having more desirable properties such as a greater half-life, than transcripts produced from the naturally occurring sequence.
  • Nucleotide sequences encoding a SLC22A12 polypeptide may be joined to a variety of other nucleotide sequences by means of well established recombinant DNA techniques.
  • Useful nucleotide sequences for joining to SLC22A12 polynucleotides include an assortment of cloning vectors such as plasmids, cosmids, lambda phage derivatives, phagemids, and the like.
  • Vectors of interest include expression vectors, replication vectors, probe generation vectors, sequencing vectors, etc. In general, vectors of interest may contain an origin of replication functional in at least one organism, convenient restriction endonuclease sensitive sites, and selectable markers for one or more host cell systems.
  • Another aspect of the subject invention is to provide for SLC22A12-specific hybridization probes capable of hybridizing with naturally occurring nucleotide sequences encoding SLC22A12. Such probes may also be used for the detection of similar organic ion transporter encoding sequences and should preferably show at least 40% nucleotide identity to SLC22A12 polynucleotides.
  • the hybridization probes of the subject invention may be derived from the nucleotide sequence presented as SEQ ID NO: 1 or from genomic sequences including promoter, enhancers or introns of the native gene. Hybridization probes may be labelled by a variety of reporter molecules using techniques well known in the art.
  • the invention relates to nucleic acid sequences that hybridize with such SLC22A12 encoding nucleic acid sequences under stringent conditions.
  • Stringent conditions refers to conditions that allow for the hybridization of substantially related nucleic acid sequences. For instance, such conditions will generally allow hybridization of sequence with at least about 85% sequence identity, preferably with at least about 90% sequence identity, more preferably with at least about 95%o sequence identity. Hybridization conditions and probes can be adjusted in well-characterized ways to achieve selective hybridization of human-derived probes. Stringent conditions, within the meaning of the invention are 65°C in a buffer containing 1 mM EDTA, 0.5 M NaHPO 4 (pH 7.2), 7 % (w/v) SDS.
  • Nucleic acid molecules that will hybridize to SLC22A12 polynucleotides under stringent conditions can be identified functionally.
  • examples of the uses for hybridization probes include: histochemical uses such as identifying tissues that express SLC22A12; measuring mRNA levels, for instance to identify a sample's tissue type or to identify cells that express abnormal levels of SLC22A12; and detecting polymorphisms of SLC22A12.
  • PCR provides additional uses for oligonucleotides based upon the nucleotide sequence which encodes SLC22A12.
  • probes used in PCR may be of recombinant origin, chemically synthesized, or a mixture of both.
  • Oligomers may comprise discrete nucleotide sequences employed under optimized conditions for identification of SLC22A12 in specific tissues or diagnostic use. The same two oligomers, a nested set of oligomers, or even a degenerate pool of oligomers may be employed under less stringent conditions for identification of closely related DNAs or RNAs.
  • PCR primers i.e., preparations of primers that are heterogeneous at given sequence locations, can be designed to amplify nucleic acid sequences that are highly homologous to, but not identical with SLC22A12.
  • Strategies are now available that allow for only one of the primers to be required to specifically hybridize with a known sequence.
  • appropriate nucleic acid primers can be ligated to the nucleic acid sought to be amplified to provide the hybridization partner for one of the primers. In this way, only one of the primers need be based on the sequence ofthe nucleic acid sought to be amplified.
  • PCR methods for amplifying nucleic acid will utilize at least two primers.
  • One of these primers will be capable of hybridizing to a first strand of the nucleic acid to be amplified and of priming enzyme-driven nucleic acid synthesis in a first direction.
  • the other will be capable of hybridizing the reciprocal sequence ofthe first strand (if the sequence to be amplified is single stranded, this sequence will initially be hypothetical, but will be synthesized in the first amplification cycle) and of priming nucleic acid synthesis from that strand in the direction opposite the first direction and towards the site of hybridization for the first primer.
  • Conditions for conducting such amplifications particularly under preferred stringent hybridization conditions, are well known.
  • RNA polymerase as T7 or SP6 RNA polymerase and the appropriate reporter molecules.
  • nucleic acid sequence can be inserted into any ofthe many available DNA vectors and their respective host cells using techniques which are well known in the art.
  • synthetic chemistry may be used to introduce mutations into the nucleotide sequence. Alternately, a portion of sequence in which a mutation is desired can be synthesized and recombined with longer portion of an existing genomic or recombinant sequence.
  • SLC22A12 polynucleotides may be used to produce a purified oligo-or polypeptide using well known methods of recombinant DNA technology.
  • the oligopeptide may be expressed in a variety of host cells, either prokaryotic or eukaryotic. Host cells may be from the same species from which the nucleotide sequence was derived or from a different species. Advantages of producing an oligonucleotide by recombinant DNA technology include obtaining adequate amounts of the protein for purification and the availability of simplified purification procedures.
  • Chromosome-based techniques such as comparative genomic hybridization (CGH) and fluorescent in situ hybridization (FISH) facilitate efforts to cytogenetically localize genomic regions that are altered in tumor cells. Regions of genomic alteration can be narrowed further using loss of heterozygosity analysis (LOH), in which disease DNA is analyzed and compared with normal DNA for the loss of a heterozygous polymorphic marker.
  • LH loss of heterozygosity analysis
  • RFLPs restriction fragment length polymorphisms [Johnson, (1989)]
  • hyper- variable minisatellite DNA Barnes, 2000].
  • a gene sequence contained in all samples at relatively constant quantity is typically utilized for sample amplification efficiency normalization.
  • This approach suffers from several drawbacks.
  • the method requires that each sample has equal input amounts of the nucleic acid and that the amplification efficiency between samples is identical until the time of analysis.
  • QC-PCR quantitative competitive PCR
  • An internal control competitor in each reaction [Piatak, (1993), BioTechniques].
  • the efficiency of each reaction is normalized to the internal competitor.
  • a known amount of internal competitor is typically added to each sample.
  • the unknown target PCR product is compared with the known competitor
  • a difficulty with this general approach lies in developing an internal control that amplifies with the same efficiency than the target molecule.
  • Fluorogenic nuclease assays are a real time quantitation method that uses a probe to monitor formation of amplification product.
  • the basis for this method of monitoring the formation of amplification product is to measure continuously PCR product accumulation using a dual-labelled fluorogenic ohgonucleotide probe, an approach frequently referred to in the literature simply as the "TaqMan method” [Piatak,(1993), Science; Heid, (1996); Gibson, (1996); Holland. (1991)].
  • the probe used in such assays is typically a short (about 20-25 bases) ohgonucleotide that is labeled with two different fluorescent dyes.
  • the 5' terminus of the probe is attached to a reporter dye and the 3' terminus is attached to a quenching dye, although the dyes could be attached at other locations on the probe as well.
  • the probe is designed to have at least substantial sequence complementarity with the probe binding site. Upstream and downstream PCR primers which bind to flanking regions of the locus are added to the reaction mixture. When the probe is intact, energy transfer between the two fluorophors occurs and the quencher quenches emission from the reporter.
  • the probe is cleaved by the 5' nuclease activity of a nucleic acid polymerase such as Taq polymerase, thereby releasing the reporter from the oligonucleotide-quencher and resulting in an increase of reporter emission intensity which can be measured by an appropriate detector.
  • a nucleic acid polymerase such as Taq polymerase
  • One detector which is specifically adapted for measuring fluorescence emissions such as those created during a fluorogenic assay is the ABI 7700 or 4700 HT manufactured by Applied Biosystems, Inc. in Foster City, Calif.
  • the ABI 7700 uses fiber optics connected with each well in a 96-or 384 well PCR tube arrangement.
  • the instrument includes a laser for exciting the labels and is capable of measuring the fluorescence spectra intensity from each tube with continuous monitoring during PCR amplification. Each tube is re-examined every 8.5 seconds.
  • Computer software provided with the instrument is capable of recording the fluorescence intensity of reporter and quencher over the course of the amplification. The recorded values will then be used to calculate the increase in normalized reporter emission intensity on a continuous basis. The increase in emission intensity is plotted versus time, i.e., the number of amplification cycles, to produce a continuous measure of amplification.
  • the amplification plot is examined at a point during the log phase of product accumulation. This is accomplished by assigning a fluorescence threshold intensity above background and determining the point at which each amplification plot crosses the threshold (defined as the threshold cycle number or Ct). Differences in threshold cycle number are used to quantify the relative amount of PCR target contained within each tube. Assuming that each reaction functions at 100% PCR efficiency, a difference of one Ct represents a two-fold difference in the amount of starting template.
  • the fluorescence value can be used in conjunction with a standard curve to determine the amount of amplification product present.
  • a variety of options are available for measuring the amplification products as they are formed.
  • One method utilizes labels, such as dyes, which only bind to double stranded DNA.
  • amplification product which is double stranded
  • dyes it is possible to distinguish between dye molecules free in solution and dye molecules bound to amplification product.
  • certain dyes fluoresce only when bound to amplification product. Examples of dyes which can be used in methods of this general type include, but are not limited to, Syber Green.TM. and Pico Green from Molecular Probes, Inc.
  • These detection methods involve some alteration to the structure or conformation of a probe hybridized to the locus between the amplification primer pair.
  • the alteration is caused by the template-dependent extension catalyzed by a nucleic acid polymerase during the amplification process.
  • the alteration generates a detectable signal which is an indirect measure ofthe amount of amplification product formed.
  • some methods involve the degradation or digestion ofthe probe during the extension reaction. These methods are a consequence of the 5 -3' nuclease activity associated with some nucleic acid polymerases. Polymerases having this activity cleave mononucleotides or small oligonucleotides from an ohgonucleotide probe annealed to its complementary sequence located within the locus.
  • the 3' end ofthe upstream primer provides the initial binding site for the nucleic acid polymerase.
  • the nucleic acid polymerase displaces a portion ofthe 5' end of the probe and through its nuclease activity cleaves mononucleotides or oligonucleotides from the probe.
  • the upstream primer and the probe can be designed such that they anneal to the complementary strand in close proximity to one another. In fact, the 3' end of the upstream primer and the 5' end of the probe may abut one another. In this situation, extension of the upstream primer is not necessary in order for the nucleic acid polymerase to begin cleaving the probe. In the case in which intervening nucleotides separate the upstream primer and the probe, extension of the primer is necessary before the nucleic acid polymerase encounters the 5' end of the probe.
  • the 5'-3' exonuclease activity ofthe nucleic acid polymerase begins cleaving mononucleotides or oligonucleotides from the 5' end of the probe. Digestion of the probe continues until the remaining portion of the probe dissociates from the complementary strand.
  • the two end sections can hybridize with each other to form a hairpin loop.
  • the reporter and quencher dye are in sufficiently close proximity that fluorescence from the reporter dye is effectively quenched by the quencher dye.
  • Hybridized probe in contrast, results in a linearized conformation in which the extent of quenching is decreased.
  • the labeled probe is selected so that its sequence is substantially complementary to a segment of the test locus or a reference locus. As indicated above, the nucleic acid site to which the probe binds should be located between the primer binding sites for the upstream and downstream amplification primers.
  • the primers used in the amplification are selected so as to be capable of hybridizing to sequences at flanking regions of the locus being amplified.
  • the primers are chosen to have at least substantial complementarity with the different strands of the nucleic acid being amplified.
  • the primers are selected in such that they flank the probe, i.e. are located upstream and downstream ofthe probe.
  • the primer must have sufficient length so that it is capable of priming the synthesis of extension products in the presence of an agent for polymerization.
  • the length and composition of the primer depends on many parameters, including, for example, the temperature at which the annealing reaction is conducted, proximity of the probe binding site to that ofthe primer, relative concentrations ofthe primer and probe and the particular nucleic acid composition of the probe.
  • the primer typically includes 15-30 nucleotides.
  • the length ofthe primer may be more or less depending on the complexity ofthe primer binding site and the factors listed above.
  • Labels for Probes and Primers The labels used for labeling the probes or primers ofthe current invention and which can provide the signal corresponding to the quantity of amplification product can take a variety of forms. As indicated above with regard to the 5' fluorogenic nuclease method, a fluorescent signal is one signal which can be measured. However, measurements may also be made, for example, by monitoring radioactivity, colorimetry, abso ⁇ tion, magnetic parameters, or enzymatic activity. Thus, labels which can be employed include, but are not limited to, fluorophors, chromophores, radioactive isotopes, electron dense reagents, enzymes, and ligands having specific binding partners (e.g., biotin-avidin).
  • a number of labels useful for attachment to probes or primers are commercially available including fluorescein and various fluorescein derivatives such as FAM, HEX, TET and JOE (all which are available from Applied Biosystems, Foster City, Calif.); lucifer yellow, and coumarin derivatives.
  • Labels may be attached to the probe or primer using a variety of techniques and can be attached at the 5' end, and/or the 3' end and/or at an internal nucleotide.
  • the label can also be attached to spacer arms of various sizes which are attached to the probe or primer. These spacer arms are useful for obtaining a desired distance between multiple labels attached to the probe or primer.
  • a single label may be utilized; whereas, in other instances, such as with the 5' fluorogenic nuclease assays for example, two or more labels are attached to the probe. Ln cases wherein the probe includes multiple labels, it is generally advisable to maintain spacing between the labels which is sufficient to permit separation of the labels during digestion of the probe through the 5'-3' nuclease activity ofthe nucleic acid polymerase.
  • a number of diseases are associated with changes in the copy number of a certain gene.
  • the real-time PCR method can be used to determine if the patient has copy number alterations which are known to be linked with diseases that are associated with the symptoms the patient has.
  • Fusion proteins are useful for generating antibodies against SLC22A12 polypeptides and for use in various assay systems. For example, fusion proteins can be used to identify proteins which interact with portions of SLC22A12 polypeptides. Protein affinity chromatography or library-based assays for protein-protein interactions, such as the yeast two-hybrid or phage display systems, can be used for this purpose. Such methods are well known in the art and also can be used as drug screens.
  • a SLC22A12 fusion protein comprises two polypeptide segments fused together by means of a peptide bond.
  • the first polypeptide segment can comprise at least 54, 75,
  • the first polypeptide segment also can comprise full-length SLC22A12.
  • the second polypeptide segment can be a full-length protein or a protein fragment.
  • Proteins commonly used in fusion protein construction include, but are not limited to jS-galactosidase, jS-glucuronidase, green fluorescent protein (GFP), autofluorescent proteins, including blue fluorescent protein (BFP), glutathione-S-transferase (GST), luciferase, horseradish peroxidase (HRP), and chloramphenicol acetyltransferase (CAT).
  • epitope tags are used in fusion protein constructions, including histidine (His) tags, FLAG tags, influenza hemagglutinin (HA) tags, Myc tags, VSV- G tags, and thioredoxin (Trx) tags.
  • fusion constructions can include maltose binding protein (MBP), S-tag, Lex a DNA binding domain (DBD) fusions, GAL4 DNA binding domain fusions, or herpes simplex virus (HSV) BP16 protein fusions.
  • MBP maltose binding protein
  • S-tag S-tag
  • DBD Lex a DNA binding domain
  • GAL4 GAL4 DNA binding domain
  • HSV herpes simplex virus
  • a naturally occurring SLC22A12 polynucleotide can be isolated free of other cellular components such as membrane components, proteins, and lipids.
  • Polynucleotides can be made by a cell and isolated using standard nucleic acid purification techniques, or synthesized using an amplification technique, such as the polymerase chain reaction (PCR), or by using an automatic synthesizer. Methods for isolating polynucleotides are routine and are known in the art. Any such technique for obtaining a polynucleotide can be used to obtain isolated SLC22A12 polynucleotides.
  • restriction enzymes and probes can be used to isolate polynucleotide fragments which comprise SLC22A12 nucleotide sequences.
  • Isolated polynucleotides are in preparations which are free or at least 70, 80, or 90% free of other molecules.
  • SLC22A12 cDNA molecules can be made with standard molecular biology techniques, using SLC22A12 mRNA as a template. SLC22A12 cDNA molecules can thereafter be replicated using molecular biology techniques known in the art. An amplification technique, such as PCR, can be used to obtain additional copies of polynucleotides of the invention, using either human genomic DNA dr cDNA as a template.
  • PCR-based methods can be used to extend nucleic acid sequences encoding human SLC22A12, for example to detect upstream sequences of SLC22A12 gene such as promoters and regulatory elements.
  • restriction-site PCR uses universal primers to retrieve unknown sequence adjacent to a known locus. Genomic DJSTA is first amplified in the presence of a primer to a linker sequence and a primer specific to the known region. The amplified sequences are then subjected to a second round of PCR with the same linker primer and another specific primer internal to the first one. Products of each round of PCR are transcribed with an appropriate RNA polymerase and sequenced using reverse transcriptase.
  • Inverse PCR also can be used to amplify or extend sequences using divergent primers based on a known region.
  • Primers can be designed using commercially available software, such as OLIGO 4.06 Primer Analysis software (National Biosciences Inc., Madison, Minn.), to be 22-30 nucleotides in length, to have a GC content of 50% or more, and to anneal to the target sequence at temperatures about 68-72°C.
  • the method uses several restriction enzymes to generate a suitable fragment in the known region of a gene. The fragment is then circularized by intramolecular ligation and used as a PCR template.
  • capture PCR which involves PCR amplification of DNA fragments adjacent to a known sequence in human and yeast artificial chromosome DNA.
  • multiple restriction enzyme digestions and ligations also can be used to place an engineered double-stranded sequence into an unknown fragment ofthe DNA molecule before performing PCR.
  • Randomly-primed libraries are preferable, in that they will contain more sequences which contain the 5' regions of genes. Use of a randomly primed library may be especially preferable for situations in which an oligo d(T) library does not yield a full-length cDNA. Genomic libraries can be useful for extension of sequence into 5' non-transcribed regulatory regions.
  • capillary electrophoresis systems can be used to analyze the size or confirm the nucleotide sequence of PCR or sequencing products.
  • capillary sequencing can employ flowable polymers for electrophoretic separation, four different fluorescent dyes (one for each nucleotide) which are laser activated, and detection of the emitted wavelengths by a charge coupled device camera.
  • Output/light intensity can be converted to electrical signal using appropriate equipment and software (e.g., GENOTYPER and Sequence NAVIGATOR, Perkin Elmer), and the entire process from loading of samples to computer analysis and electronic data display can be computer controlled.
  • Capillary electrophoresis is especially preferable for the sequencing of small pieces of DNA which might be present in limited amounts in a particular sample.
  • SLC22A.12 can be obtained, for example, by purification from human cells, by expression of SLC22A12 polynucleotides, or by direct chemical synthesis.
  • SLC22A.12 can be purified from any human cell which expresses the receptor, including those which have been transfected with expression constructs which express SLC22A12.
  • a purified SLC22A12 is separated from other compounds which normally associate with SLC22A12 in the cell, such as certain proteins, carbohydrates, or lipids, using methods well-known in the art. Such methods include, but are not limited to, size exclusion chromatography, ammonium sulfate fractionation, ion exchange chromatography, affinity chromatography, and preparative gel electrophoresis.
  • SLC22A12 polynucleotides can be inserted into an expression vector which contains the necessary elements for the transcription and translation ofthe inserted coding sequence.
  • Methods which are well known to those skilled in the art can be used to construct expression vectors containing sequences encoding SLC22A12 and appropriate transcriptional and translational control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination.
  • a variety of expression vector/host systems can be utilized to contain and express sequences encoding SLC22A12. These include, but are not limited to, microorganisms, such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors, insect cell systems infected with virus expression vectors (e.g., baculovirus), plant cell systems transformed with virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g., Ti orpBR322 plasmids), or animal cell systems.
  • microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors
  • yeast transformed with yeast expression vectors insect cell systems infected with virus expression vectors (e.g., baculovirus)
  • plant cell systems transformed with virus expression vectors e.g., cauliflower mosaic virus, CaMV; tobacco mosaic
  • control elements or regulatory sequences are those non-translated regions of the vector - enhancers, promoters, 5' and 3' untranslated regions - which interact with host cellular proteins to carry out transcription and translation. Such elements can vary in their strength and specificity. Depending on the vector system and host utilized, any number of suitable transcription and translation elements, including constitutive and inducible promoters, can be used. For example, when cloning in bacterial systems, inducible promoters such as the hybrid lacZ promoter of the BLUESCRIPT phagemid (Stratagene, LaJolla, Calif.) or pSPORTl plasmid (Life Technologies) and the like can be used. The baculovirus polyhedrin promoter can be used in insect cells. Promoters or enhancers derived from the genomes of plant cells
  • vectors e.g., heat shock, RUBISCO, and storage protein genes
  • plant viruses e.g., viral promoters or leader sequences
  • promoters from mammalian genes or from mammalian viruses are preferable. If it is necessary to generate a cell line that contains multiple copies of a nucleotide sequence encoding SLC22A12, vectors based on SV40 or EBV can be used with an appropriate selectable marker.
  • a number of expression vectors can be selected.
  • vectors which direct high level expression of fusion proteins that are readily purified can be used.
  • Such vectors include, but are not limited to, multifunctional E. coli cloning and expression vectors such as BLUESCRJJPT (Stratagene).
  • BLUESCRIPT vector a sequence encoding SLC22A12 can be ligated into the vector in frame with sequences for the amino-terminal Met and the subsequent 7 residues of ⁇ -galactosidase so that a hybrid protein is produced.
  • pL vectors or pGEX vectors also can be used to express foreign polypeptides as fusion proteins with glutathione
  • GST S-transferase
  • fusion proteins are soluble and can easily be purified from lysed cells by adsorption to glutathione-agarose beads followed by elution in the presence of free glutathione.
  • Proteins made in such systems can be designed to include heparin, thrombin, or factor Xa protease cleavage sites so that the cloned polypeptide of interest can be released from the GST moiety at will.
  • sequences encoding SLC22A12 can be driven by any of a number of promoters.
  • viral promoters such as the 35S and 19S promoters of CaMV can be used alone or in combination with the omega leader sequence from TMV.
  • plant pro- moters such as the small subunit of RUBISCO or heat shock promoters can be used. These constructs can be introduced into plant cells by direct DNA transformation or by pathogen-mediated transfection.
  • An insect system also can be used to express SLC22A12.
  • Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes in Spodoptera frugiperda cells or in Trichoplusia larvae.
  • Sequences encoding SLC22A12 can be cloned into a non-essential region of the virus, such as the polyhedrin gene, and placed under control of the polyhedrin promoter.
  • Successful insertion of SLC22A12 will render the polyhedrin gene inactive and produce recombinant virus lacking coat protein.
  • the recombinant viruses can then be used to infect S. frugiperda cells or Trichoplusia larvae in which SLC22A12 can be expressed.
  • a number of viral-based expression systems can be used to express SLC22A12 in mammalian host cells.
  • sequences encoding SLC22A12 can be ligated into an adenovirus transcription/- translation complex comprising the late promoter and tripartite leader sequence. Insertion in a non-essential El or E3 region ofthe viral genome can be used to obtain a viable virus which is capable of expressing SLC22A12 in infected host cells [Engelhard, 1994)].
  • transcription enhancers such as the Rous sarcoma virus (RSV) enhancer, can be used to increase expression in mammalian host cells.
  • RSV Rous sarcoma virus
  • HACs Human artificial chromosomes
  • HACs also can be used to deliver larger fragments of DNA than can be contained and expressed in a plasmid.
  • HACs of 6M to 10M are constructed and delivered to cells via conventional delivery methods (e.g., liposomes, polycationic amino polymers, or vesicles).
  • Specific initiation signals also can be used to achieve more efficient translation of sequences encoding SLC22A12. Such signals include the ATG initiation codon and adjacent sequences. In cases where sequences encoding SLC22A12, its initiation codon, and upstream sequences are inserted into the appropriate expression vector, no additional transcriptional or translational control signals may be needed.
  • exogenous translational control signals including the ATG initiation codon
  • the initiation codon should be in the correct reading frame to ensure translation of the entire insert.
  • Exogenous translational elements and initiation codons can be of various origins, both natural and synthetic.
  • a host cell strain can be chosen for its ability to modulate the expression of the inserted sequences or to process the expressed SLC22A12 in the desired fashion.
  • modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation.
  • Post- translational processing which cleaves a "prepro" form ofthe polypeptide also can be used to facilitate correct insertion, folding and/or function.
  • Different host cells which have specific cellular machinery and characteristic mechanisms for post-translational activities (e.g., CHO, HeLa, MDCK, HEK293, and WI38), are available from the American Type Culture Collection (ATCC; 10801 University Boulevard, Manassas, VA 20110-2209) and can be chosen to ensure the correct modification and processing of the foreign protein.
  • ATCC American Type Culture Collection
  • Stable expression is preferred for long-term, high-yield production of recombinant proteins.
  • cell lines which stably express SLC22A12 can be transformed using expression vectors which can contain viral origins of replication and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector. Following the introduction of the vector, cells can be allowed to grow for 1 -2 days in an enriched medium before they are switched to a selective medium.
  • the purpose of the selectable marker is to confer resistance to selection, and its presence allows growth and recovery of cells which successfully express the introduced SLC22A12 sequences.
  • Resistant clones of stably transformed cells can be proliferated using tissue culture techniques appropriate to the cell type. Any number of selection systems can be used to recover transformed cell lines.
  • herpes simplex virus thymidme kinase [Logan, (1984)] and adenine phosphoribosyltransferase [Wigler, (1977)] genes which can be employed in tk ⁇ or aprf cells, respectively.
  • antimetabolite, antibiotic, or herbicide resistance can be used as the basis for selection.
  • dhfr confers resistance to methotrexate [Lowy, (1980)]
  • npt confers resistance to the aminoglycosides, neomycin and G-418 [Wigler, (1980)]
  • als and pat confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively [Colbere-Garapin, 1981]. Additional selectable genes have been described.
  • trpB allows cells to utilize indole in place of tryptophan, or hisD, which allows cells to utilize histinol in place of histidine.
  • Visible markers such as anthocyanins, /3-glucuronidase and its substrate GUS, and luciferase and its substrate luciferin, can be used to identify transformants and to quantify the amount of transient or stable protein expression attributable to a specific vector system
  • marker gene expression suggests that a SLC22A12 poly- nucleotide is also present, its presence and expression may need to be confirmed. For example, if a sequence encoding SLC22A12 is inserted within a marker gene sequence, transformed cells containing sequences which encode SLC22A12 can be identified by the absence of marker gene function. Alternatively, a marker gene can be placed in tandem with a sequence encoding SLC22A12 under the control of a single promoter. Expression ofthe marker gene in response to induction or selection usually indicates expression of SLC22A12 polynucleotide.
  • host cells which contain a SLC22A12 polynucleotide and which express SLC22A12 can be identified by a variety of procedures known to those of skill in the art. These procedures include, but are not limited to, DNA-DNA or
  • DNA-RNA hybridizations and protein bioassay or immunoassay techniques which include membrane, solution, or chip-based technologies for the detection and/or quantification of nucleic acid or protein.
  • the presence of a polynucleotide sequence encoding SLC22A12 can be detected by DNA-DNA or DNA- RNA hybridization or amplification using probes or fragments or fragments of polynucleotides encoding SLC22A12.
  • Nucleic acid amplification-based assays involve the use of oligonucleotides selected from sequences encoding SLC22A12 to detect transformants which contain a SLC22A12 polynucleotide.
  • a variety of protocols for detecting and measuring the expression of SLC22A12, using either polyclonal or monoclonal antibodies specific for the polypeptide, are known in the art. Examples include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence activated cell sorting (FACS).
  • ELISA enzyme-linked immunosorbent assay
  • RIA radioimmunoassay
  • FACS fluorescence activated cell sorting
  • a two- site, monoclonal-based immunoassay using monoclonal antibodies reactive to two non-interfering epitopes on SLC22A12 can be used, or a competitive binding assay can be employed.
  • Means for producing labeled hybridization or PCR probes for detecting sequences related to polynucleotides encoding SLC22A12 include oligolabeling, nick translation, end- labeling, or PCR amplification using a labeled nucleotide.
  • sequences encoding SLC22A12 can be cloned into a vector for the production of an mRNA probe.
  • RNA probes are known in the art, are commercially available, and can be used to synthesize RNA probes in vitro by addition of labeled nucleotides and an appropriate RNA polymerase such as T7, T3, or SP6. These procedures can be conducted using a variety of commercially available kits (Amersham Pharmacia Biotech, Promega, and US Biochemical). Suitable reporter molecules or labels which can be used for ease of detection include radionuclides, enzymes, and fluorescent, chemiluminescent, or chromogenic agents, as well as substrates, cofactors, inhibitors, magnetic particles, and the like. Expression and Purification of Polypeptides
  • Host cells transformed with SLC22A12 polynucleotides can be cultured under conditions suitable for the expression and recovery of the protein from cell culture.
  • the polypeptide produced by a transformed cell can be secreted or contained intracellularly depending on the sequence and/or the vector used.
  • expression vectors containing SLC22A12 polynucleotides can be designed to contain signal sequences which direct secretion of soluble SLC22A12 through a prokaryotic or eukaryotic cell membrane or which direct the membrane insertion of membrane-bound SLC22A12.
  • purification facilitating domains include, but are not limited to, metal chelating peptides such as histidine-tryptophan modules that allow purification on immobilized metals, protein A domains that allow purification on immobilized immunoglobulin, and the domain utilized in the FLAGS extension/affinity purification system (Immunex Corp., Seattle, Wash.).
  • cleavable linker sequences such as those specific for Factor XA or enterokinase (Invitrogen, San Diego, CA) between the purification domain and SLC22A12 also can be used to facilitate purification.
  • One such expression vector provides for expression of a fusion protein containing SLC22A12 and 6 histidine residues preceding a thioredoxin or an enterokinase cleavage site. The histidine residues facilitate purification by L AC (immobilized metal ion affinity chromatography) Maddox, (1983)], while the enterokinase cleavage site provides a means for purifying
  • Sequences encoding SLC22A12 can be synthesized, in whole or in part, using chemical methods well known in the art.
  • SLC22A12 itself can be produced using chemical methods to synthesize its amino acid sequence, such as by direct peptide synthesis using solid-phase techniques. Protein synthesis can either be performed using manual techniques or by automation. Automated synthesis can be achieved, for example, using Applied Biosystems 431 A Peptide Synthesizer (Perkin Elmer).
  • fragments of SLC22A12 can be separately synthesized and combined using chemical methods to produce a full-length molecule.
  • the newly synthesized peptide can be substantially purified by preparative high performance liquid chromatography.
  • the composition of a synthetic SLC22A12 can be confirmed by amino acid analysis or sequencing. Additionally, any portion ofthe amino acid sequence of SLC22A12 can be altered during direct synthesis and/or combined using chemical methods with sequences from other proteins to produce a variant polypeptide or a fusion protein.
  • codons preferred by a particular prokaryotic or eukaryotic host can be selected to increase the rate of protein expression or to produce an RNA transcript having desirable properties, such as a half-life which is longer than that of a transcript generated from the naturally occurring sequence.
  • nucleotide sequences referred to herein can be engineered using methods generally known in the art to alter SLC22A12 polynucleotides for a variety of reasons, including but not limited to, alterations which modify the cloning, processing, and/or expression of the polypeptide or mRNA product.
  • DNA shuffling by random fragmentation and PCR reassembly of gene fragments and synthetic oligonucleotides can be used to engineer the nucleotide sequences.
  • site-directed mutagenesis can be used to insert new restriction sites, alter glycosyla- tion patterns, change codon preference, produce splice variants, introduce mutations, and so forth.
  • Any type of antibody known in the art can be generated to bind specifically to an epitope of SLC22A12.
  • Antibody as used herein includes intact immunoglobulin molecules, as well as fragments thereof, such as Fab, F(ab') , and Fv, which are capable of binding an epitope of SLC22A12. Typically, at least 6, 8, 10, or 12 contiguous amino acids are required to form an epitope. However, epitopes which involve non-contiguous amino acids may require more, e.g., at least 15, 25, or 50 amino acid.
  • An antibody which specifically binds to an epitope of SLC22A12 can be used therapeutically, as well as in immunochemical assays, such as Western blots, ELISAs, radioimmuno- assays, immunohistochemical assays, immunoprecipitations, or other immunochemical assays known in the art.
  • immunochemical assays such as Western blots, ELISAs, radioimmuno- assays, immunohistochemical assays, immunoprecipitations, or other immunochemical assays known in the art.
  • Various immunoassays can be used to identify antibodies having the desired specificity. Numerous protocols for competitive binding or immunoradiometric assays are well known in the art. Such immunoassays typically involve the measurement of complex formation between an immunogen and an antibody which specifically binds to the SLC22A12 immunogen.
  • an antibody which specifically binds to SLC22A12 provides a detection signal at least 5-, 10-, or 20-fold higher than a detection signal provided with other proteins when used in an immunochemical assay.
  • antibodies which specifically bind to SLC22A12 do not detect other proteins in immunochemical assays and can immunoprecipitate SLC22A12 from solution.
  • SLC22A12 can be used to immunize a mammal, such as a mouse, rat, rabbit, guinea pig, monkey, or human, to produce polyclonal antibodies. If desired, SLC22A12 can be conjugated to a carrier protein, such as bovine serum albumin, thyroglobulm, and keyhole limpet hemocyanin. Depending on the host species, various adjuvants can be used to increase the immunological response.
  • a carrier protein such as bovine serum albumin, thyroglobulm, and keyhole limpet hemocyanin.
  • various adjuvants can be used to increase the immunological response.
  • Such adjuvants include, but are not limited to, Freund's adjuvant, mineral gels (e.g., aluminum hydroxide), and surface active substances (e.g., lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol).
  • BCG Bacilli Calmette-Gueri
  • Corynebacterium parvum axe especially useful.
  • Monoclonal antibodies which specifically bind to SLC22A12 can be prepared using any technique which provides for the production of antibody molecules by continuous cell lines in culture. These techniques include, but are not limited to, the hybridoma technique, the human B-cell hybridoma technique, and the EBV- hybridoma technique [Roberge, (1995)].
  • chimeric antibodies the splicing of mouse antibody genes to human antibody genes to obtain a molecule with appropriate antigen specificity and biological activity
  • Monoclonal and other antibodies also can be "humanized” to prevent a patient from mounting an immune response against the antibody when it is used therapeutically.
  • Such anti- bodies may be sufficiently similar in sequence to human antibodies to be used directly in therapy or may require alteration of a few key residues. Sequence differences between rodent antibodies and human sequences can be minimized by replacing residues which differ from those in the human sequences by site directed mutagenesis of individual residues or by grating of entire complementarity determining regions.
  • Antibodies which specifically bind to SLC22A12 can contain antigen binding sites which are either partially or fully humanized, as disclosed in U.S. 5,565,332.
  • single chain antibodies can be adapted using methods known in the art to produce single chain antibodies which specifically bind to SLC22A12.
  • Antibodies with related specificity, but of distinct idiotypic composition can be generated by chain shuffling from random combinatorial immunoglobin libraries.
  • Single-chain antibodies also can be constructed using a DNA amplification method, such as PCR, using hybridoma cDNA as a template.
  • Single-chain antibodies can be mono- or bispecific, and can be bivalent or tetravalent. Construction of tetravalent, bispecific single-chain antibodies is taught.
  • a nucleotide sequence encoding a single-chain antibody can be constructed using manual or automated nucleotide synthesis, cloned into an expression construct using standard recombinant DNA methods, and introduced into a cell to express the coding sequence, as described below.
  • single-chain antibodies can be produced directly using, for example, filamentous phage technology.
  • Antibodies which specifically bind to SLC22A12 also can be produced by inducing in vivo production in the lymphocyte population or by screening immunoglobulin libraries or panels of highly specific binding reagents.
  • Other types of antibodies can be constructed and used therapeutically in methods of the invention.
  • chimeric antibodies can be constructed as disclosed in WO 93/03151.
  • Antibodies according to the invention can be purified by methods well known in the art. For example, antibodies can be affinity purified by passage over a column to which SLC22A12 is bound. The bound antibodies can then be eluted from the column using a buffer with a high salt concentration.
  • Antisense oligonucleotides are nucleotide sequences which are complementary to a specific DNA or RNA sequence. Once introduced into a cell, the complementary nucleotides combine with natural sequences produced by the cell to form complexes and block either transcription or translation.
  • an antisense ohgonucleotide is at least 11 nucleotides in length, but can be at least 12, 15, 20, 25, 30, 35, 40, 45, or 50 or more nucleotides long. Longer sequences also can be used.
  • Antisense ohgonucleotide molecules can be provided in a DNA construct and introduced into a cell as described above to decrease the level of SLC22A12 gene products in the cell.
  • Antisense oligonucleotides can be deoxyribonucleotides, ribonucleotides, or a combination of both. Oligonucleotides can be synthesized manually or by an automated synthesizer, by covalently linking the 5' end of one nucleotide with the 3' end of another nucleotide with non-phosphodiester internucleotide linkages such alkyl- phosphonates, phosphorothioates, phosphorodithioates, alkylphosphonothioates, alkylphosphonates, phosphoramidates, phosphate esters, carbamates, acetamidate, carboxymethyl esters, carbonates, and phosphate triesters.
  • Modifications of SLC22A12 gene expression can be obtained by designing antisense oligonucleotides which will form duplexes to the control, 5', or regulatory regions of the SLC22A12 gene. Oligonucleotides derived from the transcription initiation site, e.g., between positions -10 and +10 from the start site, are preferred. Similarly, inhibition can be achieved using "triple helix" base-pairing methodology. Triple helix pairing is useful because it causes inhibition ofthe ability ofthe double helix to open sufficiently for the binding of polymerases, transcription factors, or chaperons. Therapeutic advances using triplex DNA have been described in the literature [Nicholls, (1993)]. An antisense ohgonucleotide also can be designed to block translation of mRNA by preventing the transcript from binding to ribosomes.
  • Antisense oligonucleotides which comprise, for example, 2, 3, 4, or 5 or more stretches of contiguous nucleotides which are precisely complementary to a SLC22A12 polynucleotide, each separated by a stretch of contiguous nucleotides which are not complementary to adjacent SLC22A12 nucleotides, can provide sufficient targeting specificity for SLC22A12 mRNA.
  • each stretch of complementary contiguous nucleotides is at least 4, 5, 6, 7, or 8 or more nucleotides in length.
  • Non-complementary intervening sequences are preferably 1, 2, 3, or 4 nucleotides in length.
  • One skilled in the art can easily use the calculated melting point of an antisense-sense pair to determine the degree of mismatching which will be tolerated between a particular antisense ohgonucleotide and a particular SLC22A12 polynucleotide sequence.
  • Antisense oligonucleotides can be modified without affecting their ability to hybridize to a SLC22A12 polynucleotide. These modifications can be internal or at one or both ends of the antisense molecule.
  • internucleoside phosphate linkages can be modified by adding cholesteryl or diamine moieties with varying numbers of carbon residues between the amino groups and terminal ribose.
  • Modified bases and/or sugars such as arabinose instead of ribose, or a 3', 5'-substituted ohgonucleotide in which the 3' hydroxyl group or the 5' phosphate group are substituted, also can be employed in a modified antisense ohgonucleotide.
  • These modified oligonucleotides can be prepared by methods well known in the art.
  • Ribozymes are RNA molecules with catalytic activity [Uhlmann, (1987)].
  • Ribozymes can be used to inhibit gene function by cleaving an RNA sequence, as is known in the art.
  • the mechanism of ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage. Examples include engineered hammerhead motif ribozyme molecules that can specifically and efficiently catalyze endonucleolytic cleavage of specific nucleotide sequences.
  • the coding sequence of a SLC22A12 polynucleotide can be used to generate ribozymes which will specifically bind to mRNA transcribed from a SLC22A12 polynucleotide.
  • ribozymes which can cleave other RNA molecules in trans in a highly sequence specific manner have been developed and described in the art.
  • the cleavage activity of ribozymes can be targeted to specific RNAs by engineering a discrete "hybridization" region into the ribozyme.
  • the hybridization region contains a sequence complementary to the target RNA and thus specifically hybridizes with the target RNA.
  • Specific ribozyme cleavage sites within a SLC22A12 RNA target can be identified by scanning the target molecule for ribozyme cleavage sites which include the following sequences: GUA, GUU, and GUC. Once identified, short RNA sequences of between 15 and 20 ribonucleotides corresponding to the region of the target RNA containing the cleavage site can be evaluated for secondary structural features which may render the target inoperable. Suitability of candidate SLC22A12 RNA targets also can be evaluated by testing accessibility to hybridization with complementary oligonucleotides using ribonuclease protection assays. The nucleotide sequences shown in SEQ ID NO: 1 and its complement provide sources of suitable hybridization region sequences.
  • hybridizing and cleavage regions of the ribozyme can be integrally related such that upon hybridizing to the target RNA through the complementary regions, the catalytic region of the ribozyme can cleave the target.
  • Ribozymes can be introduced into cells as part of a DNA construct. Mechanical methods, such as microinjection, liposome-mediated transfection, electroporation, or calcium phosphate precipitation, can be used to introduce a ribozyme-containing DNA construct into cells in which it is desired to decrease SLC22A12 expression. Alternatively, if it is desired that the cells stably retain the DNA construct, the construct can be supplied on a plasmid and maintained as a separate element or integrated into the genome ofthe cells, as is known in the art.
  • a ribozyme-encoding DNA construct can include transcriptional regulatory elements, such as a promoter element, an enhancer or UAS element, and a transcriptional terminator signal, for controlling transcription of ribozymes in the cells (U.S. 5,641,673). Ribozymes also can be engineered to provide an additional level of regulation, so that destruction of mRNA occurs only when both a ribozyme and a target gene are induced in the cells. Screening / Screening Assays
  • Regulators as used herein refer to compounds that affect the activity of a SLC22A12 in vivo and/or in vivo. Regulators can be agonists and antagonists of a SLC22A12 polypeptide and can be compounds that exert their effect on the SLC22A12 activity via the expression, via post-translational modifications or by other means.
  • Agonists of SLC22A12 are molecules which, when bound to SLC22A12, increase or prolong the activity of SLC22A12.
  • Agonists of SLC22A12 include proteins, nucleic acids, carbohydrates, small molecules, or any other molecule which activate SLC22A12.
  • Antagonists of SLC22A12 are molecules which, when bound to SLC22A12, decrease the amount or the duration of the activity of SLC22A12. Antagonists include proteins, nucleic acids, carbohydrates, antibodies, small molecules, or any other molecule which decrease the activity of SLC22A12.
  • modulate refers to a change in the activity of SLC22A12 polypeptide. For example, modulation may cause an increase or a decrease in protein activity, binding characteristics, or any other biological, functional, or immunological properties of SLC22A12.
  • the terms “specific binding” or “specifically binding” refer to that interaction between a protein or peptide and an agonist, an antibody, or an antagonist. The interaction is dependent upon the presence of a particular structure of the protein recognized by the binding molecule (i.e., the antigenic determinant or epitope). For example, if an antibody is specific for epitope "A" the presence of a polypeptide containing the epitope A, or the presence of free unlabeled A, in a reaction containing free labeled A and the antibody will reduce the amount of labeled A that binds to the antibody.
  • the invention provides methods (also referred to herein as "screening assays") for identifying compounds which can be used for the treatment of cardiovascular diseases, reproduction diseases, urological diseases and cancer.
  • the methods entail the identification of candidate or test compounds or agents (e.g., peptides, peptidomimetics, small molecules or other molecules) which bind to SLC22A12 and/or have a stimulatory or inhibitory effect on the biological activity of SLC22A12 or its expression and then determining which of these compounds have an effect on symptoms or diseases regarding the cardiovascular diseases, reproduction diseases, urological diseases and cancer in an in vivo assay.
  • candidate or test compounds or agents e.g., peptides, peptidomimetics, small molecules or other molecules
  • Candidate or test compounds or agents which bind to SLC22A12 and/or have a stimulatory or inhibitory effect on the activity or the expression of SLC22A12 are identified either in assays that employ cells which express SLC22A12 on the cell surface (cell-based assays) or in assays with isolated SLC22A12 (cell-free assays).
  • the various assays can employ a variety of variants of SLC22A12 (e.g., full-length
  • SLC22A12 a biologically active fragment of SLC22A12, or a fusion protein which includes all or a portion of SLC22A12).
  • SLC22A12 can be derived from any suitable mammalian species (e.g., human SLC22A12, rat SLC22A12 or murine SLC22A12).
  • the assay can be a binding assay entailing direct or indirect measurement of the binding of a test compound or a known SLC22A12 ligand to
  • the assay can also be an activity assay entailing direct or indirect measurement of the activity of SLC22A12.
  • the assay can also be an expression assay entailing direct or indirect measurement of the expression of SLC22A12 mRNA or SLC22A12 protein.
  • the various screening assays are combined with an in vivo assay entailing measuring the effect of the test compound on the symptoms of cardiovascular diseases, reproduction diseases, urological diseases and cancer.
  • the invention provides assays for screening candidate or test. compounds which bind to or modulate the activity of a membrane-bound (cell surface expressed) form of SLC22A12.
  • assays can employ full-length
  • test compound can be obtained by any suitable means, e.g., from conventional compound libraries. Determining the ability ofthe test compound to bind to a membrane-bound form of SLC22A12 can be accomplished, for example, by coupling the test compound with a radioisotope or enzymatic label such that binding of the test compound to the SLC22A12 -expressing cell can be measured by detecting the labeled compound in a complex.
  • the test compound can be labelled with 125 1, 35 S, 14 C, or 3 H, either directly or indirectly, and the radioisotope detected by direct counting of radioemmission or by scintillation counting.
  • the test compound can be enzymatically labelled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label detected by determination of conversion of an appropriate substrate to product.
  • the assay comprises contacting SLC22A12 expressing cell with a known compound which binds to SLC22A12 to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with the SLC22A12 expressing cell, wherein determimng the ability of the test compound to interact with the SLC22A12 expressing cell comprises determining the ability of the test compound to preferentially bind the SLC22A12 expressing cell as compared to the known compound.
  • the assay is a cell-based assay comprising contacting a cell expressing a membrane-bound form of SLC22A12 (e.g., full-length SLC22A12, a biologically active fragment of SLC22A12, or a fusion protein which includes all or a portion of SLC22A12) expressed on the cell surface with a test compound and determining the ability of the test compound to modulate (e.g., stimulate or inhibit) the activity of the membrane-bound form of SLC22A12. Determining the ability of the test compound to modulate the activity of the membrane-bound form of SLC22A12 can be accomplished by any method suitable for measuring the activity of
  • SLC22A12 e.g., any method suitable for measuring the activity of an organic ion transporter (described in greater detail below).
  • the activity of a seven- transmembrane receptor can be measured in a number of ways, not all of which are suitable for any given receptor. Among the measures of activity are: alteration in intracellular Ca 2+ concentration, activation of phospholipase C, alteration in intracellular inositol triphosphate (IP 3 ) concentration, alteration in intracellular diacylglycerol (DAG) concentration, and alteration in intracellular adenosine cyclic 3', 5'-monophosphate (cAMP) concentration.
  • IP 3 inositol triphosphate
  • DAG diacylglycerol
  • cAMP adenosine cyclic 3', 5'-monophosphate
  • Determining the ability of the test compound to modulate the activity of SLC22A12 can be accomplished, for example, by determining the ability of SLC22A12 to bind to or interact with a target molecule.
  • the target molecule can be a molecule with which SLC22A12 binds or interacts with in nature, for example, a molecule on the surface of a cell which expresses SLC22A12, a molecule on the surface of a second cell, a molecule in the extracellular milieu, a molecule associated with the internal surface of a cell membrane or a cytoplasmic molecule.
  • the target molecule can be a component of a signal transduction pathway which facilitates transduction of an extracellular signal (e.g., a signal generated by binding of a SLC22A12 ligand, through the cell membrane and into the cell.
  • the target SLC22A12 molecule can be, for example, a second intracellular protein which has catalytic activity or a protein which facilitates the association of downstream signaling molecules with SLC22A12.
  • Determining the ability of SLC22A12 to bind to or interact with a target molecule can be accomplished by one of the methods described above for determining direct binding. In one embodiment, determining the ability of a polypeptide of the invention to bind to or interact with a target molecule can be accomplished by determining the activity ofthe target molecule.
  • the activity ofthe target molecule can be determined by detecting induction of a cellular second messenger of the target (e.g., intracellular Ca 2+ , diacylglycerol, JP 3 , etc.), detecting catalytic/enzymatic activity of the target on an appropriate substrate, detecting the induction of a reporter gene (e.g., a regulatory element that is responsive to a polypeptide of the invention operably linked to a nucleic acid encoding a detectable marker, e.g., luciferase), or detecting a cellular response.
  • a reporter gene e.g., a regulatory element that is responsive to a polypeptide of the invention operably linked to a nucleic acid encoding a detectable marker, e.g., luciferase
  • the present mvention also includes cell-free assays.
  • Such assays involve contacting a form of SLC22A12 (e.g., full-length SLC22A12, a biologically active fragment of
  • the assay includes contacting SLC22A12 with a known compound which binds SLC22A12 to forrri an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with SLC22A12, wherein determining the ability of the test compound to interact with SLC22A12 comprises determining the ability of the test compound to preferentially bind to SLC22A12 as compared to the known compound.
  • the cell-free assays of the present invention are amenable to use of either a membrane-bound form of SLC22A12 or a soluble fragment thereof.
  • a solubilizing agent such that the membrane-bound form of the polypeptide is maintained in solution.
  • solubilizing agents include but are not limited to non-ionic detergents such as n-octylglucoside, n- dodecylglucoside, n-dodecylmaltoside, octanoyl-N-methylglucamide, decanoyl-N- methylglucamide, Triton X-100, Triton X-l 14, Thesit, Isotridecypoly(ethylene glycol ether)n, 3-[(3-cholamidopropyl)dimethylamminio]-l -propane sulfonate (CHAPS), 3-
  • non-ionic detergents such as n-octylglucoside, n- dodecylglucoside, n-dodecylmaltoside, octanoyl-N-methylglucamide, decanoyl-N- methylglucamide, Triton X-100, Triton X-l
  • SLC22A12 or a SLC22A12 target molecule
  • binding of a test compound to SLC22A12, or interaction of SLC22A12 with a target molecule in the presence and absence of a candidate compound can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtitre plates, test tubes, and micro-centrifuge tubes.
  • a fusion protein can be provided which adds a domain that allows one or both ofthe proteins to be bound to a matrix.
  • glutathione-S-transferase (GST) fusion proteins or gluta- thione-S-transferase fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical; St. Louis, Mo.) or glutathione derivatized microtitre plates, which are then combined with the test compound or the test compound and either the non-adsorbed target protein or SLC22A12, and the mixture incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads or microtitre plate wells are washed to remove any unbound components and complex formation is measured either directly or indirectly, for example, as described above. Alternatively, the complexes can be dissociated from the matrix, and the level of binding or activity of SLC22A12 can be determined using standard techniques.
  • SLC22A12 or its target molecule can be immobilized utilizing conjugation of biotin and streptavidin.
  • Biotinylated polypeptide of the invention or target molecules can be prepared from biotin-NHS (N-hydroxy-succinimide) using techniques well known in the art (e.g., biotinylation kit, Pierce Chemicals; Rockford, 111.), and immobilized in the wells of streptavidin-coated plates (Pierce Chemical).
  • biotin-NHS N-hydroxy-succinimide
  • Pierce Chemicals Pierce Chemicals
  • Rockford, 111. Rockford, 111.
  • streptavidin-coated plates Piereptavidin-coated plates
  • SLC22A12 or target molecules but which do not interfere with binding of the polypeptide of the invention to its target molecule can be derivatized to the wells of the plate, and unbound target or polypeptide ofthe invention trapped in the wells by antibody conjugation.
  • Methods for detecting such complexes include immunodetection of complexes using antibodies reactive with SLC22A12 or target molecule, as well as enzyme-linked assays which rely on detecting an enzymatic activity associated with SLC22A12 or target molecule.
  • the screening assay can also involve monitoring the expression of SLC22A12.
  • regulators of expression of SLC22A12 can be identified in a method in which a cell is contacted with a candidate compound and the expression of SLC22A12 protein or mRNA in the cell is determined. The level of expression of SLC22A12 protein or mRNA the presence of the candidate compound is compared to the level of expression of SLC22A12 protein or mRNA in the absence of the candidate compound. The candidate compound can then be identified as a regulator of expression of SLC22A12 based on this comparison.
  • the candidate compound when expression of SLC22A12 protein or mRNA protein is greater (statistically significantly greater) in the presence ofthe candidate compound than in its absence, the candidate compound is identified as a stimulator of SLC22A12 protein or mRNA expression.
  • the candidate compound when expression of SLC22A12 protein or mRNA is less (statistically significantly less) in the presence ofthe candidate compound than in its absence, the candidate compound is identified as an inhibitor of SLC22A12 protein or mRNA expression.
  • the level of SLC22A12 protein or mRNA expression in the cells can be determined by methods described below.
  • the test compound is preferably a small molecule which binds to and occupies the active site of SLC22A12 polypeptide, thereby making the ligand binding site inaccessible to substrate such that normal biological activity is prevented.
  • small molecules include, but are not limited to, small peptides or peptide-like molecules.
  • Potential ligands which bind to a polypeptide of the invention include, but are not limited to, the natural ligands of known SLC22A12 ion transporter and analogues or derivatives thereof.
  • either the test compound or the SLC22A12 polypeptide can comprise a detectable label, such as a fluorescent, radioisotopic, chemiluminescent, or enzymatic label, such as horseradish peroxidase, alkaline phosphatase, or luciferase. Detection of a test compound which is bound to SLC22A12 polypeptide can then be accomplished, for example, by direct counting of radioemmission, by scintillation counting, or by determining conversion of an appropriate substrate to a detectable product. Alternatively, binding of a test compound to a SLC22A12 polypeptide can be determined without labeling either ofthe interactants.
  • a detectable label such as a fluorescent, radioisotopic, chemiluminescent, or enzymatic label, such as horseradish peroxidase, alkaline phosphatase, or luciferase.
  • a microphysiometer can be used to detect binding of a test compound with a SLC22A12 polypeptide.
  • a microphysiometer e.g., CytosensorTM
  • LAPS light-addressable potentiometric sensor
  • BIA Bimolecular Interaction Analysis
  • a SLC22A12-like polypeptide can be used as a "bait protein" in a two-hybrid assay or three-hybrid assay [Szabo, (1995); U.S. 5,283,317), to identify other proteins which bind to or interact with SLC22A12 and modulate its activity.
  • the two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains.
  • the assay utilizes two different DNA constructs.
  • polynucleotide encoding SLC22A12 can be fused to a polynucleotide encoding the DNA binding domain of a known transcription factor (e.g., GAL-4).
  • a DNA sequence that encodes an unidentified protein (“prey" or "sample” can be fused to a polynucleotide that codes for the activation domain ofthe known transcription factor.
  • the DNA-binding and activation domains of the transcription factor are brought into close proximity. This proximity allows transcription of a reporter gene (e.g., LacZ), which is operably linked to a transcriptional regulatory site responsive to the transcription factor. Expression of the reporter gene can be detected, and cell colonies containing the functional transcription factor can be isolated and used to obtain the DNA sequence encoding the protein which interacts with SLC22A12.
  • a reporter gene e.g., LacZ
  • either the SLC22A12 (or polynucleotide) or the test compound can be bound to a solid support.
  • Suitable solid supports include, but are not limited to, glass or plastic slides, tissue culture plates, microtiter wells, tubes, silicon chips, or particles such as beads (including, but not limited to, latex, polystyrene, or glass beads).
  • Any method known in the art can be used to attach SLC22A12-like polypeptide (or polynucleotide) or test compound to a solid support, including use of covalent and non-covalent linkages, passive absorption, or pairs of binding moieties attached respectively to the polypeptide (or polynucleotide) or test compound and the solid support.
  • Test compounds are preferably bound to the solid support in an anay, so that the location of individual test compounds can be tracked. Binding of a test compound to SLC22A12 (or a polynucleotide encoding for SLC22A12) can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtiter plates, test tubes, and microcentrifuge tubes.
  • SLC22A12 is a fusion protein comprising a domain that allows binding of SLC22A12 to a solid support.
  • glutathione-S-transf erase fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) or glutathione derivatized microtiter plates, which are then combined with the test compound or the test compound and the non-adsorbed SLC22A12; the mixture is then incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads or microtiter plate wells are washed to remove any unbound components. Binding of the interactants can be determined either directly or indirectly, as described above. Alternatively, the complexes can be dissociated from the solid support before binding is determined.
  • SLC22A12 or a polynucleotide encoding SLC22A12
  • a test compound can be immobilized utilizing conjugation of biotin and streptavidin.
  • Biotinylated SLC22A12 (or a poly- nucleotide encoding biotinylated SLC22A12) or test compounds can be prepared from biotin-NHS (N-hydroxysuccinimide) using techniques well known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, 111.) and immobilized in the wells of streptavidin-coated plates (Pierce Chemical).
  • antibodies which specifically bind to SLC22A12, polynucleotide, or a test compound, but which do not interfere with a desired binding site, such as the active site of SLC22A12 can be derivatized to the wells ofthe plate. Unbound target or protein can be trapped in the wells by antibody conjugation.
  • Methods for detecting such complexes include immunodetection of complexes using antibodies which specifically bind to SLC22A12 polypeptide or test compound, enzyme- linked assays which rely on detecting an activity of SLC22A12 polypeptide, and SDS gel electrophoresis under non-reducing conditions.
  • Screening for test compounds which, bind to a SLC22A12 polypeptide or polynucleotide also can be carried out in an intact cell.
  • Any cell which comprises a SLC22A12 polypeptide or polynucleotide can be used in a cell-based assay system.
  • a SLC22A12 polynucleotide can be naturally occurring in the cell or can be introduced using techniques such as those described above. Binding of the test compound to SLC22A12 or a polynucleotide encoding SLC22A12 is determined as described above.
  • Test compounds can be tested for the ability to increase or decrease SLC22A12 activity of a SLC22A12 transporter polypeptide.
  • the SLC22A12 activity can be measured, for example, using methods described in the specific examples, below.
  • SLC22A12 activity can be measured after contacting either a purified SLC22A12, a cell membrane preparation, or an intact cell with a test compound.
  • a test compound which decreases SLC22A12 activity by at least about 10, preferably about 50, more preferably about 75, 90, or 100% is identified as a potential agent for decreasing
  • a test compound which increases SLC22A12 activity by at least about 10, preferably about 50, more preferably about 75, 90, or 100% is identified as a potential agent for increasing SLC22A12 activity.
  • Such a screening procedure involves the use of melanophores which are transfected to express SLC22A12.
  • Such a screening technique is described in PCT WO 92/01810 published Feb. 6, 1992.
  • such an assay may be employed for screening for a compound which inhibits activation of the transporter polypeptide of the present invention by contacting the melanophore cells which encode the transporter with both the transporter ligand and a compound to be screened. Inhibition of the signal generated by the ligand indicates that a compound is a potential antagonist for the transporter, i.e., inhibits activation ofthe transporter.
  • the screen may be employed for identifying a compound which activates the transporter by contacting such cells with compounds to be screened and determining whether each compound generates a signal, i.e., activates the transporter.
  • Other screening techniques include the use of cells which express SLC22A12 (for example, transfected CHO cells) in a system which measures extracellular pH changes caused by transporter activation [Iwabuchi et al. (1993)].
  • compounds may be contacted with a cell which expresses the transporter polypeptide ofthe present invention and a second messenger response, e.g., signal transduction or pH changes, can be measured to determine whether the potential compound activates or inhibits the transporter.
  • Another such screening technique involves introducing RNA encoding SLC22A12 into Xenopus oocytes to transiently express the transporter.
  • the transporter oocytes can then be contacted with the transporter ligand and a compound to be screened, followed by detection of inhibition or activation of a calcium signal in the case of screening for compounds which are thought to inhibit activation ofthe transporter.
  • test compounds which increase or decrease SLC22A12 gene expression are identified.
  • the term "conelates with expression of a polynucleotide” indicates that the detection ofthe presence of nucleic acids, the same or related to a nucleic acid sequence encoding SLC22A12, by northern analysis or relatime PCR is indicative ofthe presence of nucleic acids encoding SLC22A12 in a sample, and thereby conelates with expression of the transcript from the polynucleotide encoding SLC22A12.
  • microanay refers to an anay of distinct polynucleotides or oligonucleotides arrayed on a substrate, such as paper, nylon or any other type of membrane, filter, chip, glass slide, or any other suitable solid support.
  • a SLC22A12 polynucleotide is contacted with a test compound, and the expression of an RNA or polypeptide product of SLC22A12 polynucleotide is determined.
  • the level of expression of appropriate mRNA or polypeptide in the presence of the test compound is compared to the level of expression of mRNA or polypeptide in the absence of the test compound.
  • the test compound can then be identified as a regulator of expression based on this comparison.
  • test compound when expression of mRNA or polypeptide is greater in the presence ofthe test compound than in its absence, the test compound is identified as a stimulator or enhancer ofthe mRNA or polypeptide expression.
  • test compound when expression of the mRNA or polypeptide is less in the presence of the test compound than in its absence, the test compound is identified as an inhibitor of the mRNA or polypeptide expression.
  • the level of SLC22A12 mRNA or polypeptide expression in the cells can be determined by methods well known in the art for detecting mRNA or polypeptide. Either qualitative or quantitative methods can be used.
  • the presence of polypeptide products of SLC22A12 polynucleotide can be determined, for example, using a variety of techniques known in the art, including immunochemical methods such as radioimmunoassay, Western blotting, and immunohistochemistry.
  • polypeptide synthesis can be determined in vivo, in a cell culture, or in an in vitro translation system by detecting incorporation of labelled amino acids into SLC22A12.
  • Such screening can be carried out either in a cell-free assay system or in an intact cell.
  • Any cell which expresses SLC22A12 polynucleotide can be used in a cell-based assay system.
  • the SLC22A12 polynucleotide can be naturally occurring in the cell or can be introduced using techniques such as those described above. Either a primary culture or an established cell line can be used.
  • test compounds for use in the screening assays of the invention can be obtained from any suitable source, e.g., conventional compound libraries.
  • the test compounds can also be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the "one-bead one-compound” library method; and synthetic library methods using affinity chromatography selection.
  • the biological library approach is limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds [Lam, (1997)]. Examples of methods for the synthesis of molecular libraries can be found in the art. Libraries of compounds may be presented in solution or on beads, bacteria, spores, plasmids or phage.
  • Computer modeling and searching technologies permit identification of compounds, or the improvement of already identified compounds, that can modulate SLC22A12 expression or activity. Having identified such a compound or composition, the active sites or regions are identified. Such active sites might typically be ligand binding sites, such as the interaction domain of the ligand with SLC22A12.
  • the active site can be identified using methods known in the art including, for example, from the amino acid sequences of peptides, from the nucleotide sequences of nucleic acids, or from study of complexes of the relevant compound or composition with its natural ligand. In the latter case, chemical or X-ray crystallographic methods can be used to find the active site by finding where on the factor the complexed ligand is found.
  • the three dimensional geometric structure ofthe active site is determined. This can be done by known methods, including X-ray crystallography, which can determine a complete molecular structure. On the other hand, solid or liquid phase NMR can be used to determine certain intramolecular distances. Any other experimental method of structure determination can be used to obtain partial or complete geometric structures.
  • the geometric structures may be measured with a complexed ligand, natural or artificial, which may increase the accuracy ofthe active site structure determined.
  • the methods of computer based numerical modeling can be used to complete the structure or improve its accuracy.
  • Any recognized modeling method may be used, including parameterized models specific to particular biopolymers such as proteins or nucleic acids, molecular dynamics models based on computing molecular motions, statistical mechanics models based on thermal ensembles, or combined models.
  • standard molecular force fields representing the forces between constituent atoms and groups, are necessary, and can be selected from force fields known in physical chemistry.
  • the incomplete or less accurate experimental structures can serve as constraints on the complete and more accurate structures computed by these modeling methods.
  • candidate modulating compounds can be identified by searching databases containing compounds along with information on their molecular structure. Such a search seeks compounds having structures that match the determined active site structure and that interact with the groups defining the active site. Such a search can be manual, but is preferably computer assisted. These compounds found from this search are potential SLC22A12 modulating compounds.
  • these methods can be used to identify improved modulating compounds from an already known modulating compound or ligand.
  • the composition of the known compound can be modified and the structural effects of modification can be determined using the experimental and computer modeling methods described above applied to the new composition.
  • the altered structure is then compared to the active site structure of the compound to determine if an improved fit or interaction results. In this manner systematic variations in composition, such as by varying side groups, can be quickly evaluated to obtain modified modulating compounds or ligands of improved specificity or activity.
  • Heart failure is defined as a pathophysiological state in which an abnormality of cardiac function is responsible for the failure of the heart to pump blood at a rate commensurate with the requirement of the metabolizing tissue. It includes all forms of pumping failures such as high-output and low-output, acute and chronic, right- sided or left-sided, systolic or diastolic, independent ofthe underlying cause.
  • MI Myocardial infarction
  • Ischemic diseases are conditions in which the coronary flow is restricted resulting in a perfusion which is inadequate to meet the myocardial requirement for oxygen.
  • This group of diseases includes stable angina, unstable angina and asymptomatic ischemia.
  • Arrhythmias include all forms of atrial and ventricular tachyarrhythmias, atrial tachycardia, atrial flutter, atrial fibrillation, atrio-ventricular reentrant tachycardia, preexitation syndrome, ventricular tachycardia, ventricular flutter, ventricular fibrillation, as well as bradycardic forms of arrhythmias.
  • Hypertensive vascular diseases include primary as well as all kinds of secondary arterial hypertension, renal, endocrine, neurogenic, others.
  • the genes may be used as drug targets for the treatment of hypertension as well as for the prevention of all complications arising from cardiovascular diseases.
  • Peripheral vascular diseases are defined as vascular diseases in which arterial and/or venous flow is reduced resulting in an imbalance between blood supply and tissue oxygen demand. It includes chronic peripheral arterial occlusive disease (PAOD), acute arterial thrombosis and embolism, inflammatory vascular disorders, Raynaud's phenomenon and venous disorders.
  • PAOD peripheral arterial occlusive disease
  • acute arterial thrombosis and embolism inflammatory vascular disorders
  • Raynaud's phenomenon Raynaud's phenomenon
  • Atherosclerosis is a cardiovascular disease in which the vessel wall is remodeled, compromising the lumen of the vessel.
  • the atherosclerotic remodeling process involves accumulation of cells, both smooth muscle cells and monocyte/macrophage inflammatory cells, in the intima of the vessel wall. These cells take up lipid, likely from the circulation, to form a mature atherosclerotic lesion.
  • the formation of these lesions is a chronic process, occurring over decades of an adult human life, the majority of the morbidity associated with atherosclerosis occurs when a lesion ruptures, releasing thrombogenic debris that rapidly occludes the artery. When such an acute event occurs in the coronary artery, myocardial infarction can ensue, and in the worst case, can result in death.
  • the formation of the atherosclerotic lesion can be considered to occur in five overlapping stages such as migration, lipid accumulation, recruitment of inflammatory cells, proliferation of vascular smooth muscle cells, and extracellular matrix deposition.
  • stages such as migration, lipid accumulation, recruitment of inflammatory cells, proliferation of vascular smooth muscle cells, and extracellular matrix deposition.
  • Each of these processes can be shown to occur in man and in animal models of atherosclerosis, but the relative contribution of each to the pathology and clinical significance ofthe lesion is unclear.
  • Cardiovascular diseases include but are not limited to disorders of the heart and the vascular system like congestive heart failure, myocardial infarction, ischemic diseases of the heart, all kinds of atrial and ventricular anhythmias, hypertensive vascular diseases, peripheral vascular diseases, and atherosclerosis. Too high or too low levels of fats in the bloodstream, especially cholesterol, can cause long-term problems. The risk to develop atherosclerosis and coronary artery or carotid artery disease (and thus the risk of having a heart attack or stroke) increases with the total cholesterol level increasing. Nevertheless, extremely low cholesterol levels may not be healthy.
  • disorders of lipid metabolism are hyper- lipidemia (abnormally high levels of fats (cholesterol, triglycerides, or both) in the blood, may be caused by family history of hyperlipidemia), obesity, a high-fat diet, lack of exercise, moderate to high alcohol consumption, cigarette smoking, poorly controlled diabetes, and an underactive thyroid gland), hereditary hyperlipidemias (type I hyperlipoproteinemia (familial hyperchylomicronemia), type II hyperlipo- proteinemia (familial hypercholesterolemia), type HI hyperlipoproteinemia, type TV hyperlipoproteinemia, or • type V hyperlipoproteinemia), hypolipoproteinemia, lipidoses (caused by abnormalities in the enzymes that metabolize fats), Gaucher's disease, Niemann-Pick disease, Fabry's disease, Wolman's disease, cerebrotendinous xanthomatosis, sitosterolemia, Refsum's disease, or Tay-Sachs disease.
  • hyper- lipidemia abnormal
  • Kidney disorders may lead to hypertension or hypotension. Examples for kidney problems possibly leading to hypertension are renal artery stenosis, pyelonephritis, glomerulonephritis, kidney tumors, polycistic kidney disease, injury to the kidney, or radiation therapy affecting the kidney. Excessive urination may lead to hypotension.
  • the human SLC22A12 is highly expressed in the following cardiovascular related tissues: pericardium, fetal kidney, kidney, kidney tumor. Expression in the above mentioned tissues demonstrates that the human SLC22A12 or mRNA can be utilized to diagnose of cardiovascular diseases. Additionally the activity of the human
  • SLC22A12 can be modulated to treat cardiovascular diseases.
  • the human SLC22A12 is highly expressed in kidney tissues : fetal kidney, kidney, kidney tumor. Expression in kidney tissues demonstrates that the human SLC22A12 or mRNA can be utilized to diagnose of blood pressure disorders as an cardiovascular disorder. Additionally the activity of the human SLC22A12 can be modulated to treat - but not limited to - blood pressure disorders as hypertension or hypotension.
  • Cancer disorders within the scope of this definition comprise any disease of an organ or tissue in mammals characterized by poorly controlled or uncontrolled multiplication of normal or abnormal cells in that tissue and its effect on the body as a whole.
  • Cancer diseases within the scope of the definition comprise benign neoplasms, dysplasias, hyperplasias as well as neoplasms showing metastatic growth or any other transformations like e.g. leukoplakias which often precede a breakout of cancer.
  • Cells and tissues are cancerous when they grow more rapidly than normal cells, displacing or spreading into the sunounding healthy tissue or any other tissues ofthe body described as metastatic growth, assume abnormal shapes and sizes, show changes in their nucleocytoplasmatic ratio, nuclear polychromasia, and finally may cease.
  • Cancerous cells and tissues may affect the body as a whole when causing paraneoplastic syndromes or if cancer occurs within a vital organ or tissue, normal function will be impaired or halted, with possible fatal results.
  • the ultimate involvement of a vital organ by cancer, either primary or metastatic, may lead to the death of the mammal affected. Cancer tends to spread, and the extent of its spread is usually related to an individual's chances of surviving the disease.
  • Cancers are generally said to be in one of three stages of growth: early, or localized, when a tumor is still confined to the tissue of origin, or primary site; direct extension, where cancer cells from the tumour have invaded adjacent tissue or have spread only to regional lymph nodes; or metastasis, in which cancer cells have migrated to distant parts of the body from the primary site, via the blood or lymph systems, and have established secondary sites of infection.
  • Cancer is said to be malignant because of its tendency to cause death if not treated. Benign tumors usually do not cause death, although they may if they interfere with a normal body function by virtue of their location, size, or paraneoplastic side effects. Hence benign tumors fall under the definition of cancer within the scope of this definition as well.
  • cancer cells divide at a higher rate than do normal cells, but the distinction between the growth of cancerous and normal tissues is not so much the rapidity of cell division in the former as it is the partial or complete loss of growth restraint in cancer cells and their failure to differentiate into a useful, limited tissue of the type that characterizes the functional equilibrium of growth of normal tissue.
  • Cancer tissues may express certain molecular receptors and probably are influenced by the host's susceptibility and immunity and it is known that certain cancers ofthe breast and prostate, for example, are considered dependent on specific hormones for their existence.
  • the term "cancer" under the scope of the definition is not limited to simple benign neoplasia but comprises any other benign and malign neoplasia like 1) Carcinoma, 2) Sarcoma, 3)
  • Carcinosarcoma 4) Cancers of the blood-forming tissues, 5) tumors of nerve tissues including the brain, 6) cancer of skin cells. Cancer according to 1) occurs in epithelial tissues, which cover the outer body (the skin) and line mucous membranes and the inner cavitary structures of organs e.g. such as the breast, lung, the respiratory and gastrointestinal tracts, the endocrine glands, and the genitourinary system. Ductal or glandular elements may persist in epithelial tumors, as in adenocarcinomas like e.g. thyroid adenocarcinoma, gastric adenocarcinoma, uterine adenocarcinoma.
  • adenocarcinomas like e.g. thyroid adenocarcinoma, gastric adenocarcinoma, uterine adenocarcinoma.
  • Cancers of the pavement-cell epithelium of the skin and of certain mucous membranes may be termed epidermoid or squamous-cell carcinomas ofthe respective tissues and are in the scope of the definition of cancer as well.
  • Cancer according to 2) develops in connective tissues, including fibrous tissues, adipose (fat) tissues, muscle, blood vessels, bone, and cartilage like e.g. osteogenic sarcoma; liposarcoma, fibrosarcoma, synovial sarcoma.
  • Cancer according to 3 is cancer that develops in both epithelial and connective tissue.
  • Cancer disease within the scope of this definition may be primary or secondary, whereby primary indicates that the cancer originated in the tissue where it is found rather than was established as a secondary site through metastasis from another lesion.
  • Cancers and tumor diseases within the scope of this definition may be benign or malign and may affect all anatomical structures of the body of a mammal.
  • they comprise cancers and tumor diseases of I) the bone manow and bone marrow derived cells (leukemias), H) the endocrine and exocrine glands like e.g. thyroid, parathyroid, pituitary, adrenal glands, salivary glands, pancreas 111) the breast, like e.g.
  • malignant osteogenic sarcoma benign osteoma, cartilage tumors; like malignant chondrosarcoma or benign chondroma; bone manow tumors like malignant myeloma or benign eosinophilic granuloma, as well as metastatic tumors from bone tissues at other locations of the body;
  • X) the mouth, throat, larynx, and the esophagus XI) the urinary bladder and the internal and external organs and structures of the urogenital system of male and female like ovaries, uterus, cervix of the uterus, testes, and prostate gland, XII) the prostate, XTTT) the pancreas, like ductal carcinoma of the pancreas;
  • XIV) the lymphatic tissue like lymphomas and other tumors of lymphoid origin, XV) the skin,
  • XVI) cancers and tumor diseases of all anatomical structures belonging to the respiration and respiratory systems including thoracal muscles and linings, XVLT) primary or secondary cancer of the lymph nodes XVTTT) the tongue and of the bony structures of the hard palate or sinuses, XVTV) the mouth, cheeks, neck and salivary glands, XX) the blood vessels including the heart and their linings, XXI) the smooth or skeletal muscles and their ligaments and linings, XXIl) the peripheral, the autonomous, the central nervous system including the cerebellum, XXIIJ) the adipose tissue.
  • the human SLC22A12 is highly expressed in the following cancer tissues: kidney tumor.
  • the expression in the above mentioned tissues and in particular the differential expression between diseased tissue kidney tumor and healthy tissue kidney demonstrates that the human SLC22A12 or mRNA can be utilized to diagnose of cancer. Additionally the activity of the human SLC22A12 can be modulated to treat cancer. Disorders Related to Urology
  • Genitourinary disorders comprise benign and malign disorders of the organs constituting the genitourinary system of female and male, renal diseases like acute or chronic renal failure, immimologically mediated renal diseases like renal transplant rejection, lupus nephritis, immune complex renal diseases, glomerulopathies, nephritis, toxic nephropathy, obstructive uropathies like benign prostatic hyperplasia (BPH), neurogenic bladder syndrome, urinary incontinence like urge-, stress-, or overflow incontinence, pelvic pain, and erectile dysfunction.
  • renal diseases like acute or chronic renal failure
  • immimologically mediated renal diseases like renal transplant rejection, lupus nephritis, immune complex renal diseases, glomerulopathies, nephritis, toxic nephropathy, obstructive uropathies like benign prostatic hyperplasia (BPH), neurogenic bladder syndrome, urinary incontinence like urge-, stress-, or
  • the human SLC22A12 is highly expressed in the following urological tissues: fetal kidney, kidney, kidney tumor.
  • the expression in the above mentioned tissues demonstrates that the human SLC22A12 or mRNA can be utilized to diagnose of urological disorders. Additionally the activity of the human SLC22A12 can be modulated to treat urological disorders.
  • disorders of the male reproductive system include but are not limited to balanoposthitis, balanitis xerotica obliterans, phimosis, paraphimosis, erythroplasia of Queyrat, skin cancer of the penis, Bowen's and Paget's diseases, syphilis, herpes simplex infections, genital warts, molluscum contagiosum, priapism, peyronie's disease, benign prostatic hyperplasia (BPH), prostate cancer, prostatitis, testicular cancer, testicular torsion, inguinal hernia, epididymo-orchitis, mumps, hydroceles, spermatoceles, or varicoceles.
  • Impotence may results from vascular impairment, neurologic disorders, drugs, abnormalities of the perns, or psychologic problems.
  • disorders of the female reproductive include premature menopause, pelvic pain, vaginitis, vulvitis, vulvovaginitis, pelvic inflammatory disease, fibroids, menstrual disorders (premenstrual syndrome (PMS), dysmenonhea, amenonhea, primary amenonhea, secondary amenonhea, menonhagia, hypomenonhea, poly- menonhea, oligomenonhea, metronhagia, menometronhagia, Postmenopausal bleeding), bleeding caused by a physical disorder, dysfunctional uterine bleeding, polycystic ovary syndrome (Stein-Leventhal syndrome), endometriosis, cancer ofthe uterus, cancer of the cervix, cancer of the ovaries, cancer of the vulva, cancer of the vagina, cancer ofthe fallopian tubes, or hydatidiform mole.
  • PMS menstrual syndrome
  • dysmenonhea amenonhea, primary amenonhe
  • Infertility may be caused by problems with sperm, ovulation, the fallopian tubes, and the cervix as well as unidentified factors.
  • Complications of pregnancy include miscarriage and stillbirth, ectopic pregnancy, anemia, Rh incompatibility, problems with the placenta, excessive vomiting, preeclampsia, eclampsia, and skin rashes (e.g. herpes gestationis, urticaria of pregnancy) as well as preterm labor and premature rupture ofthe membranes.
  • Breast disorders may be noncancerous (benign) or cancerous (malignant). Examples of breast disorders are but are not limited to breast pain, cysts, fibrocystic breast disease, fibrous lumps, nipple discharge, breast infection, breast cancer (ductal carcinoma, lobular carcinoma, medullary carcinoma, tubular carcinoma, and inflammatory breast cancer), Paget's disease ofthe nipple or Cystosarcoma phyllodes.
  • the human SLC22A12 is highly expressed in the following tissues of the reproduction system: testis, breast.
  • the expression in the above mentioned tissues demonstrates that the human SLC22A12 or mRNA can be utilized to diagnose of reproduction disorders. Additionally the activity of the human SLC22A12 can be modulated to treat reproduction disorders.
  • the present invention provides for both prophylactic and therapeutic methods for cardiovascular diseases, reproduction diseases, urological diseases and cancer.
  • the regulatory method of the invention involves contacting a cell with an agent that modulates one or more of the activities of SLC22A12.
  • An agent that modulates activity can be an agent as described herein, such as a nucleic acid or a protein, a naturally-occurring cognate ligand ofthe polypeptide, a peptide, a peptidomimetic, or any small molecule.
  • the agent stimulates one or more of the biological activities of SLC22A12. Examples of such stimulatory agents include the active SLC22A12 and nucleic acid molecules encoding a portion of SLC22A12.
  • the agent inhibits one or more of the biological activities of SLC22A12. Examples of such inhibitory agents include antisense nucleic acid molecules and antibodies.
  • the present invention provides methods of treating an individual afflicted with a disease or disorder characterized by unwanted expression or activity of SLC22A12 or a protein in the SLC22A12 signaling pathway.
  • the method involves administering an agent like any agent identified or being identifiable by a screening assay as described herein, or combination of such agents that modulate say upregulate or downregulate the expression or activity of SLC22A12 or of any protein in the SLC22A12 signaling pathway.
  • the method involves administering a regulator of SLC22A12 as therapy to compensate for reduced or undesirably low expression or activity of SLC22A12 or a protein in the SLC22A12 signaling pathway.
  • Stimulation of activity or expression of SLC22A12 is desirable in situations in which activity or expression is abnormally low and in which increased activity is likely to have a beneficial effect. Conversely, inhibition of activity or expression of SLC22A12 is desirable in situations in which activity or expression of SLC22A12 is abnormally high and in which decreasing its activity is likely to have a beneficial effect.
  • This invention is further illustrated by the following examples which should not be construed as limiting. The contents of all references, patents and published patent applications cited throughout this application are hereby incorporated by reference.
  • This invention further pertains to novel agents identified by the above-described screening assays and uses thereof for treatments as described herein.
  • compositions suitable for administration typically comprise the nucleic acid molecule, protein, or antibody and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical admimstration.
  • pharmaceutically acceptable carrier is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical admimstration.
  • the use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conven- tional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.
  • the invention includes pharmaceutical compositions comprising a regulator of SLC22A12 expression or activity (and/or a regulator ofthe activity or expression of a protein in the SLC22A12 signaling pathway) as well as methods for preparing such compositions by combining one or more such regulators and a pharmaceutically acceptable carrier.
  • pharmaceutical compositions comprising a regulator identified using the screening assays of the invention packaged with instructions for use.
  • the instructions would specify use of the pharmaceutical composition for treatment of cardiovascular diseases, reproduction diseases, urological diseases and cancer.
  • regulators that are agonists of SLC22A12 activity or increase SLC22A12 expression the instructions would specify use of the pharmaceutical composition for treatment of cardiovascular diseases, re- production diseases, urological diseases and cancer.
  • An antagonist of SLC22A12 may be produced using methods which are generally known in the art.
  • purified SLC22A12 may be used to produce antibodies or to screen libraries of pharmaceutical agents to identify those which specifically bind SLC22A12.
  • Antibodies to SLC22A12 may also be generated using methods that are well known in the art.
  • Such antibodies may include, but are not limited to, polyclonal, monoclonal, chimeric, single chain antibodies, Fab fragments, and fragments produced by a Fab expression library. Neutralizing antibodies like those which inhibit dimer formation are especially prefened for therapeutic use.
  • the polynucleotides encoding SLC22A12, or any fragment or complement thereof may be used for therapeutic purposes.
  • the complement of the polynucleotide encoding SLC22A12 may be used in situations in which it would be desirable to block the transcription of the mRNA.
  • cells may be transformed with sequences complementary to polynucleotides encoding SLC22A12.
  • complementary molecules or fragments may be used to modulate SLC22A12 activity, or to achieve regulation of gene function.
  • sense or antisense oligonucleotides or larger fragments can be designed from various locations along the coding or control regions of sequences encoding SLC22A12.
  • Expression vectors derived from retroviruses, adenoviruses, or herpes or vaccinia viruses, or from various bacterial plasmids may be used for delivery of nucleotide sequences to the targeted organ, tissue, or cell population. Methods which are well known to those skilled in the art can be used to construct vectors which will express nucleic acid sequence complementary to the polynucleotides of the gene encoding SLC22A12. These techniques are described, for example, in [Scott and Smith (1990) Science 249:386-390].
  • any of the therapeutic methods described above may be applied to any subject in need of such therapy, including, for example, mammals such as dogs, cats, cows, horses, rabbits, monkeys, and most preferably, humans.
  • An additional embodiment of the invention relates to the administration of a pharmaceutical composition containing SLC22A12 in conjunction with a pharma- ceutically acceptable carrier, for any ofthe therapeutic effects discussed above.
  • Such pharmaceutical compositions may consist of SLC22A12, antibodies to SLC22A12, and mimetics, agonists, antagonists, or inhibitors of SLC22A12.
  • the compositions may be administered alone or in combination with at least one other agent, such as a stabilizing compound, which may be admimstered in any sterile, biocompatible pharmaceutical carrier including, but not limited to, saline, buffered saline, dextrose, and water.
  • the compositions may be administered to a patient alone, or in combination with other agents, drugs or hormones.
  • a pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration.
  • routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration.
  • Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylene- diaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
  • the parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
  • compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
  • suitable carriers include physiological saline, bacterio- static water, Cremophor EMTM (BASF, Parsippany, NJ.) or phosphate buffered saline (PBS).
  • the composition must be sterile and should be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, a pharmaceutically acceptable polyol like glycerol, propylene glycol, liquid polyetheylene glycol, and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition.
  • Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions can be prepared by incorporating the active compound (e.g., a polypeptide or antibody) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above, hi the case of sterile powders for the preparation of sterile injectable solutions, the prefened methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed.
  • compositions can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
  • a binder such as microcrystalline cellulose, gum tragacanth or gelatin
  • an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch
  • a lubricant such as magnesium stearate or sterotes
  • a glidant such as colloidal silicon dioxide
  • a sweetening agent such as sucrose or saccharin
  • the compounds are delivered in the form of an aerosol spray from a pressurized container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.
  • a suitable propellant e.g., a gas such as carbon dioxide, or a nebulizer.
  • Systemic administration can also be by transmucosal or transdermal means.
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives.
  • Transmucosal administration can be accomplished through the use of nasal sprays or suppositories.
  • the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.
  • the compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.
  • the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.
  • a controlled release formulation including implants and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid.
  • Liposomal suspensions including liposomes targeted to infected cells with monoclonal antibodies to viral antigens
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • the specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.
  • the pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.
  • the instructions for administration will specify use ofthe composition for cardiovascular diseases, reproduction diseases, urological diseases and cancer.
  • the instructions for administration will specify use of the composition for cardiovascular diseases, reproduction diseases, urological diseases and cancer.
  • antibodies which specifically bind SLC22A12 may be used for the diagnosis of disorders characterized by the expression of SLC22A12, or in assays to monitor patients being treated with SLC22A12 or agonists, antagonists, and inhibitors of SLC22A12.
  • Antibodies useful for diagnostic purposes may be prepared in the same manner as those described above for therapeutics. Diagnostic assays for SLC22A12 include methods which utilize the antibody and a label to detect SLC22A12 in human body fluids or in extracts of cells or tissues.
  • the antibodies may be used with or without modification, and may be labeled by covalent or non- covalent joining with a reporter molecule.
  • a wide variety of reporter molecules, several of which are described above, are known in the art and may be used.
  • SLC22A12 A variety of protocols for measuring SLC22A12, including ELISAs, RIAs, and FACS, are known in the art and provide a basis for diagnosing altered or abnormal levels of SLC22A12 expression.
  • Normal or standard values for SLC22A12 expression are established by combining body fluids or cell extracts taken from normal mammalian subjects, preferably human, with antibody to SLC22A12 under conditions suitable for complex formation The amount of standard complex formation may be quantified by various methods, preferably by photometric means.
  • the polynucleotides encoding SLC22A12 may be used for diagnostic purposes.
  • the polynucleotides which may be used include ohgonucleotide sequences, complementary RNA and DNA molecules, and PNAs.
  • the polynucleotides may be used to detect and quantitate gene expression in biopsied tissues in which expression of SLC22A12 may be conelated with disease.
  • the diagnostic assay may be used to distinguish between absence, presence, and excess expression of SLC22A12, and to monitor regulation of SLC22A12 levels during therapeutic intervention.
  • Polynucleotide sequences encoding SLC22A12 may be used for the diagnosis of cardiovascular diseases, reproduction diseases, urological diseases and cancer associated with expression of SLC22A12.
  • SLC22A12 may be used in Southern, Northern, or dot-blot analysis, or other membrane-based technologies; in PCR technologies; in dipstick, pin, and ELISA assays; and in microanays utilizing fluids or tissues from patient biopsies to detect altered SLC22A12 expression. Such qualitative or quantitative methods are well known in the art.
  • the nucleotide sequences encoding SLC22A12 may be useful in assays that detect the presence of associated disorders, particularly those mentioned above.
  • the nucleotide sequences encoding SLC22A12 may be labelled by standard methods and added to a fluid or tissue sample from a patient under conditions suitable for the formation of hybridization complexes. After a suitable incubation period, the sample is washed and the signal is quantitated and compared with a standard value.
  • nucleotide sequences have hybridized with nucleotide sequences in the sample, and the presence of altered levels of nucleotide sequences encoding SLC22A12 in the sample indicates the presence of the associated disorder.
  • assays may also be used to evaluate the efficacy of a particular therapeutic treatment regimen in animal studies, in clinical trials, or in monitoring the treatment of an individual patient.
  • a normal or standard profile for expression is established. This may be accomplished by combining body fluids or cell extracts taken from normal subjects, either animal or human, with a sequence, or a fragment thereof, encoding SLC22A12, under conditions suitable for hybridization or amplification. Standard hybridization may be quantified by comparing the values obtained from normal subjects with values from an experiment in which a known amount of a substantially purified polynucleotide is used. Standard values obtained from normal samples may be compared with values obtained from samples from patients who are symptomatic for a disorder. Deviation from standard values is used to establish the presence of a disorder.
  • Another technique for drug screening which may be used provides for high throughput screening of compounds having suitable binding affinity to the protein of interest as described in published PCT application WO84/03564.
  • large numbers of different small test compounds are synthesized on a solid substrate, such as plastic pins or some other surface.
  • the test compounds are reacted with SLC22A12, or fragments thereof, and washed. Bound SLC22A12 is then detected by methods well known in the art.
  • Purified SLC22A12 can also be coated directly onto plates for use in the aforementioned drug screening techniques.
  • non- neutralizing antibodies can be used to capture the peptide and immobilize it on a solid support.
  • a therapeutically effective dose refers to that amount of active ingredient which increases or decreases SLC22A12 activity relative to SLC22A12 activity which occurs in the absence ofthe therapeutically effective dose.
  • the therapeutically effective dose can be estimated initially either in cell culture assays or in animal models, usually mice, rabbits, dogs, or pigs. The animal model also can be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.
  • Therapeutic efficacy and toxicity e.g., ED 50 (the dose therapeutically effective in 50% of the population) and LD 50 (the dose lethal to 50% of the population), can be determined by standard pharmaceutical procedures in cell cultures or experimental animals.
  • the dose ratio of toxic to therapeutic effects is the therapeutic index, and it can be expressed as the ratio, LD 50 /ED 5 o.
  • Pharmaceutical compositions which exhibit large therapeutic indices are prefened. The data obtained from cell culture assays and animal studies is used in formulating a range of dosage for human use.
  • the dosage contained in such compositions is preferably within a range of circulating concentrations that include the ED 50 with little or no toxicity. The dosage varies within this range depending upon the dosage form employed, sensitivity of the patient, and the route of administration.
  • Dosage and administration are adjusted to provide sufficient levels ofthe active ingredient or to maintain the desired effect. Factors which can be taken into account include the severity of the disease state, general health of the subject, age, weight, and gender of the subject, diet, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy. Long-acting pharmaceutical compositions can be administered every 3 to 4 days, every week, or once every two weeks depending on the half-life and clearance rate ofthe particular formulation.
  • Normal dosage amounts can vary from 0.1 micrograms to 100,000 micrograms, up to a total dose of about 1 g, depending upon the route of administration.
  • Guidance as to particular dosages and methods of delivery is provided in the literature and generally available to practitioners in the art. Those skilled in the art will employ different formulations for nucleotides than for proteins or their inhibitors. Similarly, delivery of polynucleotides or polypeptides will be specific to particular cells, conditions, locations, etc.
  • polynucleotides encoding the antibody can be constructed and introduced into a cell either ex vivo or in vivo using well-established techniques including, but not limited to, transferrin-polycation- mediated DNA transfer, transfection with naked or encapsulated nucleic acids, liposome-mediated cellular fusion, intracellular transportation of DNA-coated latex beads, protoplast fusion, viral infection, electroporation, "gene gun", and DEAE- or calcium phosphate-mediated transfection.
  • the reagent is preferably an antisense oligo- nucleotide or a ribozyme.
  • Polynucleotides which express antisense oligonucleotides or ribozymes can be introduced into cells by a variety of methods, as described above.
  • a reagent reduces expression of SLC22A12 gene or the activity of SLC22A12 by at least about 10, preferably about 50, more preferably about 75, 90, or 100% relative to the absence of the reagent. The effectiveness of the mechanism chosen to decrease the level of expression of SLC22A12 gene or the activity of
  • SLC22A12 can be assessed using methods well known in the art, such as hybridization of nucleotide probes to SLC22A12-specific mRNA, quantitative RT- PCR, immunologic detection of SLC22A12, or measurement of SLC22A12 activity.
  • any ofthe pharmaceutical compositions of the invention can be administered in combination with other appropriate therapeutic agents. Selection of the appropriate agents for use in combination therapy can be made by one of ordinary skill in the art, according to conventional pharmaceutical principles.
  • the combination of therapeutic agents can act synergistically to effect the treatment or prevention of the various disorders described above. Using this approach, one may be able to achieve therapeutic efficacy with lower dosages of each agent, thus reducing the potential for adverse side effects.
  • Any of the therapeutic methods described above can be applied to any subject in need of such therapy, including, for example, mammals such as dogs, cats, cows, horses, rabbits, monkeys, and most preferably, humans.
  • Nucleic acid molecules of the invention are those nucleic acid molecules which are contained in a group of nucleic acid molecules consisting of (i) nucleic acid molecules encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 2, (ii) nucleic acid molecules comprising the sequence of SEQ ID NO: 1, (iii) nucleic acid molecules having the sequence of SEQ ID NO: 1, (iv)nucleic acid molecules the complementary strand of which hybridizes under stringent conditions to a nucleic acid molecule of (i), (ii), or (iii); and (v) nucleic acid molecules the sequence of which differs from the sequence of a nucleic acid molecule of (iii) due to the degeneracy of the genetic code, wherein the polypeptide encoded by said nucleic acid molecule has SLC22A12 activity.
  • Polypeptides of the invention are those polypeptides which are contained in a group of polypeptides consisting of (i) polypeptides having the sequence of SEQ ID NO: 2, (ii) polypeptides comprising the sequence of SEQ ID NO: 2, (iii) polypeptides encoded by nucleic acid molecules ofthe invention and (iv) polypeptides which show at least 99%, 98%, 95%, 90%, or 80% homology with a polypeptide of (i), (ii), or (iii), wherein said purified polypeptide has SLC22A12 activity.
  • An object of the invention is a method of screening for therapeutic agents useful in the treatment of a disease comprised in a group of diseases consisting of cardiovascular diseases, reproduction diseases, urological diseases and cancer in a mammal comprising the steps of (i) contacting a test compound with a SLC22A12 polypeptide, (ii) detect binding of said test compound to said SLC22A12 polypeptide.
  • a test compound with a SLC22A12 polypeptide
  • detect binding of said test compound to said SLC22A12 polypeptide are identified potential therapeutic agents for such a disease.
  • Another object ofthe invention is a method of screening for therapeutic agents useful in the treatment of a disease comprised in a group of diseases consisting of cardiovascular diseases, reproduction diseases, urological diseases and cancer in a mammal comprising the steps of (i) determining the activity of a SLC22A12 polypeptide at a certain concentration of a test compound or in the absence of said test compound, (ii) determining the activity of said polypeptide at a different concentration of said test compound.
  • compounds that lead to a difference in the activity of the SLC22A12 polypeptide in (i) and (ii) are identified potential therapeutic agents for such a disease.
  • Another object ofthe invention is a method of screening for therapeutic agents useful in the treatment of a disease comprised in a group of diseases consisting of cardiovascular diseases, reproduction diseases, urological diseases and cancer in a mammal comprising the steps of (i) determining the activity of a SLC22A12 polypeptide at a certain concentration of a test compound, (ii) determimng the activity of a SLC22A12 polypeptide at the presence of a compound known to be a regulator of a SLC22A12 polypeptide.
  • compounds that show similar effects on the activity of the SLC22A12 polypeptide in (i) as compared to compounds used in (ii) are identified potential therapeutic agents for such a disease.
  • test compound displaces a ligand which is first bound to the polypeptide.
  • Another object ofthe invention is a method of screening for therapeutic agents useful in the treatment of a disease comprised in a group of diseases consisting of cardiovascular diseases, reproduction diseases, urological diseases and cancer in a mammal comprising the steps of (i) contacting a test compound with a SLC22A12 polynucleotide, (ii) detect binding of said test compound to said SLC22A12 polynucleotide.
  • Compounds that, e.g., bind to the SLC22A12 polynucleotide are potential therapeutic agents for the treatment of such diseases.
  • Another object of the invention is the method of the above, wherein the nucleic acid molecule is RNA.
  • Another object ofthe invention is a method ofthe above, wherein the contacting step is in or at the surface of a cell. Another object of the invention is a method ofthe above, wherein the contacting step is in a cell-free system.
  • Another object ofthe invention is a method ofthe above, wherein the polynucleotide is coupled to a detectable label.
  • Another object ofthe invention is a method ofthe above, wherein the test compound is coupled to a detectable label.
  • Another object of the invention is a method of diagnosing a disease comprised in a group of diseases consisting of cardiovascular diseases, reproduction diseases, urological diseases and cancer in a mammal comprising the steps of (i) determining the amount of a SLC22A12 polynucleotide in a sample taken from said mammal, (ii) determining the amount of SLC22A12 polynucleotide in healthy and/or diseased mammal.
  • a disease is diagnosed, e.g., if there is a substantial similarity in the amount of SLC22A12 polynucleotide in said test mammal as compared to a diseased mammal.
  • Another object of the invention is a pharmaceutical composition for the treatment of a disease comprised in a group of diseases consisting of cardiovascular diseases, reproduction diseases, urological diseases and cancer in a mammal comprising a therapeutic agent which binds to a SLC22A12 polypeptide.
  • Another object of the invention is a pharmaceutical composition for the treatment of a disease comprised in a group of diseases consisting of cardiovascular diseases, reproduction diseases, urological diseases and cancer in a mammal comprising a therapeutic agent which regulates the activity of a SLC22A12 polypeptide.
  • Another object of the invention is a pharmaceutical composition for the treatment of a disease comprised in a group of diseases consisting of cardiovascular diseases, reproduction diseases, urological diseases and cancer in a mammal comprising a therapeutic agent which regulates the activity of a SLC22A12 polypeptide, wherein said therapeutic agent is (i) a small molecule, (ii) an RNA molecule, (iii) an antisense ohgonucleotide, (iv) a polypeptide, (v) an antibody, or (vi) a ribozyme.
  • Another object ofthe invention is a pharmaceutical composition for the treatment of a disease comprised in a group of diseases consisting of cardiovascular diseases, reproduction diseases, urological diseases and cancer in a mammal comprising a SLC22A12 polynucleotide.
  • Another object of the invention is a pharmaceutical composition for the treatment of a disease comprised in a group of diseases consisting of cardiovascular diseases, reproduction diseases, urological diseases and cancer in a mammal comprising a SLC22A12 polypeptide.
  • Another object of the invention is the use of regulators of a SLC22A12 for the preparation of a pharmaceutical composition for the treatment of a disease comprised in a group of diseases consisting of cardiovascular diseases, reproduction diseases, urological diseases and cancer in a mammal.
  • Another object of the invention is a method for the preparation of a pharmaceutical composition useful for the treatment of a disease comprised in a group of diseases consisting of cardiovascular diseases, reproduction diseases, urological diseases and cancer in a mammal comprising the steps of (i) identifying a regulator of SLC22A12, (ii) determimng whether said regulator ameliorates the symptoms of a disease comprised in a group of diseases consisting of cardiovascular diseases, reproduction diseases, urological diseases and cancer in a mammal; and (iii) combining of said regulator with an acceptable pharmaceutical carrier.
  • Another object of the invention is the use of a regulator of SLC22A12 for the regulation of SLC22A12 activity in a mammal having a disease comprised in a group of diseases consisting of cardiovascular diseases, reproduction diseases, urological diseases and cancer.
  • SLC22A12 for diagnosis and modulation of urological diseases and cardiovascular diseases. Furthermore the above described expression suggest a - but not limited to - utilization SLC22A12 to reproduction diseases and cancer.
  • Example 1 Search for homologous sequences in public sequence data bases
  • the degree of homology can readily be calculated by known methods. Prefened methods to determine homology are designed to give the largest match between the sequences tested. Methods to determine homology are codified in publicly available computer programs such as BestFit, BLASTP, BLASTN, and FASTA. The BLAST programs are publicly available from NCBI and other sources in the internet.
  • RNA from each cell or tissue source was first reverse transcribed. 85 ⁇ g of total RNA was reverse transcribed using 1 ⁇ mole random hexamer primers, 0.5 mM each of dATP, dCTP, dGTP and dTTP (Qiagen, Hilden, Germany), 3000 U RnaseQut (Invitrogen, Groningen, Netherlands) in a final volume of 680 ⁇ l.
  • the first strand synthesis buffer and Omniscript reverse transcriptase (2 u/ ⁇ l) were from (Qiagen, Hilden,
  • the reaction was incubated at 37°C for 90 minutes and cooled on ice. The volume was adjusted to 6800 ⁇ l with water, yielding a final concentration of 12.5 ng ⁇ l of starting RNA.
  • SLC22A12 were designed using the Perkin Elmer ABI Primer ExpressTM software and were synthesized by TibMolBiol (Berlin, Germany).
  • the SLC22A12 forward primer sequence was: Primerl (SEQ ID NO: 3).
  • the SLC22A12 reverse primer sequence was Primer2 (SEQ ID NO: 4).
  • Probel SEQ ID NO: 5
  • FAM carboxyfluorescein succinimidyl ester
  • SLC22A12 (carboxytetramethylrhodamine) as the quencher, is used as a probe for SLC22A12.
  • the following reagents were prepared in a total of 25 ⁇ l : lx TaqMan buffer A, 5.5 mM MgCl 2 , 200 nM of dATP, dCTP, dGTP, and dUTP, 0.025 U/ ⁇ l AmpliTaq GoldTM, 0.01 U/ ⁇ l AmpErase and Probel (SEQ ID NO: 4), SLC22A12 forward and reverse primers each at 200 nM, 200 nM SLC22A12 FAM/TAMRA-labelled probe, and 5 ⁇ l of template cDNA. Thermal cycling parameters were 2 min at 50°C, followed by 10 min at 95°C, followed by 40 cycles of melting at 95°C for 15 sec and annealing/extending at 60°C for 1 min.
  • the CT (threshold cycle) value is calculated as described in the "Quantitative determination of nucleic acids" section.
  • the CF- value (factor for threshold cycle correction) is calculated as follows :
  • PCR reactions were set up to quantitate the housekeeping genes (HKG) for each cDNA sample.
  • CTnK G -values were calculated as described in the "Quantitative determination of nucleic acids" section.
  • CTHK G -n-mean value (CT HKGI -value + CT HKG2 -value +... + CT ⁇ KG - n -value) / n
  • CTpannei mean value (CT mean value of all HKG in all tested cDNAs)
  • CT C D NA -n CT value ofthe tested gene for the cDNA n
  • CF C DNA- ⁇ CT cor -cDNA-n (conected CT value for a gene on cDNA n)
  • SLC22A12 The expression of SLC22A12 was investigated in the tissues listed in table 1.
  • Table 1 Relative expression ofSLC22A12 in various human tissues.
  • Purkinj e fibers 1 interventricular septum 1 fetal aorta 1 aorta 1 aorta sclerotic 1 artery 1 coronary artery 1 coronary artery sclerotic 1 pulmonary artery 1 carotid artery 1 mesenteric artery 1 vein 1 pulmonic valve 1 coronary artery endothel cells 1 coronary artery smooth muscle primary cells 1 aortic smooth muscle cells 1 pulmonary artery smooth muscle cells 1 aortic endothel cells 1
  • HEP G2 cells 1 leukocytes (peripheral blood) 1
  • HEK CNS + APP 1 neural progenitor cells 1 astrocytes 1 retina 1 fetal lung 1 fetal lung fibroblast IMR-90 cells 1 fetal lung fibroblast MRC-5 cells 1 lung 1 lung right upper lobe 1 lung right mid. lobe 1 lung right lower lobe 1 lung lupus disease 1 lung tumor 1 lung COPD 1 trachea 1 primary bronchia 1 secondary bronchia 1 bronchial epithelial cells 1 bronchial smooth muscle cells 1 small airway epithelial cells 1 cervix 1 testis 14
  • HeLa cells (cervix tumor) 1 placenta 1 uterus 1 uterus tumor 1 ovary 1 ovary tumor 1 breast 6 breast tumor 1
  • MDA MB 231 cells (breast tumor) 1 mammary gland 1 prostate 1 prostate BPH 1 prostate tumor 1 bladder 1 ureter 1 penis 1 corpus cavernosum 1 fetal kidney 307 kidney 17439 kidney tumor 1 renal epithelial cells 1
  • Oligonucleotides, cDNA or genomic fragments comprising the antisense strand of a polynucleotide coding for SLC22A12 are used either in vitro or in vivo to inhibit translation of the mRNA.
  • Such technology is how well known in the art, and antisense molecules can be designed at various locations along the nucleotide sequences.
  • the gene of interest is effectively turned off.
  • the function of the gene is ascertained by observing behavior at the intracellular, cellular, tissue or organismal level (e.g., lethality, loss of differentiated function, changes in morphology, etc.).
  • modifications of gene expression is obtained by designing antisense sequences to intron regions, promoter/enhancer elements, or even to transacting regulatory genes.
  • Expression of SLC22A12 is accomplished by subcloning the cDNAs into appropriate expression vectors and transfecting the vectors into expression hosts such as, e.g., E. coli.
  • the vector is engineered such that it contains a promoter for
  • /3-galactosidase upstream of the cloning site, followed by sequence containing the amino-terminal Methionine and the subsequent seven residues of /3-galactosidase.
  • an engineered bacteriophage promoter useful for artificial priming and transcription and for providing a number of unique endonuclease restriction sites for cloning.
  • IPTG Isopropyl- ⁇ -D-thio- galactopyranoside
  • cDNA clone inserts are generated by an essentially random process, there is probability of 33% that the included cDNA will lie in the conect reading frame for proper translation. If the cDNA is not in the pro er reading frame, it is obtained by deletion or insertion ofthe appropriate number of bases using well known methods including in vitro mutagenesis, digestion with exonuclease in or mung bean nuclease, or the inclusion of an ohgonucleotide linker of appropriate length.
  • the SLC22A12 cDNA is shuttled into other vectors known to be useful for expression of proteins in specific hosts.
  • Ohgonucleotide primers containing cloning sites as well as a segment of DNA (about 25 bases) sufficient to hybridize to stretches at both ends of the target cDNA is synthesized chemically by standard methods. These primers are then used to amplify the desired gene segment by PCR. The resulting gene segment is digested with appropriate restriction enzymes under standard conditions and isolated by gel electrophoresis. Alternately, similar gene segments are produced by digestion of the cDNA with appropriate restriction enzymes. Using appropriate primers, segments of coding sequence from more than one gene are ligated together and cloned in appropriate vectors. It is possible to optimize expression by construction of such chimeric sequences.
  • Suitable expression hosts for such chimeric molecules include, but are not limited to, mammalian cells such as Chinese Hamster Ovary (CHO) and human 293 cells., insect cells such as Sf9 cells, yeast cells such as Saccharomyces cerevisiae and bacterial cells such as -£ . coli.
  • a useful expression vector also includes an origin of replication to allow propagation in bacteria, and a selectable marker such as the /3-lactamase antibiotic resistance gene to allow plasmid selection in bacteria.
  • the vector may include a second selectable marker such as the neomycin phosphotransferase gene to allow selection in transfected eukaryotic host cells.
  • Vectors for use in eukaryotic expression hosts require RNA processing elements such as 3' polyadenylation sequences if such are not part of the cDNA of interest.
  • the vector contains promoters or enhancers which increase gene expression.
  • promoters are host specific and include MMTV, SV40, and metallothionine promoters for CHO cells; tip, lac, tac and T7 promoters for bacterial hosts; and alpha factor, alcohol oxidase and PGH promoters for yeast.
  • Transcription enhancers such as the rous sarcoma virus enhancer, are used in mammalian host cells.
  • Lipofectamine (Gibco BRL catolog no. 18324-020) mediated gene transfer.
  • SLC22A12 is expressed as a chimeric protein with one or more additional polypeptide domains added to facilitate protein purification.
  • purification facilitating domains include, but are not limited to, metal chelating peptides such as histidine- tryptophan modules that allow purification on immobilized metals [Appa Rao, 1997] and the domain utilized in the FLAGS extension/affinity purification system
  • This denatured protein is used to immunize mice or rabbits using standard protocols; about 100 ⁇ .g are adequate for immunization of a mouse, while up to 1 mg might be used to immunize a rabbit.
  • the denatured protein is radioiod nated and used to screen potential murine B-cell hybridomas for those which produce antibody. This procedure requires only small quantities of protein, such that 20 mg is sufficient for labeling and screening of several thousand clones.
  • the amino acid sequence of an appropriate SLC22A12 domain is analyzed to determine regions of high antigenicity.
  • Oligopeptides comprising appropriate hydrophilic regions are synthesized and used in suitable immunization protocols to raise antibodies.
  • the optimal amino acid sequences for immunization are usually at the C-terminus, the N- terminus and those intervening, hydrophilic regions of the polypeptide which are likely to be exposed to the external environment when the protein is in its natural conformation.
  • selected peptides typically, about 15 residues in length, are synthesized using an Applied Biosystems Peptide Synthesizer Model 431 A using finoc-chemistry and coupled to keyhole limpet hemocyanin (KLH; Sigma, St. Louis, MO) by reaction with M-maleimidobenzoyl-N-hydroxysuccinimide ester, MBS. If necessary, a cysteine is introduced at the N-terminus of the peptide to permit coupling to KLH. Rabbits are inimunized with the peptide-KLH complex in complete Freund's adjuvant.
  • KLH keyhole limpet hemocyanin
  • the resulting antisera are tested for antipeptide activity by binding the peptide to plastic, blocking with 1% bovine serum albumin, reacting with antisera, washing and reacting with labeled (radioactive or fluorescent), affinity purified, specific goat anti-rabbit IgG.
  • Hybridomas are prepared and screened using standard techniques. Hybridomas of interest are detected by screening with labeled SLC22A12 to identify those fusions producing the monoclonal antibody with the desired specificity.
  • wells of plates FAST; Becton-Dickinson, Palo Alto, CA
  • affinity purified, specific rabbit anti-mouse (or suitable antispecies 1 g) antibodies at 10 mg/ml.
  • the coated wells are blocked with 1% bovine serum albumin, (BSA), washed and incubated with supernatants from hybridomas. After washing the wells are incubated with labeled SLC22A12 at 1 mg/ml.
  • BSA bovine serum albumin
  • 10 M " preferably 10 to 10 M " or stronger, are typically made by standard procedures.
  • Particular SLC22A12 antibodies are useful for investigating signal transduction and the diagnosis of infectious or hereditary conditions wliich are characterized by differences in the amount or distribution of SLC22A12 or downstream products of an active signaling cascade.
  • Diagnostic tests for SLC22A12 include methods utilizing antibody and a label to detect SLC22A12 in human body fluids, membranes, cells, tissues or extracts of such.
  • the polypeptides and antibodies of the present invention are used with or without modification. Frequently, the polypeptides and antibodies are labeled by joining them, either covalently or noncovalently, with a substance which provides for a detectable signal.
  • labels and conjugation techniques are known and have been reported extensively in both the scientific and patent literature. Suitable labels include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent agents, chemiluminescent agents, chromogenic agents, magnetic particles and the like.
  • a variety of protocols for measuring soluble or membrane-bound SLC22A12, using either polyclonal or monoclonal antibodies specific for the protein, are known in the art. Examples include enzyme-linked immunosorbent assay (ELISA), radioimmuno- assay (RIA) and fluorescent activated cell sorting (FACS).
  • ELISA enzyme-linked immunosorbent assay
  • RIA radioimmuno- assay
  • FACS fluorescent activated cell sorting
  • Native or recombinant SLC22A12 is purified by immunoaffinity chromatography using antibodies specific for SLC22A12.
  • an immunoaffinity column is constructed by covalently coupling the anti-TRH antibody to an activated chromatographic resin.
  • Polyclonal immunoglobulins are prepared from immune sera either by precipitation with ammonium sulfate or by purification on immobilized Protein A (Pharmacia).
  • monoclonal antibodies are prepared from mouse ascites fluid by ammonium sulfate precipitation or chromatography on immobilized Protein A. Partially purified immunoglobulin is covalently attached to a chromatographic resin such as CnBr-activated Sepharose (Pharmacia LKB Biotechnology). The antibody is coupled to the resin, the resin is blocked, and the derivative resin is washed according to the manufacturer's instructions.
  • a chromatographic resin such as CnBr-activated Sepharose (Pharmacia LKB Biotechnology).
  • Such immunoaffinity columns are utilized in the purification of SLC22A12 by pre- paring a fraction from cells containing SLC22A12 in a soluble form.
  • This preparation is derived by solubilization of whole cells or of a subcellular fraction obtained via differential centrifugation (with or without addition of detergent) or by other methods well known in the art.
  • soluble SLC22A12 containing a signal sequence is secreted in useful quantity into the medium in which the cells are grown.
  • a soluble SLC22A12-containing preparation is passed over the immunoaffinity column, and the column is washed under conditions that allow the preferential absorbance of SLC22A12 (e.g., high ionic strength buffers in the presence of detergent). Then, the column is eluted under conditions that disrupt antibody/protein binding (e.g., a buffer of pH 2-3 or a high concentration of a chaotrope such as urea or thiocyanate ion), and SLC22A12 is collected.
  • a buffer of pH 2-3 or a high concentration of a chaotrope such as urea or thiocyanate ion
  • Test compounds can be screened for the ability to bind to OAT-like polypeptides or polynucleotides or to affect OAT-like activity or OAT-like gene expression using high throughput screening.
  • high throughput screening many discrete compounds can be tested in parallel so that large numbers of test compounds can be quickly screened.
  • the most widely established techniques utilize 96-well microtiter plates. The wells of the microtiter plates typically require assay volumes that range from 50 to 500 microliter.
  • instruments, materials, pipettors, robotics, plate washers, and plate readers are commercially available to fit the 96-well format. Altematively, "free format assays," or assays that have no physical barrier between samples, can be used.
  • an assay using pigment cells (melanocytes) in a simple homogeneous assay for combinatorial peptide libraries is described by [Jayawickreme et al, (1994)].
  • the cells are placed under agarose in petri dishes, then beads that cany combinatorial compounds are placed on the surface of the agarose.
  • the combinatorial compounds are partially released the compounds from the beads. Active compounds can be visualized as dark pigment areas because, as the compounds diffuse locally into the gel matrix, the active compounds cause the cells to change colors.
  • Chelsky "Strategies for Screening Combinatorial Libraries: Novel and Traditional Approaches," reported at the First Annual Conference of The Society for Biomolecular Screening in Philadelphia, Pa. (Nov. 7-10, 1995).
  • Chelsky placed a simple homogenous enzyme assay for carbonic anhydrase inside an agarose gel such that the enzyme in the gel would cause a color change throughout the gel.
  • beads carrying combinatorial compounds via a photolinker were placed inside the gel and the compounds were partially released by UV-light. Compounds that inhibited the enzyme were observed as local zones of inhibition having less color change.
  • test samples are placed in a porous matrix.
  • One or more assay components are then placed within, on top of, or at the bottom of a matrix such as a gel, a plastic sheet, a filter, or other form of easily manipulated solid support.
  • a matrix such as a gel, a plastic sheet, a filter, or other form of easily manipulated solid support.
  • the test compound is preferably a small molecule which binds to a OAT-like polypeptide, thereby reducing the normal biological activity ofthe OAT- like polypeptide.
  • small molecules include, but are not limited to, small peptides or peptide-like molecules.
  • either the test compound or the OAT-like polypeptide can comprise a detectable label, such as a fluorescent, radioisotopic, chemiluminescent, or enzymatic label, such as horseradish peroxidase, alkaline phosphatase, or luciferase. Detection of a test compound which is bound to the OAT-like polypeptide can then be accomplished, for example, by direct counting of radioemmission, by scintillation counting, or by determining conversion of an appropriate substrate to a detectable product.
  • binding of a test compound to a OAT-like polypeptide can be determined without labeling either of the interactants.
  • a microphysiometer can be used to detect binding of a test compound with a OAT-like polypeptide.
  • a microphysiometer e.g., CytosensorTM
  • a microphysiometer is an analytical instrument that measures the rate at which a cell acidifies its environment using a light-addressable potentiometric sensor (LAPS). Changes in this acidification rate can be used as an indicator of the interaction between a test compound and a OAT-like polypeptide
  • Determining the ability of a test compound to bind to a OAT-like polypeptide also can be accomplished using a technology such as real-time Bimolecular Interaction Analysis (BIA) [Sjolander & Urbaniczky, (1991), and Szabo et al, (1995)].
  • BIA is a technology for studying biospecific interactions in real time, without labeling any of the interactants (e.g., BIAcoreTM). Changes in the optical phenomenon surface plasmon resonance (SPR) can be used as an indication of real-time reactions between biological molecules.
  • a OAT-like polypeptide in yet another aspect ofthe invention, can be used as a "bait protein" in a two-hybrid assay or three-hybrid assay (see, e.g., U.S. Patent 5,283,317; [Iwabuchi et al, (1993)]; and W094/10300), to identify other proteins which bind to or interact with the OAT-like polypeptide and modulate its activity.
  • the two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains.
  • the assay utilizes two different DNA constructs.
  • polynucleotide encoding a OAT-like polypeptide can be fused to a polynucleotide encoding the DNA binding domain of a known transcription factor (e.g., GAL-4).
  • a DNA sequence that encodes an unidentified protein (“prey" or "sample” can be fused to a polynucleotide that codes for the activation domain of the known transcription factor.
  • the DNA-binding and activation domains ofthe transcription factor are brought into close proximity. This proximity allows transcription of a reporter gene (e.g., LacZ), which is operably linked to a transcriptional regulatory site responsive to the transcription factor. Expression ofthe reporter gene can be detected, and cell colonies containing the functional transcription factor can be isolated and used to obtain the DNA sequence encoding the protein which interacts with the OAT-like polypeptide.
  • a reporter gene e.g., LacZ
  • either the OAT-like polypeptide (or polynucleotide) or the test compound can be bound to a solid support.
  • Suitable solid supports include, but are not limited to, glass or plastic slides, tissue culture plates, microtiter wells, tubes, silicon chips, or particles such as beads (including, but not limited to, latex, polystyrene, or glass beads).
  • Any method known in the art can be used to attach the OAT-like polypeptide (or polynucleotide) or test compound to a solid support, including use of covalent and non-covalent linkages, passive abso ⁇ tion, or pairs of binding moieties attached respectively to the polypeptide (or polynucleotide) or test compound and the solid support.
  • Test compounds are preferably bound to the solid support in an anay, so that the location of individual test compounds can be tracked. Binding of a test compound to a OAT-like polypeptide (or polynucleotide) can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtiter plates, test tubes, and microcentrifuge tubes.
  • the OAT-like polypeptide is a fusion protein comprising a domain that allows the OAT-like polypeptide to be bound to a solid support.
  • glutathione-S-transferase fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) or glutathione derivatized microtiter plates, which are then combined with the test compound or the test compound and the non-adsorbed OAT-like polypeptide; the mixture is then incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads or microtiter plate wells are washed to remove any unbound components. Binding of the interactants can be determined either directly or indirectly, as described above. Alternatively, the complexes can be dissociated from the solid support before binding is determined.
  • OAT-like polypeptide or polynucleotide
  • test compound can be immobilized utilizing conjugation of biotin and streptavidin.
  • Biotinylated OAT-like polypeptides (or polynucleotides) or test compounds can be prepared from biotin-NHS(N-hydroxysuccin- imide) using techniques well known in the art (e.g., biotinylation kit, Pierce
  • antibodies which specifically bind to a OAT-like polypeptide, polynucleotide, or a test compound, but which do not interfere with a desired binding site, such as the active site of the OAT-like polypeptide, can be derivatized to the wells of the plate. Unbound target or protein can be trapped in the wells by antibody conjugation.
  • Methods for detecting such complexes include immunodetection of complexes using antibodies which specifically bind to the OAT-like polypeptide or test compound, enzyme-linked assays which rely on detecting an activity of the OAT-like polypeptide, and SDS gel electrophoresis under non-reducing conditions.
  • Any cell which comprises a OAT- like polypeptide or polynucleotide can be used in a cell-based assay system.
  • a OAT- like polynucleotide can be naturally occurring in the cell or can be introduced using techniques such as those described above. Binding of the test compound to a OAT- like polypeptide or polynucleotide is determined as described above.
  • OAT-like polypeptides comprising a glutathione-S-transferase protein and absorbed onto glutathione-derivatized wells of 96-well microtiter plates are contacted with test compounds from a small molecule library at pH 7.0 in a physiological buffer solution.
  • OAT-like polypeptides comprise an amino acid sequence shown in any one or more of SEQ ID NO:6 to 10.
  • the test compounds comprise a fluorescent tag.
  • the samples are incubated for 5 minutes to one hour. Control samples are incubated in the absence of a test compound.
  • the buffer solution containing the test compounds is washed from the wells. Binding of a test compound to a OAT-like polypeptide is detected by fluorescence measurements of the contents of the wells.
  • a test compound which increases the fluorescence in a well by at least 15% relative to fluorescence of a well in which a test compound is not incubated is identified as a compound wliich binds to an ADO- ribosylation factor-related polypeptide.
  • Example ll Functional activity
  • Functional assays can be carried out as described in the specific examples, after contacting either a purified OAT-like polypeptide or an intact cell with a test compound.
  • a test compound which decreases a functional activity of a human OAT- like polypeptide by at least about 10, preferably about 50, more preferably about 75, 90, or 100% is identified as a potential agent for decreasing OAT-like protein activity.
  • a test compound which increases a functional activity by at least about 10, preferably about 50, more preferably about 75, 90, or 100% is identified as a potential agent for increasing OAT-like protein activity.
  • Example 12 Effect of test compound on anoinic transport
  • H-labeled substrate in the presence or absence of a test compound can be measured as described in [Hsiang et al (1999, supra)]. Briefly, 293cl8 cells are transfected with human OATP2-like expression constructs using LipofectAMINE Plus (Life Technologies, Inc.) according to the manufacturer's instructions. The medium is removed, and the cells are washed once in serum-free DMEM. 3 H-labeled substrate, either alone or in the presence of a test compound, is added in the same medium and incubated at room temperature for 5-10 minutes. The cells are quickly washed once with ice-cold DMEM containing 5% bovine serum albumin, then washed three times with ice-cold DMEM. Cells are lysed in 0.1 N NaOH. Radiolabel incorporation is determined by liquid scintillation counting.
  • the goal of rational drug design is to produce structural analogs of biologically active polypeptides of interest or of small molecules with which they interact, agonists, antagonists, or inhibitors. Any of these examples are used to fashion drugs which are more active or stable forms ofthe polypeptide or which enhance or interfere with the function of a polypeptide in vivo.
  • the three-dimensional stracture of a protein of interest, or of a protein-inhibitor complex is determined by x-ray crystallography, by computer modeling or, most typically, by a combination ofthe two approaches. Both the shape and charges of the polypeptide must be ascertained to elucidate the structure and to determine active site(s) ofthe molecule. Less often, useful information regarding the structure of a polypeptide is gained by modeling based on the structure of homologous proteins. In both cases, relevant structural information is used to design efficient inhibitors. Useful examples of rational drug design include molecules which have improved activity or stability or which act as inhibitors, agonists, or antagonists of native peptides.
  • a target-specific antibody selected by functional assay, as described above, and then to solve its crystal structure.
  • This approach in principle, yields a pharmacore upon which subsequent drug design is based. It is possible to bypass protein crystallography altogether by generating anti-idiotypic antibodies (anti-ids) to a functional, pharmacologically active antibody. As a minor image of a minor image, the binding site of the anti-ids is expected to be an analog of the original receptor. The anti-id is then used to identify and isolate peptides from banks of chemically or biologically produced peptides. The isolated peptides then act as the pharmacore.
  • anti-ids anti-idiotypic antibodies
  • Animal model systems which elucidate the physiological and behavioral roles of the SLC22A12 are produced by creating nonhuman transgenic animals in which the activity of the SLC22A12 is either increased or decreased, or the amino acid sequence of the expressed SLC22A12 is altered, by a variety of techniques.
  • Examples of these techniques include, but are not limited to: 1) Insertion of normal or mutant versions of DNA encoding a SLC22A12, by microinjection, electroporation, retroviral transfection or other means well known to those skilled in the art, into appropriately fertilized embryos in order to produce a transgenic animal or 2) homologous recombination of mutant or normal, human or animal versions of these genes with the native gene locus in transgenic animals to alter the regulation of expression or the structure of these SLC22A12 sequences.
  • the technique of homologous recombination is well known in the art.
  • transgenic animal One means available for producing a transgenic animal, with a mouse as an example, is as follows: Female mice are mated, and the resulting fertilized eggs are dissected out of their oviducts. The eggs are stored in an appropriate medium such as cesiumchloride M2 medium. DNA or cDNA encoding SLC22A12 is purified from a vector by methods well known to the one skilled in the art. Inducible promoters may be fused with the coding region of the DNA to provide an experimental means to regulate expression of the transgene. Alternatively or in addition, tissue specific regulatory elements may be fused with the coding region to permit tissue-specific expression of the transgene.
  • microinjection needle which may be made from capillary tubing using a piper puller
  • the egg to be injected is put in a depression slide.
  • the needle is inserted into the pronucleus of the egg, and the DNA solution is injected.
  • the injected egg is then transfened into the oviduct of a pseudopregnant mouse which is a mouse stimulated by the appropriate hormones in order to maintain false pregnancy, where it proceeds to the uterus, implants, and develops to term.
  • micro- injection is not the only method for inserting DNA into the egg but is used here only for exemplary purposes.
  • Kellogg et al. 1990, Anal. Biochem. 189:202-208 Konig et al, 278, Am. J. Physiol. Gastrointest. Liver Physiol. Gl 56-64, 2000

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Abstract

L'invention concerne un SLC22A12 humain associé aux maladies cardio-vasculaires, aux maladies liées à la reproduction, aux maladies urologiques et au cancer. L'invention concerne également des dosages permettant d'identifier des composés utiles dans le traitement ou la prévention des maladies cardio-vasculaires, des maladies liées à la reproduction, des maladies urologiques et du cancer. L'invention concerne également des composés se liant au SLC22A12 et/ou activant ou inhibant son activité, ainsi que des compositions pharmaceutiques contenant ces composés.
PCT/EP2004/010111 2003-09-22 2004-09-10 Agents diagnostiques et therapeutiques pour des maladies liees au transporteur slc22a12 (slc22a12) de cations organiques WO2005029084A1 (fr)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001049728A2 (fr) * 2000-01-06 2001-07-12 Protegene Inc. Proteines humaines a domaines hydrophobes et adn codant ces proteines
WO2001062923A2 (fr) * 2000-02-25 2001-08-30 Incyte Genomics, Inc. Transporteurs et canaux ioniques
WO2001077329A2 (fr) * 2000-04-07 2001-10-18 Millenium Pharmaceuticals, Inc. 12303, molecule twik humaine et utilisations correspondantes
WO2003089583A2 (fr) * 2002-04-16 2003-10-30 Origene Technologies, Inc. Genes et batteries de genes specifiques de tissus

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001049728A2 (fr) * 2000-01-06 2001-07-12 Protegene Inc. Proteines humaines a domaines hydrophobes et adn codant ces proteines
WO2001062923A2 (fr) * 2000-02-25 2001-08-30 Incyte Genomics, Inc. Transporteurs et canaux ioniques
WO2001077329A2 (fr) * 2000-04-07 2001-10-18 Millenium Pharmaceuticals, Inc. 12303, molecule twik humaine et utilisations correspondantes
WO2003089583A2 (fr) * 2002-04-16 2003-10-30 Origene Technologies, Inc. Genes et batteries de genes specifiques de tissus

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