Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
  • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/158—Expression markers

Definitions

• the present invention relates to methods and compositions for the detection, diagnosis, prevention and treatment of disease states and related disorders.
• the disease states of the present invention include cardiac, kidney and inflammatory disease.
• genes that are differentially expressed in the cells, tissues, or peripheral blood of a subject suffering from, or predisposed to, such disease states may be identified through the methods of the present invention.
• the present invention also relates to compositions and methods useful in the diagnosis, prevention and thera- Commissionic treatment of disease states through the use of the differentially expressed genes of the present invention.
• Methods and compositions are provided for the diagnostic evaluation and prognosis of conditions involving such disease states, for the identification of subjects exhibiting a predisposition to such conditions, for therapeutic uses, e.g., modulating the effect of such differentially expressed genes, for monitoring subjects undergoing clinical evaluation for the prevention and treatment of a disease and its disorders, and for monitoring the efficacy of compounds used in clinical trials.
• the present invention relates generally to methods and compositions for the detection, diagnosis, prevention and treatment of a disease, specifically cardiac, kidney or inflammatory disease, and related disorders. Particularly, the present invention relates to methods useful in diagnosing, identifying, monitoring, preventing, and treating the onset and progression of such disease states through the use of genes and gene products differentially expressed in a disease, specifically cardiac, kidney or inflammatory disease, along with modulators thereof.
• CHF congestive heart failure
• Traditionally CHF has been treated by a series of agents including diuretics, vasodilators, angiotensin converting enzyme inhibitors, ⁇ -adrenergic antagonists, and positive inotropes like digoxin.
• diuretics vasodilators, angiotensin converting enzyme inhibitors, ⁇ -adrenergic antagonists, and positive inotropes like digoxin.
• these drugs principally provide symptomatic relief and typically only extend the life of one suffering from the disease for periods ranging from 6-12 months.
• adult ventricular muscle cells can adapt to increased workloads through the activation of a hypertrophic process.
• This process is characterized by an increase in the contractile protein content of cardiac muscle cells without a proliferative response because the adult cardiomyocyte is terminally differentiated and has lost its ability to divide. Cardiac growth during the hypertrophic process therefore results primarily from an increase in protein content per individual cardiomyocyte, with little or no change in cell number.
• the acquisition of the cardiac hypertrophic phenotype is in part dependent upon the activation of cardiac muscle gene program.
• ventricular hypertrophy is also characterized by alterations in the expression of certain non-contractile proteins, such as atrial natriuretic peptide (ANP, also known as ANF)
• ANP atrial natriuretic peptide
• ANP expression is down regulated in the ventricle and expression is mainly confined to the atrium Following induction of hypertrophy, ANP is reexpressed in the vent ⁇ culum.
• ANP expression can be considered to be a non contractile protein marker of cardiac ventricular hypertrophy.
• Ventricular hypertrophy is initially a compensatory mechanism by which the heart is attempting to counteract the effects of conditions like pressure overload, loss of contractile tissue, obstruction of blood flow, or increased peripheral demand for blood flow, all of which can be generated by a variety of physiological or pathological stimuli.
• a typically short term, compensated hypertrophic response is desirable.
• cardiac, e.g. left ventricular, hypertrophy physiological hypertrophy
• hypertrophic response may eventually contribute to cardiac dysfunction.
• hypertrophy is termed decompe ⁇ sated hypertrophy, and antagonism of cardiac hypertrophy is considered desirable.
• CHF Heart failure affects approximately five million Americans. New cases of heart failure number about 400,000 each year.
• the pathophysiology of CHF is rather complex.
• the central hallmark of the disease is the inability of the heart to pump sufficient oxygenated blood to meet the demands of peripheral tissues.
• Numerous etiologies contribute to the development of CHF, including primary diseases of, or insults to, the myocardium itself, cardiac defects, hypertension, inflammation, kidney disease and vascular disease.
• renm angiotensm converting enzyme (ACE) inhibitors Factors known to contribute centrally to the pathophysiology of heart disease are biosynthesized in the heart itself. These factors are produced in cardiac myocytes, fibroblasts, smooth muscle and endothelial cells, and inflammatory cells associated with the myocardium.
• the heart has been shown to contain its own renm angiotensm system. Blockade of the cardiac renm angiotensm system is believed to contribute significantly to the therapeutic efficacy of the therapeutic class of agents known as angiotensm converting enzyme (ACE) inhibitors.
• ACE angiotensm converting enzyme
• the heart also produces other factors including, but not limited to, endothelins, bradykinin, adre ⁇ omeduliin, tumor necrosis factor, transforming growth factors, and natriuretic peptides.
• therapeutic strategies are limited to the modulation of such substances, which are already known to contribute to the disease. Indeed, it is estimated that the functional contributions of only a minor fraction of all known secreted factors encoded by the human genome have been defined.
• differentially expressed genes related to disease states in addition to methods and compositions for the diagnostic evaluation and prognosis of conditions involving such diseases, for the identification of subjects exhibiting a predisposition to such conditions, for modulating the effect of these differentially expressed genes and their expression products, for monitoring patients undergoing clinical evaluation for the prevention and treatment of a disease, specifically cardiac, kidney or inflammatory disease, and its disorders, and for monitoring the efficacy of compounds used in clinical trials.
• a disease specifically cardiac, kidney or inflammatory disease, and its disorders
• a strategy aimed at the identification of genes which are differentially expressed in association with a disease, specifically cardiac, kidney or inflammatory disease, will likely elucidate expression products or other factors that can contribute to or ameliorate the symptoms of the disease state and are potential candidate targets for therapeutic modulation or which are potentially therapeutic themselves.
• Such genes can also contribute to methodologies for diagnosing, evaluating, preventing and treating such diseases.
• the primary goal of therapy for cardiac diseases has been the relief of symptoms associated with reduced cardiac output.
• current drugs provide some improvement in cardiac output, they fail to address the underlying mechanisms that lead to heart failure. A lack of understanding of the mechanisms responsible for progressive heart failure has made it difficult to devise long term strategies for treatment.
• the heart tissue In addition to changes in mass, the heart tissue also remodels the cellular architecture of the cardiomyocyte, evident as alterations in sarcome ⁇ c structure and contractile fiber formation. Following initial compensatory changes, the myocardium can ultimately fail due to irreversible enlargement and dilation. To afford the cellular changes in the tissues of the remodeling heart, there are many documented molecular changes, which are controlled by changes in cardiac gene expression ( Komuro et a/., Ann. Rev. Physiol. 55:55 75 (1993)). Such changes are not, however, confined to the cardiac myoc ⁇ te. As important are the alterations and remodeling of the interstitial compartment. For example, proliferation and activation of cardiac myocytes in the failing heart lead to extracellular matrix deposition, which negatively affects the contractility of the ventricle wall.
• a compensated hypertrophic heart can maintain diasto c and systolic function, but eventually the LVH response is exhausted, and continued cell loss and fibrosis leads to a demise of the heart.
• the rat LVH model is well suited to examine cellular and molecular changes associated with early responses to pressure overload, long term compensation, and late stage failure.
• a particularly important application of the microarray method allows for the assessment of differential gene expression in pairs of mRNA samples from two different tissues, or in the same tissue comparing normal versus disease states or time progression of the disease.
• Microarray analysis allows one to analyze the expression of known genes of interest, or for the discovery of novel genes expressed differentially in tissue pairs of interest.
• an attractive application of this technology is as a fundamental discovery tool to identify new genes, and their corresponding expression products, which contribute to the pathogenesis of disease and related conditions.
• Microarray technology has been successfully applied to large-scale analysis of human gene expression to identify cancer-specific genes and inflammatory-specific genes (DeRisi et al., Nat. Genet 14(41:457-60 (1996); Heller et a/., Proc. Natl.
• DeRisi et a/ examined a pre selected set of 870 different genes for their expression in a melanoma cell line and a non-tumo ⁇ ge ⁇ ic version of the same cell line. The microarray analysis revealed a decrease in expression for 15/870 (1.7%) and an increase in expression for 63/870 (7.3%) of the genes in non-tumo ⁇ genic relative to tumorigenic cells (only signals ⁇ 0.52 or > 2.4 were deemed significant).
• Heller et al. employed microarrays to evaluate the expression of 1000 genes in cells taken from normal and inflamed human tissues.
• genes which are differentially expressed in association with a disease, specifically cardiac, kidney or inflammatory disease, are identified using the methods of the present invention.
• DNA microarrays are utilized to identify the genes of the present invention.
• the present invention emphasizes the importance of gene regulation in association with a disease, specifically cardiac, kidney or inflammatory disease.
• One skilled in the art in view of the present disclosure, recognizes that the expression products of these genes have application as therapeutic agents, or targets for therapeutic modulation in a disease and its related conditions.
• the present invention also relates to the use of these genes, their expression products, and their modulators, in the detection, diagnosis, prevention, and treatment of disease.
• the present invention addresses deficiencies in the prior art by providing methods for identifying specific genes that are differentially expressed in subjects in response to a disease, specifically a cardiac, kidney or inflammatory disease, state, at a different level than such genes are expressed in a biological sample [e.g., cells, tissue or peripheral blood) obtained from a normal subject (i.e., a subject who is not suffering from or predisposed to the disease, e.g., a control subject).
• a disease state associated with the differentially expressed genes of the present invention may be detected, or diagnosed, by examining a blood sample rather than relying on a more invasive or less sensitive test to derive a prognosis.
• a subject may be monitored for disease progression, status, and response to therapies through monitoring of the expression of differentially expressed genes.
• a "patient,” “individual,” or “subject” are interchangeable terms and may be an animal, including a laboratory animal or other animal species, or a human
• cardiac diseases include CHF, dilated congestive cardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy, mitral valve disease, aortic valve disease, tncuspid valve disease, angina pecto ⁇ s, myocardial infarction, cardiac arrhythmia, pulmonary hypertension, arterial hypertension, renovascular hypertension, arteriosclerosis, atherosclerosis, and cardiac tumors.
• kidney diseases include acute renal failure, glomerulonephritis, chronic renal failure, azotemia, uremia, immune renal disease; acute nephritic syndrome, rapidly progressive nephritic syndrome, ⁇ ephrotic syndrome, Berger's Disease, chronic nephntic/proteinuric syndrome, tubulointerstital disease, nephrotoxic disorders, renal infarction, atheroembolic renal disease, renal cortical necrosis, malignant nephroangiosclerosis, renal vein thrombosis, renal tubular acidosis, renal glucosuna, nephrogenic diabetes sipidus, Bartter's Syndrome, Liddle's Syndrome, polycystic renal disease, interstitial nephritis, acute hemolytic uremic syndrome, medullary cystic disease, medullary sponge kidney, hereditary nephritis, and nail-patella syndrome.
• Such inflammatory diseases include myocarditis, asthma, chronic inflammation, autoimmune diabetes, tumor angiogenesis, rheumatoid arthritis, rheumatoid spondylitis, osteoarthntis, gouty arthritis and other arthritic conditions, sepsis, septic shock, endotoxic shock, Gram-negative sepsis, toxic shock syndrome, asthma, adult respiratory distress syndrome, stroke, reperfusion injury, CNS injuries such as neural trauma and ischemia, psoriasis restenosis, cerebral malaria, chronic pulmonary inflammatory disease, silicosis, pulmonary sarcosis, bone resorption diseases such as osteoporosis, graft versus host reaction, Crohn's Disease, ulcerative colitis including inflammatory bowel disease (IBD), and pyresis.
• IBD inflammatory bowel disease
• the present invention relates to methods and compositions for the detection, diagnosis, prevention, and treatment of a disease, specifically cardiac, kidney or inflammatory disease.
• genes are identified and described which are differentially expressed in cells, tissue or peripheral blood relative to normal cells, tissue or peripheral blood and/or to cells, tissue or peripheral blood at a different stage of a disease, specifically cardiac, kidney or inflammatory disease.
• genes are identified which are differentially expressed in subjects suffering from a disease, specifically cardiac, kidney or inflammatory disease, relative to normal subjects.
• the modulation of the expression of the identified genes and/or the activity of the identified gene products can be utilized therapeutically to prevent or treat a disease, specifically cardiac, kidney or inflammatory disease, and related disorders.
• methods and compositions are described for the identification of novel therapeutic compounds for the inhibition of such diseases.
• the identified genes and/or gene products and/or modulators can be used to identify cells exhibiting or predisposed to a disorder involving a disease phenotype, thereby diagnosing individuals having, or at risk for developing, such disorders. Additionally, the identified genes and/or gene products can be used to determine severity or duration of such diseases. Furthermore, the detection of the differential expression of identified genes can be used to devise treatments for a disease, specifically cardiac, kidney or inflammatory disease. Still further, the detection of differential expression of identified genes can be used to design a preventive intervention for subjects at risk of such diseases.
• One such method for the treatment of a disease comprises the administration to a subject of an effective amount of a modulator of one or more genes encoding human proteins of the group consisting of native sequence 1-8U, native sequence prostacyciin-stimulating factor, native sequence osf-2, native sequence tissue specific mRNA protein, native sequence IGFBP-6, native sequence 0SF-1, native sequence gas-1, native sequence YMP, native sequence BTG2, native sequence SDFI a, native sequence peripheral benzodiazepine receptor, and native sequence cellular ligand of annexin II.
• Another such method comprises the administration to a subject of an effective amount of a modulator of one or more human proteins of the group consisting of native sequence 1-8U, native sequence prostacyciin-stimulating factor, native sequence osf-2, native sequence tissue specific mRNA protein, native sequence IGFBP-6, native sequence OSF- 1 , native sequence gas-1, native sequence YMP, native sequence BTG2, native sequence SDFI a, native sequence peripheral benzodiazepine receptor, and native sequence cellular ligand of annexin II.
• the subject may preferably be a human patient.
• This modulator may be positive or negative; consist of one or more human proteins of the group consisting of 1 -8U, prostacyciin-stimulating factor, osf-2, tissue specific mRNA protein, IGFBP-6, 0SF-1 , gas-1, YMP, BTG2, SDFI a, peripheral benzodiazepine receptor, and cellular ligand of annexin II; and be selected from the group consisting of peptides, phosphopeptides, small organic or inorganic molecules, antibodies, and epitope-binding fragments.
• a modulator may be selected from the group consisting of antisense, ribozyme, and triple helix molecules.
• Yet another such method for the treatment of a disease, specifically cardiac, kidney or inflammatory disease, and most specifically cardiac disease comprises the administration to a human patient of an effective amount of one or more isolated human proteins of the group consisting of native sequence 1-8U, native sequence prostacyciin- stimulating factor, native sequence osf-2, native sequence tissue specific mRNA protein, native sequence IGFBP-6, native sequence OSF-1, native sequence gas-1, native sequence YMP, native sequence BTG2, native sequence SDFI a, native sequence peripheral benzodiazepine receptor, and native sequence cellular ligand of annexin II.
• Further methods of the present invention comprise the administration to a human patient of an effective dose of an antibody to a cellular receptor of, an organic molecule inhibitor capable of binding to a cellular receptor of, an expression product of an isolated nucleotide sequence encoding, or a syngeneic host cell transformed with an isolated nucleotide sequence encoding one or more human proteins.
• This isolated nucleotide sequence may comprise an antisense oligonucleotide capable of hybridizing with, and inhibiting the translation of, the mRNA encoded by a gene encoding one or more of the human proteins of the group consisting of 1-8U, prostacyciin-stimulating factor, osf-2, tissue specific mRNA protein, IGFBP-6, OSF-1, gas-1, YMP, BTG2, SDFI a, peripheral benzodiazepine receptor, and cellular ligand of annexin II.
• inventions of the present invention may use this DNA molecule as a vector or operably linked to a regulatory sequence that controls expression of the coding sequence in a host cell, said host cell preferably comprising a human cell such as a cardiac cell, more preferably a left ventricle cell.
• a host cell preferably comprising a human cell such as a cardiac cell, more preferably a left ventricle cell.
• Another embodiment of the present invention provides for the screening of a subject suspected of having a disease, specifically cardiac, kidney, or inflammatory disease, and more specifically cardiac disease.
• the expression of one or more proteins selected from the group consisting of 1 -8U, prostacyciin-stimulating factor, osf-2, tissue specific mRNA protein, IGFBP-6, OSF-1, gas-1, YMP, BTG2, SDFIa, peripheral benzodiazepine receptor, and cellular ligand of annexin II is determined in a subject suspected of having, or being predisposed to, a cardiac disease, and compared to the expression levels of the one or more proteins in a normal subject. Further, this difference in expression is preferably at least about two-fold or more in the subject, and the subject is preferably a human patient.
• an array comprising one or more oligonucleotides complementary to reference DNA or RNA sequences encoding one or more human proteins selected from the group consisting of 1-8U, prostacyciin- stimulating factor, osf-2, tissue specific mRNA protein, IGFBP-6, OSF-1 , gas-1 , YMP, BTG2, SDFI a, peripheral benzodiazepine receptor, and cellular ligand of annexin II is used for detecting disease, specifically cardiac, kidney, or inflammatory disease.
• the reference DNA or RNA preferably is obtained from a biological sample from a normal subject and from a subject exhibiting a disease, specifically cardiac, disease. Such subjects are preferably humans.
• the biological sample preferably comprises peripheral blood or tissue, preferably a cell such as a cardiac cell, and more preferably a left ventricle cell.
• a disease specifically cardiac, kidney, or inflammatory disease, and more specifically cardiac disease, in a human patient.
• the expression level of one or more proteins selected from the group consisting of 1 -8U, prostacyciin-stimulating factor, osf-2, tissue specific mRNA protein, IGFBP-6, OSF-1, gas-1, YMP, BTG2, SDFI a, peripheral benzodiazepine receptor, and cellular ligand of annexin Ii is determined in the subject and compared to the expression levels of the one or more proteins in a normal subject.
• a tissue sample from the human patient may be obtained from cardiac tissue, specifically left ventricle tissue, or from the subject's blood.
• cDNA probes are hybridized on the array to create fluorometric, colorimetric or such identifying emissions, which are then compared with the existing encoded proteins.
• a diagnostic kit comprising said array is contemplated and used for detecting and diagnosing a disease, specifically cardiac, kidney or inflammatory disease.
• This kit may comprise control oligonucleotide probes, PCR reagents and detectable labels.
• this kit may comprise biological samples taken from human subjects, said samples comprising blood or tissue, preferably cardiac tissue, more preferably left ventricle cells.
• Such diagnostic kits may also comprise antibodies to the differentially expressed disease state genes of the present invention, which may be monoclonal.
• a method for identifying a modulator of a differentially expressed disease state gene comprising contacting a biological sample from a subject having a disease, specifically cardiac, kidney or inflammatory disease, with a compound and determining the expression level of said differentially expressed gene. Comparison may be made between the expression level of the differentially expressed gene in a normal subject or said subject prior to contact with a compound and the expression level of the differentially expressed gene after contact with a compound, said compound selected from the group consisting of small molecules, active polypeptides and antibodies.
• Figure 1 shows RNA blot analysis of ANP and BNP in LVH rats.
• Aortic banded and sham operated control rats were sacrificed at 10 weeks and 20 weeks post surgery.
• RNA was extracted from the left ventricle of each animal and probed on Northern blots for ANP and BNP transcripts using specific oligonucleotide probes.
• Figure 2 shows PCR amplified DNA from 96 random clones of rat left ventricle.
• PCR product (10 % of total) from 96 clones was loaded onto a 1.0 % agarose gel and visualized by ethidium bromide staining.
• Figure 3 shows a microarray analysis of 96 clones expressed in rat heart. Randomly chosen clones from a rat left ventricle cDNA library were printed onto a microarray and hybridized with Cy5-labeled rat left ventricle cDNA. The intensity of each probe is expressed in pseudo-color according to the scale shown. Blank spots resulted from lack of PCR amplifiabie insert DNA from the corresponding clone.
• Figure 4 shows the differential expression data of representative genes obtained through the disease models of the present invention and determined via microarray analysis.
• Those representative disease model differentially expressed genes (clone ID nos. P0204 06, P0237 02, P0248 D1 1, P0228 H09, P0246_H10, P0237_B09, P0207 C03, P0214_A1 1, P0182_F08, P0219_H09, P0242_B03, P02B8 G09) were found to correspond to human genes encoding 1-8U, prostacyciin-stimulating factor, osf-2, tissue specific mRNA, insulin-like growth factor binding protein 6, OSF-1, gas-1 , YMP, BTG2, pre-B cell stimulating factor homolog (SDFI a), peripheral benzodiazepine receptor, and cellular ligand of annexin II (p11), respectively.
• SDFI a pre-B cell stimulating factor homolog
• Figures 51 - 5L show alignment data comparing the cDNA encoding the differentially expressed animal disease model genes with human cDNA corresponding to 1 -8U (SEQ ID N0:1; SEQ ID N0:2), prostacyciin-stimulating factor (SEQ ID N0:3; SEQ ID N0:4), osf-2 (SEQ ID N0:5; SEQ ID N0:6), tissue specific mRNA (SEQ ID N0:7; SEQ ID N0:8), insuhn-like growth factor binding protein 6 (SEQ ID N0:9; SEQ ID N0:10), OSF-1 (SEQ ID N0:1 1 ; SEQ ID N0:12), gas-1 (SEQ ID N0:13; SEQ ID N0:14), YMP (SEQ ID N0:15; SEQ ID N0:16), BTG2 (SEQ ID N0:17; SEQ ID N0:18), pre-B cell stimulating factor homolog (SDFI a) (SEQ
• Figures 6A-6E show alignment data comparing human cDNA sequences from the GenBank database with multiple cDNA clones encoding the differentially expressed animal disease model genes of the present invention, corresponding to 1-8U, tissue specific mRNA, YMP, pre-B cell stimulating factor homolog (SDFI a), peripheral benzodiazepine receptor, and cellular ligand of annexin II (p11).
• Figures 7A L show the nucleotide sequences encoding the polypeptides corresponding to 1-8U (SEQ ID N0:25), prostacyciin-stimulating factor (SEQ ID N0:26), osf-2 (SEQ ID N0:27), tissue specific mRNA (SEQ ID N0:28), insulin-like growth factor binding protein 6 (SEQ ID N0:29), OSF-1 (SEQ ID N0:30), gas-1 (SEQ ID N0:31 ), YMP (SEQ ID N0:32), BTG2 (SEQ ID N0:33), pre-B cell stimulating factor homolog (SDFI a) (SEQ ID N0:34), peripheral benzodiazepine receptor (SEQ ID N0:35), and cellular ligand of annexin II (p1 1 ) (SEQ ID N0:36).
• SEQ ID N0:25 shows the nucleotide sequences encoding the polypeptides corresponding to 1-8U (SEQ ID N0
• Figures 8A-L show the ammo acid sequences encoding the polypeptides corresponding to 1 -8U (SEQ ID N0:37), prostacyciin-stimulating factor (SEQ ID N0:38), osf-2 (SEQ ID N0:39), tissue specific mRNA (SEQ ID N0:40), insuhn-like growth factor binding protein 6 (SEQ ID N0:41 ), OSF-1 (SEQ ID N0:42), gas-1 (SEQ ID N0:43), YMP (SEQ ID N0:44), BTG2 (SEQ ID N0:45), pre-B cell stimulating factor homolog (SDFI a) (SEQ ID N0:46), peripheral benzodiazepine receptor (SEQ ID N0:47), and cellular ligand of annexin II (pi 1 ) (SEQ ID N0:48).
• SDFI a SEQ ID N0:46
• peripheral benzodiazepine receptor SEQ ID N0:47
• Figure 9 shows characteristics of the human cDNA corresponding to 1 -8U, prostacyciin-stimulating factor, osf 2, tissue specific mRNA, insu n-like growth factor binding protein 6, OSF-1, gas-1, YMP, BTG2, pre-B cell stimulating factor homolog (SDFI a), peripheral benzodiazepine receptor, and cellular ligand of annexin II (p1 1 ), as well as characteristics of the proteins themselves.
• FIG 10 shows OSF-2 gene expression is neonatal rat cardiac myocytes treated with various stimuli known to induce a hypertrophic response.
• Figure 11 shows that inhibition of p38 ⁇ prevents induction of prostacyclm stimulating factor (mac25, IGFBP 7) in human peripheral blood mononuclear cells (HPBMNC)
• Figure 12 shows the ANP and GAPDH transcript levels in rat neonatal cardiac myocytes treated with 0, 0.2 or 1 ⁇ g/ml doses of OSF 1
• Figures 13 A and B illustrate the up regulation of COX 2 and IL 1 ⁇ in human peripheral blood mononuclear cells treated with 0, 0.1 or 5 ⁇ g/ml IGFBP 6.
• Figure 14 illustrates the induction of IL 1 ⁇ synthesis in human peripheral blood mononuclear cells (HPBMC) treated with 0, 0.4, 0.8, 2 and 4 nM concentrations of IGFBP 6 V. DETAILED DESCRIPTION OF THE INVENTION A. DEFINITIONS
• Kidney disease includes acute renal failure, glomerulonephritis, chronic renal failure, azotemia, uremia, immune renal disease, acute nephritic syndrome, rapidly progressive nephritic syndrome, nephrotic syndrome, Berger's Disease, chronic nephritic/proteinu ⁇ c syndrome, tubulomterstital disease, nephrotoxic disorders, renal infarction, atheroembolic renal disease, renal cortical necrosis, malignant nephroangiosclerosis, renal vein thrombosis, renal tubular acidosis, renal glucosuna, nephrogemc diabetes insipidus, Banter's Syndrome, ⁇ ddle's Syndrome, polycystic renal disease, medullary cystic disease, medullary sponge kidney, hereditary nephritis, and nail patella syndrome, along with any disease or disorder that relates to the renal system and related disorders, as well as symptoms indicative of, or related to,
• “Inflammatory disease” includes myocarditis, asthma, chronic inflammation, autoimmune diabetes, tumor angiogenesis, rheumatoid arthritis, rheumatoid spondylitis, osteoarth ⁇ tis, gouty arthritis and other arthritic conditions, sepsis, septic shock, endotoxic shock, Gram negative sepsis, toxic shock syndrome, asthma, adult respiratory distress syndrome, stroke, reperfusion injury, CNS injuries such as neural trauma and ischemia, psoriasis restenosis, cerebral malaria, chronic pulmonary inflammatory disease, silicosis, pulmonary sarcosis, bone resorption diseases such as osteoporosis, graft versus host reaction, Crohn's Disease, ulcerative colitis including inflammatory bowel disease (IBD), and pyresis, along with any disease or disorder that relates to inflammation and related disorders, as well as symptoms indicative of or related to, inflammation and related disorders "Cardiac disease” includes congestive heart failure, myocardit
• Gene includes differentially expressed genes and their expression products whose expression pattern can be utilized as part of a prognostic or diagnostic marker for the evaluation of a disorder involving a disease, specifically cardiac, kidney or inflammatory disease, or which can be used in methods for identifying compounds useful for the detection, diagnosis, prevention and treatment of such diseases and related disorders.
• the effect of the compound on the gene expression normally displayed in connection with disorders involving a disease, specifically cardiac, kidney or inflammatory disease can be used to evaluate the efficacy of the compound as a treatment for such a disorder, or can, additionally, be used to monitor patients undergoing clinical evaluation for the treatment of the disorder.
• Gene also includes differentially expressed genes and their expression products involved in a disease, specifically cardiac, kidney or inflammatory disease, such that modulation of the level of gene expression or of gene product activity can act to prevent or treat such a disease and related conditions.
• Compounds that modulate the expression of the gene or the activity of the gene product can be used in the treatment of such diseases in a subject, as well as the differentially expressed gene itself or functional variations thereof.
• compounds that modulate the expression of the gene or activity of the gene product can be used in treatments for a disease, specifically cardiac, kidney or inflammatory disease, and related conditions.
• compounds that modulate the expression of the gene or activity of the gene product can be used to design a preventive intervention in individuals at risk of a disease.
• Genes may also be defined via the ability of their products to interact with other gene products involved in a disease, specifically cardiac, kidney or inflammatory disease, and include the target, interactive, and diagnostic genes of the present invention.
• Genes termed "target genes” or “diagnostic genes” include genes differentially expressed in subjects with a disease, specifically cardiac, kidney or inflammatory disease, relative to their expression in normal subjects or relative to their expression at a different stage of a disease.
• Genes termed "interactive genes” include genes whose products exhibit an ability to interact with gene products involved in a disease, specifically cardiac, kidney or inflammatory disease. Interactive genes can additionally have diagnostic or target gene characteristics.
• “Expression pattern” includes the pattern generated when the expression pattern of a series (which can range from two up to all the genes which exist for a given state) of genes is determined. An expression pattern can be used in the same diagnostic, prognostic and compound identification methods as the expression of a single gene.
• An "oligonucleotide” includes a nucleic acid polymer composed of two or more nucleotides or nucleotide analogs. An oligonucleotide can be derived from natural sources but is often synthesized chemically. It is of any size.
• An "oligonucleotide array or microarray” includes a spatially defined pattern of oligonucleotide probes on a solid support. A "preselected array of oligonucleotides” is an array of spatially defined oligonucleotides on a solid support.
• nucleic acid reagent used in standard automated oligonucleotide synthesis typically carries a protected phosphate on the 3' hydroxyl of the nbose.
• nucleic acid reagents are referred to as nucleotides, nucleotide reagents, nucieoside reagents, nucleoside phosphates, nucleoside 3'-phosphates, nucleoside phosphoramidites, phosphoramidites, nucieoside phosphonates, phosphonates and the like. It is generally understood that nucleotide reagents carry a protected phosphate group in order to form a phosphodiester linkage.
• a “solid support” includes a fixed organizational support matrix, such as silica, polymeric materials, or glass.
• at least one surface of the substrate is partially planar.
• recombinant when used with reference to a cell, animal, or virus indicates that the cell, animal, or virus encodes a foreign DNA or RNA.
• recombinant cells optionally express nucieic acids (e.g., RNA) not found within the native (non-recombinant) form of the cell.
• Stringent hybridization conditions are sequence dependent and will be different with different environmental parameters (e.g., salt concentrations, and presence of organics). Generally, stringent conditions are selected to be about 5° C to 20° C lower than the thermal melting point (TJ for the specific nucleic acid sequence at a defined ionic strength and pH. Preferably, stringent conditions are about 5° C to 10° C lower than the thermal melting point for a specific nucleic acid bound to a complementary nucleic acid.
• T m is the temperature (under defined ionic strength and pH) at which 50% of a nucleic acid (e.g., tag nucleic acid) hybridizes to a perfectly matched probe.
• Stringent wash conditions are ordinarily determined empirically for hybridization of each set of tags to a corresponding probe array
• the arrays are first hybridized (typically under stringent hybridization conditions) and then washed with buffers containing successively lower concentrations of salts, or higher concentrations of detergents, or at increasing temperatures until the signal to noise ratio for specific to non-specific hybridization is high enough to facilitate detection of specific hybridization.
• Stringent temperature conditions will usually include temperatures in excess of about 30° C, more usually in excess of about 37° C, and occasionally in excess of about 45° C.
• Stringent salt conditions will ordinarily be less than about 1000 mM, usually less than about 500 mM, more usually less than about 400 mM, typically less than about 300 mM, preferably less than about 200 mM, and more preferably less than about 150 mM.
• the combination of parameters is more important than the measure of any single parameter See, e.g , Wetmur et al , J Mol. Biol 31 :349-70 (1966), and Wetmur, Critical Reviews in Biochemistry and Molecular £/o/ ⁇ $y 26(34):227 59 (1991 )
• stringent conditions or “high stringency conditions,” as defined herein, may be hybridization in 50% f ormamide, 5x SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1 % sodium p ⁇ rophosphate, 5 x Denhardt s solution, sonicated salmon sperm DNA (50 g/ml), 0.1 % SDS, and 10% dextran sulfate at 42 C, with washes at 42 C in 0.2x SSC (sodium chloride/sodium citrate) and 50% formamide at 55 C, followed by a high stringency wash consisting of 0.1 x SSC containing EDTA at 55 C.
• 5x SSC 0.75 M NaCl, 0.075 M sodium citrate
• 50 mM sodium phosphate pH 6.8
• 0.1 % sodium p ⁇ rophosphate 5 x Denhardt s solution
• sonicated salmon sperm DNA 50 g/m
• nucleic acid sequences refers to the residues that are identical, after aligning the sequences, and introducing or deleting gaps, if necessary to achieve the maximum percent identity, and not considering any conservative substitutions as part of the sequence identity.
• the local homology algorithm of Smith and Waterman can conduct optimal alignment of sequences for comparison, e.g., by the homology alignment algorithm of Needleman and Wunsch (Needleman et a/., J. Mo/. Biol. 48:443 (1970)), by the search for similarity method of Pearson and Lipman (Pearson et a/., Proc. Natl. Acad.
• blastp which compares an ammo acid query sequence against a protein sequence database
• blastn which compares a nucleotide query sequence against a nucleotide sequence database
• blastx which compares the six-frame conceptual translation products of a nucleotide query sequences (both strands) against a protein sequence database
• tblastn which compares a protein query sequence against a nucleotide sequence database dynamically translated in all six reading frames (both strands)
• tblastx which compares the six frame translations of a nucleotide query sequence against the six-frame translations of a nucleotide sequence database.
• a nucleic acid “tag” is a selected nucleic acid with a specified nucleic acid sequence.
• a nucleic acid "probe” hybridizes to a nucleic acid “tag.”
• nucleic acid tags are incorporated as labels into biological libraries, and the tag nucleic acids are detected using an array of probes.
• a "list of tag nucleic acids” is a pool of tag nucleic acids, or a representation (i.e., an electronic or paper copy) of the sequences in the pool of tag nucleic acids.
• the pool of tags can be, for instance, all possible tags of a specified length (i.e., all 20-mers), or a subset thereof
• a set of nucleic acid tags binds to a probe with "minimal cross hybridization" when a single species (or "type") of tag in the tag set accounts for the majority of all tags which bind to an array comprising a probe species under stringent conditions Typically, about 80% or more of the tags bound to the probe species are of a single species under stringent conditions. Usually about 90% or more of the tags bound to the probe species are of a single species under stringent conditions Preferably 95% or more of the tags bound to the probe species are of a single species under stringent conditions.
• the terms “differentially expressed gene,” “expression,” “gene expressions” and “expression products” include production of a gene RNA message or the RNA message produced or both
• the terms “differentially expressed gene,” “expression,” “gene expression” and “expression products” include either translation of a mRNA into proteins, polypeptides or peptides, or to the produced proteins, polypeptides, or peptides themselves.
• a differentially expressed gene may be a gene whose expression is activated to a higher or lower level in a subject suffering a disease, specifically a cardiac, kidney or inflammatory disease state, relative to its expression in a normal or control subject.
• a differentially expressed gene may be either activated or inhibited at the nucleic acid level or protein level, for example, by a modulator, or may it be subject to alternative splicing to result in a different polypeptide product. Such differences may be evidenced by a change in mRNA levels, surface expression, secretion or other partitioning of a polypeptide, for example.
• Differential gene expression may include a comparison of expression between two or more genes, or a comparison of the ratios of the expression between two or more genes, or even a comparison of two differently processed products of the same gene, which differ between normal subjects and subjects suffering from a disease, specifically a cardiac, kidney or inflammatory disease state.
• Differential expression includes both quantitative, as well as qualitative, differences in the temporal or cellular expression pattern in a gene or its expression products among, for example, normal and diseased cells, or among cells which have undergone different disease events or disease stages.
• Modulation relates to a change in the production of a differentially expressed gene RNA message or the RNA message produced or both, in particular embodiments of the invention, modulation of "differentially expressed genes," “gene expression,” and “expression products” may refer to either a change in the translation of a gene RNA message into proteins, polypeptides or peptides, or to the produced proteins, polypeptides, or peptides themselves.
• a differentially expressed gene may have its expression modulated to a higher or lower level in a subject suffering or predisposed to a disease state, thus producing the desired therapeutic or prophylactic preventative effect.
• a differentially expressed gene may be either activated or inhibited at the nucleic acid level or protein level, for example, by a modulator.
• Modulators within the context of the present invention also include an antibody to, an antibody to a cellular receptor of, an organic molecule inhibitor capable of binding to a cellular receptor of one or more of these differentially expressed genes, antisense, triple helix, or ribozyme methodologies, or the gene itself and variants thereof
• label refers to a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, or chemical means.
• useful nucleic acid labels include 32 P, 35 S, fluorescent dyes, electron dense reagents, enzymes (e g , as commonly used in an ELISA), biotin, dioxigenm, or haptens and proteins for which antisera or monoclonal antibodies are available
• “differentially expressed gene” i.e., target and diagnostic genes) or “interactive gene” also includes (a) a gene comprising at least one of the DNA sequences disclosed herein; (b) any DNA sequence that encodes the ammo acid sequence encoded by the DNA sequences disclosed herein or contained within the coding region of the gene to which the DNA sequences disclosed here belong; (c) any DNA sequence that hybridizes to the complement of the coding sequences disclosed herein or contained within the coding region of the gene to which the DNA sequences disclosed herein belong under highly stringent conditions, e.g., hybridization to filter-bound DNA in 0.5M NaHP0 4 , 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65°C, and washing in O.lx SSC/0.1 % SDS at 68°C.
• SDS sodium dodecyl sulfate
• “Negative modulation,” as used herein, refers to a reduction in the level or activity of target gene product relative to the level or activity of the target gene product in the absence of the modulatory treatment.
• “Positive modulation,” as used herein, refers to an increase in the level or activity of target gene product relative to the level or activity of target gene product in the absence of modulatory treatment.
• treating refers to reduction or alleviation of at least one adverse effect or symptom of a disease, specifically cardiac, kidney or inflammatory disease, e.g., a disorder or disease characterized by or associated with differential polypeptide activity or nucleic acid expression
• a "cell associated activity” refers to a normal or abnormal activity or function of a cell. Examples of cell associated activities include proliferation, migration, differentiation, production or secretion of molecules such as proteins, and cell survival.
• the cell may be a cardiac cell, e.g , a cardiac myocyte or fibroblast.
• altered relates to a change, e.g., an increase or decrease, of a cell associated activity.
• the agent stimulates polypeptide activity or nucleic acid expression.
• stimulatory agents include an active gene protein, a nucleic acid molecule encoding differentially expressed gene that has been introduced into the cell, and a modulatory agent which stimulates polypeptide activity or differentially expressed gene expression and which is identified using the drug screening assays described herein.
• the "1-8U”, "prostacyclin stimulating factor”, “osf 2", “tissue specific mRNA protein”, “insulin like growth factor binding protein 6 (IGFBP 6)", “OSF 1”, “gas 1 ", “YMP”, “BTG2”, “pre B cell stimulating factor homolog (SDFIa)", “peripheral benzodiazepine receptor' , and "cellular ligand of annexin II (p11 )" polypeptides can be isolated from a variety of sources, such as from a variety of human tissue types, or prepared by recombinant and/or synthetic methods; all such polypeptides are specifically within the scope of the definition, regardless of their mode of preparation, and include variants thereof.
• sequence 1 8U refers to polypeptides having the same ammo acid sequence as a respective polypeptide derived from nature.
• native sequence polypeptides can be isolated from nature or can be produced by recombinant and/or synthetic means.
• the term "native sequence" in conjunction with the designation of a particular polypeptide specifically encompasses naturally occurring truncated or secreted forms (e.g., an extracellular domain sequence), as well as naturally occurring variant forms (e.g., alternatively spliced forms), and naturally occurring allelic variants of the named polypeptides.
• the native sequence 1-8U polypeptide has the ammo acid sequence of SEQ ID NO: 37
• the native sequence prostacyclin stimulating factor has the ammo acid sequence of SEQ ID NO: 38
• the native sequence osf 2 has the ammo acid sequence of SEQ ID NO: 39
• the native sequence tissue specific mRNA protein has the ammo acid sequence of SEQ ID NO: 40
• the native sequence insulin like growth factor binding protein 6 IGFBP-2
• the native sequence OSF 1 has the ammo acid sequence of SEQ ID NO.
• the native sequence gas 1 has the ammo acid sequence of SEQ ID NO 43
• the native sequence YMP has the ammo acid sequence of SEQ ID NO: 44
• the native sequence BTG2 has the ammo acid sequence of SEQ ID NO: 45
• the native sequence SDFIa has the ammo acid sequence of SEQ ID NO 46
• the native sequence peripheral benzodiazepm receptor has the ammo acid sequence of SEQ ID NO: 47
• the native sequence cellular ligand of annexin II has the ammo acid sequence of SEQ ID NO: 48
• variants and “ammo acid sequence variant” are used interchangeably and designate polypeptides in which one or more ammo acids are added and/or substituted and/or deleted and/or inserted at the N or C-terminus or anywhere with the corresponding native sequence, and which retain at least one activity (as defined below) of the corresponding native polypeptide
• a “variant” polypeptide usually has at least about 75% ammo acid sequence identity, or at least about 80% ammo acid sequence identity, preferably at least about 85% ammo acid sequence identity, even more preferably at least about 90% ammo acid sequence identity, and most preferably at least about 95% ammo acid sequence identity with the ammo acid sequence of the corresponding native sequence polypeptide.
• Activity refers to form(s) of the "1-8U", “prostacyciin-stimulating factor”, “osf 2", “tissue specific mRNA protein", “insulin-like growth factor binding protein 6 (IGFBP-6)", “OSF-1”, “gas 1 ", “YMP”, “BTG2”, “pre B cell stimulating factor homolog (SDFI a)", “peripheral benzodiazepine receptor”, or "cellular ligand of annexin II (p11 )” polypeptides which retain a qualitative biological and/or immunological property of a native sequence "1-8U", “prostacyciin-stimulating factor”, “osf 2", “tissue specific mRNA protein”, “insuhn-like growth factor binding protein 6 (IGFBP 6)", “OSF 1”, “gas 1 ", “YMP”, “BTG2”, “pre B cell stimulating factor homolog (SDF1 a)", "peripheral benz
• “Immunological cross-reactivity” means that the candidate polypeptide is capable of competitively inhibiting the qualitative biological activity of a "1-8U", “prostacyclin stimulating factor”, “osf 2", “tissue specific mRNA protein”, “insulin-like growth factor binding protein 6 (IGFBP-6)", “OSF-1”, “gas 1”, “YMP”, “BTG2”, “pre-B cell stimulating factor homolog (SDFI a)", “peripheral benzodiazepine receptor”, or "cellular ligand of annexin II (p11)” polypeptide having this activity with poiyclonal antisera raised against the known active "1-8U", “prostacyclin stimulating factor”, “osf 2", “tissue specific mRNA protein”, “insulin-like growth factor binding protein 6 (IGFBP-6)", “OSF 1”, “gas 1 ", “YMP”, “BTG2”, “pre B cell stimulating factor homolog (SDFI a)", "peripheral benzodiazepine receptor", or
• the immunological cross-reactivity is preferably "specific", which means that the binding affinity of the immunologically cross-reactive molecule identified to the corresponding polypeptide herein is significantly higher (preferably at least about 2-t ⁇ mes, more preferably at least about 4-t ⁇ mes, most preferably at least about 6 times higher) than the binding affinity of that molecule to any other known native polypeptide.
• antagonist is used in the broadest sense and includes any molecule that partially or fully blocks, inhibits or neutralizes a biological activity of a “1 8U”, “prostacyclin stimulating factor”, “osf 2", “tissue specific mRNA protein”, “insulin like growth factor binding protein 6 (IGFBP 6)", “OSF 1 ", “gas 1 ", “YMP”, “BTG2”, “pre B cell stimulating factor homolog (SDFI a)", “peripheral benzodiazepine receptor", or "cellular ligand of annexin II (pi 1)" polypeptide disclosed herein.
• the term “agonist” is used in the broadest sense and includes any molecule that mimics the biological activity of a “1 8U”, “prostacyclin stimulating factor”, “osf 2", “tissue specific mRNA protein”, “insulin like growth factor binding protein 6 (IGFBP 6)", “OSF 1", “gas 1 ", “YMP”, “BTG2”, “pre-B cell stimulating factor homolog (SDFI a)", “peripheral benzodiazepine receptor”, or "cellular ligand of annexin II (p11 )” polypeptide disclosed herein
• the term “antibody” is used in the broadest sense and specifically covers ant ⁇ -1 -8U, anti-prostacyclin- stimulating factor, anti-osf 2, anti-tissue specific mRNA protein, ant ⁇ -IGFBP-6, a ⁇ t ⁇ -OSF-1, a ⁇ t ⁇ -gas-1, anti-YMP, anti BTG2, anti-SDFI a, anti-peripheral benzodiazepine receptor
• the monoclonal antibodies specifically includes "chimeric" antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the cha ⁇ n(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Patent No. 4,816,567; Morrison et a/., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)).
• the monoclonal antibodies further include "humanized" antibodies or fragments thereof (such as Fv, Fab, Fab', F(ab'), or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulm.
• humanized antibodies are human immunoglobulms (recipient antibody) in which residues from a CDR of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity, and capacity, in some instances, Fv FR residues of the human immunoglobulm are replaced by corresponding non-human residues.
• humanized antibodies may comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. These modifications are made to further refine and maximize antibody performance.
• the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulm and all or substantially all of the FR regions are those of a human immunoglobulm sequence.
• the humanized antibody optimally also will comprise at least a portion of an immunoglobulm constant region (Fc), typically that of a human immunoglobulm.
• Fc immunoglobulm constant region
• the humanized antibody includes a PRIMATIZED antibody wherein the antigen-binding region of the antibody is derived from an antibody produced by immunizing macaque monkeys with the antigen of interest.
• Antibody fragments comprise a portion of an intact antibody, preferably the antigen binding or variable region of the intact antibody. Examples of antibody fragments include Fab, Fab', F(ab') 2 , and Fv fragments; diabodies; linear antibodies (Zapata et al., Protein Eng. 8(10):1057-1062 (1995)); single chain antibody molecules; and multispecific antibodies formed from antibody fragments.
• the present invention relates to methods involving in vivo and in vitro models of a disease, specifically cardiac, kidney or inflammatory disease, coupled with sensitive and high throughput assays, preferably microarray assays, to identify genes differentially expressed in such diseases
• the expression of the differentially expressed genes of the present invention can be determined from peripheral blood, tissues, or cells of a subject.
• the genes of the present invention are differentially expressed in cells and peripheral blood relative to normal cells and peripheral blood or differentially expressed relative to cells and peripheral blood at different disease stages.
• a variety of methods can be utilized for the identification of genes involved in a disease, specifically cardiac, kidney or inflammatory disease. Described below are experimental models, which can be utilized for the generation of biological samples that can be used for the identification of such genes. Samples generated in model categories can be characterized for the presence of differentially expressed gene sequences, as discussed below. /.
• Models For Identifying Differentially Expressed Genes Representative models of disease, specifically cardiac, kidney or inflammatory disease, states are described herein These models can be utilized within the context of the present invention, e.g., for the identification of genes which are differentially expressed in normal cells versus cells in a disease, specifically cardiac, kidney or inflammatory disease, state, in cells within different diseases, among cells within a single given disease state, in cells within different stages of a disease, or in cells within different time stages of a disease.
• a gene may be regulated one way, i.e., the gene can exhibit one differential gene expression pattern, in a given model, but can be regulated differently in another model.
• the use, therefore, of multiple models can be helpful in distinguishing the roles and relative importance of particular genes in a disease, specifically cardiac, kidney or inflammatory disease.
• animal models of a disease, specifically cardiac, kidney or inflammatory disease, and related disorders can be utilized to discover differentially expressed gene sequences.
• RNA from both the normal and disease state model is isolated and analyzed for differentially expressed genes using microarray analysis.
• three representative / ? vivo cardiac disease models, a representative kidney disease model, and a representative inflammatory disease model have been successfully utilized to identify differentially expressed genes of the present invention. These genes are expressed at higher or lower levels in the disease state, relative to the normal state, and preferably are expressed at least about a two-fold higher or lower level relative to the normal state at at least one time point.
• Representative in vivo animal models for use in the present invention include the following: general inflammation - carrageenan induced paw edema, arachidomc acid-induced ear inflammation; arthritis - adjuvant- induced polyarthritis, collagen induced arthritis, streptococcal cell wall-induced arthritis; multiple sclerosis experimental autoimmune encephalomyehtis (EAE); Systemic Lupus Erythematosis (SLE); NZB - spontaneous SLE mouse, DNA/anti-DNA immune complex-induced SLE; insulin-dependent diabetes mellitus • NOD spontaneous diabetes mouse; inflammatory bowel disease - acetic acid or t ⁇ nitrobenzene sulf onic (TNBS) induced ulcerative colitis; respiratory disease - antigen induced bronchoconstnction (asthma), lipopolysaccha ⁇ de (LPS)- ⁇ nduced acute respiratory distress syndrome (ARDS), analgesia - acetic acid induced or phenylqumone-
• the present invention is not limited to the in vivo models recited above and that any known models can be used within the context of the present invention.
• b. In Vitro Models Another model that can be utilized within the context of the present invention to discover differentially expressed gene sequences is the in vitro specimen model.
• the specimen model uses biological samples from subjects, e.g., peripheral blood, cells and tissues, including surgical and biopsy specimens
• specimens can represent normal peripheral blood and tissue or peripheral blood and tissue from patients suffering from a disease, specifically cardiac, kidney or inflammatory disease, or having undergone surgical treatment for disorders involving a disease, such as, for example, coronary bypass surgery
• Surgical specimens can be procured under standard conditions involving freezing and storing in liquid nitrogen (see Karmali et al., Br. J. Cancer 48:689 96 (1983)).
• RNA from specimen cells is isolated by, for example, differential centrifugation of homogenized tissue, and analyzed for differential expression relative to other specimen cells, preferably using microarray analysis.
• Cell lines can also be used to identify genes that are differentially expressed in a disease, specifically cardiac, kidney or inflammatory disease. Differentially expressed genes are detected, as described herein, by comparing the pattern of gene expression between the experimental and control conditions. In such models, genetically matched disease cell lines (e.g., variants of the same cell line) may be utilized. For example, the gene expression pattern of two variant cell lines can compared, wherein one variant exhibits characteristics of one disease state while the other variant exhibits characteristics of another disease state.
• genetically matched disease cell lines e.g., variants of the same cell line
• two variant cell lines both of which exhibit characteristics of the same disease, specifically cardiac, kidney or inflammatory disease, but which exhibit differing degrees of disease disorder severity may be used.
• genetically matched cell lines can be utilized, one of which exhibits characteristics of a disease, specifically cardiac, kidney or inflammatory disease, state, while the other exhibits a normal cellular phenotype.
• the cell line variants are cultured under appropriate conditions, harvested, and RNA is isolated and analyzed for differentially expressed genes, as with the other models.
• microarray analysis is used.
• differentially expressed genes of the present invention can be identified by using a variety of methods, which are well known to those of skill in the art. For example, differential screening (Tedder et al., Proc. Natl. Acad. Sci. USA 85:208 12 (1988)), subtractive hybridization (Hednck et al., Nature 308:149 53 (1984), Lee et al., Proc. Natl. Acad. Sci. USA 88:2825 (1984)), and differential display (Liang etal., U.S. Patent No. 5,262,311 (1993), which is incorporated herein by reference in its entirety), can be utilized to identify nucleic acid sequences derived from genes that are differentially expressed.
• RNA either total or mRNA
• Any RNA isolation technique that does not select against the isolation of mRNA can be utilized for the purification of such RNA samples. See, e.g., Ausubel et al., supra. Additionally, large numbers of tissue samples can readily be processed using techniques well known to those of skill in the art, such as, for example, the single-step RNA isolation process of Chomczynski (U.S. Patent. No. 4,843,155, which is incorporated herein by reference in its entirety).
• microarrays are utilized to assess differential expression of genes.
• DNA microarrays preferably are utilized within the methods of the present invention to assess the expression profile of genes expressed in normal subjects and subjects suffering from a disease, specifically cardiac, kidney or inflammatory disease.
• Identification of the differentially expressed disease genes of the present invention can be performed by: constructing normalized and subtracted cDNA libraries from mRNA extracted from the cells or tissue of healthy animals and an animal model of disease or of healthy patients and diseased patients, i.e., using any of the in vitro or in vivo models described herein; purifying the DNA of ations from cDNA libraries representing healthy and diseased cells or tissue, microarraying the purified DNA for expression analysis; and probing microarrays to identify the genes from the clones that are differentially expressed using labeled cDNA from healthy and diseased cells or tissues.
• PCR amplified inserts of cDNA clones are applied to a substrate in a dense array.
• At least 10,000 nucleotide sequences are applied to the substrate.
• the microarrayed genes, immobilized on the microchip at 10,000 elements each, are suitable for hybridization under stringent conditions.
• Fluorescentl ⁇ labeled cDNA probes may be generated through incorporation of fluorescent nucleotides by reverse transcription of RNA extracted from tissues of interest.
• Labeled cDNA probes applied to the chip hybridize with specificity to each spot of DNA on the array. After stringent washing to remove non-specifically bound probes, the chip is scanned by confocal laser microscopy. Quantitation of hybridization of each arrayed element allows for assessment of corresponding mRNA abundance.
• in vivo models of disease states were used to detect the differentially expressed genes of the present invention.
• three representative cardiac disease models, a representative kidney disease model, and a representative inflammatory disease model were successfully utilized to identify specific differentially expressed genes.
• separate DNA libraries were constructed from mRNA extracted from disease state tissue and normal tissue. From these libraries, at least 20,000 unidentified cDNA clones were preferably chosen for analysis and microarrayed on chips. Probes generated from normal and disease tissue, from multiple time points, were hybridized to the microarray.
• genes, which are differentially expressed in normal and diseased tissue were revealed and further identified by DNA sequencing.
• the analysis of the clones for differential expression reveal genes whose expression is elevated or decreased in association with a disease, specifically cardiac, kidney or inflammatory disease, in the specific in vivo model chosen.
• LVH rats were examined for expression of ANP mRNA which, according to published data (Schunkert et al., 1995, supra), should increase in the diseased animals.
• mRNA was extracted from the left ventricle of each animal and analyzed by Northern blot ( Figure 1). ANP transcripts were significantly elevated ( " 5-fold) at 10 weeks and 20 weeks relative to normal. The levels of mRNA were examined for BNP ( Figure 1 ), cardiac ⁇ -actin (not shown) and ⁇ -myos ⁇ n heavy chain (not shown) by Northern blot and, as expected, these were also elevated in the diseased animals. Blots were probed for cyciophihn transcripts to attest to equal loading of mRNA.
• CVB3 infection in mice results in myocardial disease progression, which was used as a model for examination of the pathogenesis of virus-induced human myocarditis.
• the virus is directly injurious to myocardial cells early following infection during the preinflammatory period as determined by light and electron microscopic cytological assessment [km ⁇ * et al., J. Med. Virol 47. 251 259 (1995); Chow et al., Lab. Invest. 64: 55-64 (1991); McManus et al., Clin. Immunol. ImmunopathoL 68-159 169 (1993); Melnick et al., J. Expert. Med. 93: 247 266 (1951 )).
• mice (Jackson Laboratories, Bar Harbor, Maine) were 4 weeks of age when received at St. Paul's Hospital Animal Care Facility, University of British Columbia. Mice were acclimatised for one week in a St. Paul's Hospital Animal Care Facility level 2 biohazard containment room prior to the onset of the experiment. Any mice that died naturally during the course of the disease were not included in groups of mice to be used for RNA extraction. Mice were euthanized by CO, narcosis.
• Myocarditic CVB3 was kindly provided by Dr. Charles J. Gauntt (University of Texas, San Antonio, Texas) and was stored at -80°C. Virus was propagated in HeLa cells (American Type Tissue Culture Collection, Rockville, MD.) and is routinely titred before the onset of all experiments using the plaque assay method, with modifications as previously described (Anderson etal., J. Virol. 70: 4632-4645 (1996)).
• Adolescent A/J mice were infected with 1 x10 5 pfu of myocarditic CVB3 or PBS sham and euthanized on days 3, 9, and 30 post-infection
• Ten to fifteen mice per group (CVB3 infected or sham injected) per time-point (days 3, 9, and 30) were euthanized and heart muscle was removed.
• a small portion of the apex of the heart was removed and fixed in 4% paraformaldehyde. The remainder of the heart was flash frozen in liquid nitrogen and stored at -80°C for future RNA isolation.
• Sections from the heart were fixed in fresh DPBS-buffered 4% paraformaldehyde overnight at 4°C. Fixed tissue was dehydrated in graded alcohols, cleared in xylene, embedded in paraffin, and sectioned for hematoxylin and eosin, and Masson's t ⁇ chrome stains. Serial sections were also prepared for in situ hybridization and nick-end labelling stained. The extent and severity of virus-induced injury (including coagulation necrosis, contraction band necrosis, and cytopathic effects), inflammation, and tissue fibrosis and calcification was evaluated and scored as previously described (Chow et al., supra).
• Tissue was collected 2 week, 4 week, 8 week, 12 week and 16 week post-surgery. Blood was collected the day before surgery and the day before sacrifice for measurement of plasma ANP level. On the day of necropsy, each heart was divided transversely into two halves so that the infarcted area is bisected. One half of the heart was used for histological evaluation, and the other for mRNA microarray analysis. Poly A + mRNA was prepared from each of the animais, as described herein, for assessment of differentially expressed genes in the disease state, using microarray analysis in a preferred embodiment. A summary of the findings of the microarray analysis is provided in Figure 4, and described in detail below. b.
• septum tissue was obtained from diseased rat hearts obtained through the left ventricle rat Ml model of Pfeffer et al., as described above Poly A + mRNA was prepared from each of these septu s, as described herein, for assessment of differentially expressed genes in the disease state, using microarray analysis in a preferred embodiment.
• a summary of the findings of the microarray analysis is provided in Figure 4, and described in detail below.
• an in vivo model of kidney disease was used within the context of the present invention.
• the specific rat model used was an inherited form of autosomal dominant polycystic kidney disease (ADPKD) which develops in Ha SPRD rats (Kaspareit Rittmghaus et al.. Transplant Proc. 6: 2582 3 (1990); Cowley et al., Kidney Int. 43:522-34 (1993)). Renal cysts and renal failure were evident in six month old male heterozygous rats (Cy/ + ), whereas control rats (+/+) showed no sign of cysts or renal failure. Five diseased animals (Cy/+) and one normal ( + /+) were sacrificed and the kidneys removed.
• ADPKD autosomal dominant polycystic kidney disease
• poly A+ mRNA was prepared, as described previously, for assessment of differentially expressed genes in the disease state, using microarray analysis in a preferred embodiment.
• a summary of the findings of the microarray analysis is provided in Figure 4, and described in detail below.
• microarray analysis is performed on cDNA obtained from both the normal and disease state models to assess the presence of differentially expressed disease state genes.
• High quality DNA is important for the microarray printing process.
• DNA was generated by PCR amplification of the cDNA insert from clones. 10,000 clones per array were generally used. Indeed, it is preferable to use a robust method of template preparation, preferably accomplished in 96-well plates.
• PCR primers were synthesized that amplify insert DNA from the vector pCR2.1, which was used for library construction. After 30 cycles of amplification each PCR product is passed over a gel filtration column to remove unincorporated primers and salts. To maintain robustness, the columns are packed in 96-well filter plates and liquid handling is performed robotically. The yield, per PCR reaction, is generally 2-5 ⁇ g, enough DNA for printing several hundred chips.
• Figure 2 shows a gel containing purified PCR products from a single plate of 96 rat cDNA clones. In some samples no amplified DNA was produced (e.g., #37 and #44) and, in some cases, the size of the product indicated that the plas id lacked an insert (e.g , #49 and #61 ).
• 96 purified samples from a single microtiter plate were produced as a microarray.
• 85 vl of PCR reaction mixture was aliquoted into each well of a thin walled, 0.2 ml 96-well plate
• the reaction mixture contained 0.2 mM each dNTP, 1.25 units of Taq polymerase, and 1 X Taq buffer (Boehringer Mannheim).
• Primers, 1 ⁇ xx ⁇ each, are from vector regions, which flank the cloning site of pCR2.1 and include a 5' primary amine with a 6 carbon linker to facilitate attachment of DNA product to the glass surface of the microarray chip.
• PCR conditions are: 2 mm., 95°C to denature, then 30 cycles of 95°, 30 sec. / 65°C, 40 sec / 72°C, 1 mm. 30 sec, and a final extension of 72°C, 5 mm. using a MJResearch PTC 100 thermocycier.
• PCR products were purified by gel filtration over Sephacryl 400 (Sigma). Briefly, 400 /I of pre-swollen Sephacryl 400 was loaded into each well of a 96-well filter plate (PallBiosupport) and spun into a collection plate at 800g for 1 mm Wells were washed 5 times with 0.2x SSC. PCR reaction mixtures were loaded onto the column and purified DNA (flow-thru) was collected at 800g for 1 mm. Samples are dried down at 50° C overnight and arrayed. Fluorescent probe pairs were synthesized by reverse transcription of poly A+ RNA using, separately, Cy3 dCTP and Cy5 dCTP (Amersham).
• RNA is degraded in 0.1 M NaOH, 65°C for 10 mm.
• Labeled cDNA was purified by successive filtration with Chroma Spin 30 spin columns (Clontech) following manufacturer's instructions. Samples were dried at room temperature in the dark using a covered Speed- Vac. Probes were applied to the test chip for hybridization and the data collected essentially as described in Schena et al., supra. The intensity of hybridization signal at each element reflected the level of expression of the mRNA for each gene in the rat ventricle. Digitized signal data was stored and prepared for analysis. The data from this experiment is presented in Figure 3.
• clones may be randomly picked from a cDNA library, resulting in redundant selection of genes expressed at high and moderate abundance. It is estimated that 50% of all transcripts in a cell derive from " 400 genes (Bishop et al., Nature 250(463):199-204 (1974)). Thus, random picking of 20,000 cDNA clones would represent roughly half that number of different genes, and rare transcripts may be underrepresented. However, in a separate embodiment of the present invention, a greater number of different clones can be randomly chosen for microarray analysis if cDNA libraries produced from the models of the present invention are first normalized.
• a normalized version of a cDNA library was generated from normal tissue, cells or blood (e.g., the left ventricle of normal rat), in a particular embodiment, poly A+ RNA was purified from the tissue samples provided by the in vivo disease models described above.
• a directionally cloned cDNA library was first generated by conventional methods. Briefly, double stranded cDNA was generated by priming first strand synthesis for reverse transcription using oligo dT primers which contain a Not I restriction site.
• Xba I adapters are added to the 5' end of the cDNA, and the cDNA size was selected for > 500 bp and ligated into the corresponding restriction sites of phagemid vector pCR2.1 (Invitrogen, San Diego CA).
• Phagemid vector pCR2.1 contains an F1 origin of replication.
• the cDNA library can be propagated as single stranded phage with appropriate helper virus.
• Single stranded, circular DNA was extracted from the phage library and serves as "tester” DNA in the hybridization step of normalization.
• the other component of the hybridization, "driver" DNA was generated from the library by PCR amplification using a set of primers specific for the region of the vector, which flanks the cloned inserts.
• oligonucleotides (10 ⁇ g each), corresponding to polylmker sequence (same strand as tester DNA) which is present in the PCR product, was included in the hybridization reaction to block annealing of vector-specific sequences which are in common between tester and driver DNA.
• the reaction mixture under oil, was heated 3 mm. at 80° C, and hybridization performed at 30°C for 24 hr (calculated C 0 t " 5).
• Single stranded circles were purified from the reaction mixture by hydroxylapatite (HAP) chromatography, converted to double strand DNA, and electroporated into bacteria to yield a normalized cDNA library representative of genes expressed in the left ventricle of rat.
• HAP hydroxylapatite
• a subtracted library can be made using protocols similar to those used to generate normalized libraries Again, the method of Bonaldo et al., supra, described here briefly is used.
• a total cDNA library is generated from the tissue obtained from the disease model (e.g., left ventricle taken from a hypertrophic rat (10 week aortic banded)).
• the cDNA library is directionally cloned in pCR2.1 vector and single stranded tester DNA derived as described above for library normalization.
• the driver DNA is generated by PCR amplification of cloned inserts from the total cDNA library prepared from the left ventricle of normal rat. Hybridization occurs between sequences, which are in common to normal and diseased hearts.
• the reaction is driven more thoroughly (calculated C 0 , " 27) than normalization by using more driver (1.5 ⁇ g vs.
• 30,000 clones are picked for microarraying. 25,000 clones are taken from the normalized library generated from normal rats, and 5,000 from the subtracted library made from hypertrophic rats.
• the subtracted library should be less complex (i.e., fewer unique clones) than the normalized library, therefore, fewer clones need be picked. If, as estimated, only about 1 % of all 20,000 genes are unique to the disease state, then the complexity would be only about 200, thus picking 5000 would likely yield a representative of each.
• Preferably included on the microarray with the 30,000 unidentified genes are a set of known clones.
• Rat clones for the list of genes were isolated by PCR amplification from cDNA libraries using specific primer pairs. These known clones were included because they represent genes of particular interest and help evaluate the sensitivity of the microarray methodology. Indeed, any genes of particular interest may be included on such microarrays.
• ANP, BNP, endotheim, ⁇ myosm heavy chain, and ⁇ -actin are genes that change expression levels in the LVH model, and thus they serve as useful positive controls in the in vivo model exemplified herein
• the differentially expressed genes preferably are detected by microarray methods; however, differential expression detected by any other means, including but not limited to RNA diagnosticing methods, Northern blotting, immunodetectio ⁇ , protein-protein interactions, biological activity and other methods known in the art fall within the scope of the present invention.
• the differentially expressed cardiac genes may be identified through the use of differential screening methods. Differential screening involves the duplicate screening of a cDNA library in which one copy of the library is screened with a total cell cDNA probe corresponding to the mRNA population of one cell type, while a duplicate copy of the cDNA library is screened with a total cDNA probe corresponding to the mRNA population of a second cell type.
• one cDNA probe can correspond to a total cell cDNA probe of a cell type or tissue derived from a control subject, while the second cDNA probe can correspond to a total cell cDNA probe of the same cell type derived from an experimental subject.
• Subtractive hybridization techniques generally involve the isolation of mRNA taken from two different sources, e.g., control and experimental tissue, the hybridization of the mRNA or single-stranded cDNA reverse- transcribed from the isolated mRNA, and the removal of all hybridized, and therefore double-stranded, sequences.
• the remaining non-hybridized, single-stranded cDNAs potentially represent clones derived from genes that are differentially expressed in the two mRNA sources.
• Such single-stranded cDNAs are then used as the starting material for the construction of a library comprising clones derived from differentially expressed genes.
• the differential display technique describes a procedure, utilizing PCR (U.S. Patent No. 4,683,202, incorporated herein by reference), which allows for the identification of sequences derived from genes which are differ- entially expressed.
• isolated RNAs are reverse-transcribed into single-stranded cDNA, using standard techniques known to those of skill in the art.
• Primers for the reverse transcriptase reaction can include, but are not limited to, oligo dT-containi ⁇ g primers, preferably of the 3' primer type of oligonucleotide described below.
• this technique uses pairs of PCR primers, as described below, which allow for the amplification of clones representing a random subset of the RNA transcripts present within any given cell. Using different pairs of primers allows each of the mRNA transcripts present in a cell to be amplified. Among such amplified transcripts can be identified those which have been produced from differentially expressed genes.
• the pattern of clones resulting from the reverse transcription and amplification of the mRNA of two different cell types is displayed via sequencing gel electrophoresis and compared. Differences in the two banding patterns indicate potentially differentially expressed genes.
• the differential expression of such putatively differentially expressed genes may be corroborated. Corroboration can be accomplished via, for example, such well-known techniques as Northern analysis, quantitative RT-coupled PCR, microarrays, or RNase protection.
• the differentially expressed genes can be further characterized, and can be identified as target or diagnostic genes, as discussed below. 8. Detection of Differentially Expressed Genes Using Microarray Analysis
• microarrays were constructed and probed as described above.
• Figure 4 provides a detailed summary of the characteristics of twelve representative differentially expressed disease genes of the present invention.
• the expression data provided relates to the counterpart gene expressed in the in vivo models described supra, and shows the differential expression data of representative genes obtained through the disease models of the present invention and determined via microarray analysis.
• Figure 4 provides the clone identification number for the differentially expressed model gene.
• those representative disease model differentially expressed genes were found to correspond to human genes encoding 1-8U, prostacyciin-stimulating factor, osf 2, tissue specific mRNA, insulin-like growth factor binding protein 6, OSF 1, gas 1 , YMP, BTG2, pre-B cell stimulating factor homolog (SDFI a), peripheral benzodiazepine receptor, and cellular ligand of annexin II (p1 1).
• probes were applied to the microarrays for hybridization and the data collected essentially as described in Schena et al., supra. The intensity of hybridization signal at each element reflected the level of expression of the mRNA for each gene.
• the numeric data provided in Figure 4 reflects the relative expression level of the gene in the disease state as compared to the expression level of the gene in the normal, or non- disease state, in the five representative disease state models delineated above and as determined by microarray analysis. Specifically, the data shown in Figure 4 provides a positive or negative multiple of the expression level of the gene in the disease state, as compared to the normal state in the representative models.
• Data are reported as differential expression values with positive numbers indicative of genes expressed at higher levels in the diseased tissue relative to normal tissue, and negative values indicative of lower expression in disease.
• the data also reflects expression levels of genes in certain disease models over various time points. For example, gene expression in the myocardial infarction model was compared at 2, 4, 8, 12, and 16 weeks for the representative genes in the disease state versus the normal state. Indeed, such experimentation provides valuable data regarding the temporal relationship of gene expression levels in disease states and provides important insights regarding the treatment, diagnosis, and modulation of differentially expressed disease state genes, as discussed in detail infra.
• clones assayed on microarrays were found to be differentially expressed. Secondary chips may be used for more extensive hybridizations, including examination of individual animals, and more thorough evaluation of time points.
• clones that reproducibly scored in microarray analysis to be at least about two fold elevated or decreased were microarrayed on separate secondary chips and their expression levels determined. It is understood, however, that differentially expressed genes exhibiting less than about a two-fold change in expression, e.g., less than one, one-half, or one quarter, or greater than about a two-fold change in expression, e.g., greater than three, five, ten, twenty, one hundred-fold, or one thousand-fold, are within the scope of the present invention.
• the isolated amplified gene fragments obtained through differential display have their 5' terminal end at some random point within the gene and have 3' terminal ends at a position corresponding to the 3' end of the transcribed portion of the gene.
• the remainder of the gene i.e., the 5' end of the gene, when using differential display
• RNA can be isolated following standard procedures from an appropriate tissue or cellular source.
• a reverse transcription reaction can then be performed on the RNA using an oligonucleotide primer complementary to the mRNA that corresponds to the amplified cloned fragment, for the priming of first strand synthesis. Because the primer is anti parallel to the mRNA, extension will proceed toward the 5' end of the mRNA.
• the resulting RNA/DNA hybrid can then be tailed with guanmes using a standard terminal transferase reaction, the hybrid can be digested with RNase H, and second strand synthesis can then be primed with a poly C primer.
• Amplification of nucleic acids from samples is sometimes desirable and can be accomplished by, e.g., PCR See generally Erlich, ed v PCR Technology Principles and Applications for DNA Amplification, Freeman Press (New York, NY 1992); Innis et al , eds., PCR Protocols A Guide to Methods and Applications, Academic Press Inc. (San Diego, CA 1990); Mattila et al., Nucleic Acids Res. 19:4967 (1991); Eckert et al., PCR Methods and Applications 1:17 (1991), McPherson et al., eds., PCR, IRL Press (Oxford); and U.S. Patent No. 4,683,202.
• LCR ligase chain reaction
• NASBA nucleic acid based sequence amplification
• differentially expressed disease genes identified by the methods of the present invention were sequenced and compared with human gene nucleotide sequences in the GenBank database.
• the nucleotide sequences of those twelve representative differentially expressed genes and their human gene counterparts obtained through the GenBank database are provided in Figure 5.
• the differentially expressed genes of the present invention were identical to a known rat gene nucleotide sequence, the known sequence is used for comparison.
• Figure 5 shows alignment data comparing the cDNA encoding the representative differentially expressed genes discovered through the microarray analysis of the present invention with human cDNA sequences in the GenBank database, which correspond to the human proteins 1 -8U (SEQ ID N0:1; SEQ ID N0:2), prostacyciin-stimulating factor (SEQ ID N0:3; SEQ ID N0:4), osf-2 (SEQ ID N0:5; SEQ ID N0:6), tissue specific mRNA (SEQ ID N0:7; SEQ ID N0:8), insulin-like growth factor binding protein 6 (SEQ ID N0:9; SEQ ID N0:10), OSF-1 (SEQ ID N0:11; SEQ ID N0:12), gas-1 (SEQ ID N0:13; SEQ ID N0:14), YMP (SEQ ID N0:15; SEQ ID N0:16), BTG2 (SEQ ID N0:17; SEQ ID N0:18), pre-B cell stimulating factor
• the complementary nucleic acid sequences of these human genes and their expression products may be used in all embodiments of the present invention. Indeed, all or a portion of the DNA sequences of differentially expressed genes discovered through the methods of the present invention may be found to correspond to known genes or, for example, ESTs or SNPs. in instances where a complete human gene is not found to directly correspond to the differentially expressed genes of the present invention, yet a portion of a known gene, EST, or SNP does correspond, one skilled in the art may preferably elucidate the complete corresponding human gene and/or the differentially expressed gene, or portions thereof, using known methods. This, in turn, may allow for elucidation of the complete differentially expressed human or model gene, which may be used in the methods described infra.
• Figure 6 shows alignment data comparing human cDNA sequences in the GenBank database with multiple cDNA clones encoding the differentially expressed genes discovered through the microarray analysis of the present invention, which correspond to the human proteins 1-8U, tissue specific mRNA, YMP, pre-B cell stimulating factor homolog (SDFI a), peripheral benzodiazepine receptor, and cellular ligand of annexin II (p1 1). Start and stop codons are underlined.
• Figure 7 shows the nucleotide sequences encoding the polypeptides corresponding to the human genes 1-8U (SEQ ID N0:25), prostacyciin-stimulating factor (SEQ ID N0:26), osf-2 (SEQ ID N0:27),tissue specific mRNA (SEQ ID N0:28), insulin-like growth factor binding protein 6 (SEQ ID N0:29), OSF-1 (SEQ ID N0:30), gas-1 (SEQ ID N0:31), YMP (SEQ ID N0:32), BTG2 (SEQ ID N0:33), pre-B cell stimulating factor homolog (SDFI a) (SEQ ID N0:34), peripheral benzodiazepine receptor (SEQ ID N0:35), and cellular ligand of annexin II (p1 1 ) (SEQ ID N0:36).
• Figure 8 shows the amino acid sequences of the polypeptides corresponding to 1-8U (SEQ ID N0:37), prostacyciin-stimulating factor (SEQ ID N0:38), osf-2 (SEQ ID N0:39), tissue specific mRNA (SEQ ID N0:40), insulinlike growth factor binding protein 6 (SEQ ID N0:41 ), OSF-1 (SEQ ID N0:42), gas-1 (SEQ ID N0:43), YMP (SEQ ID N0:44), BTG2 (SEQ ID N0:45), pre-B cell stimulating factor homolog (SDFI a) (SEQ ID N0:46), peripheral benzodiazepine receptor (SEQ ID N0:47), and cellular ligand of annexin II (pi 1 ) (SEQ ID N0:48).
• Figure 9 shows the characteristics of the human cDNA corresponding to the human proteins 1-8U, prostacyciin-stimulating factor, osf-2, tissue specific mRNA, insulin-like growth factor binding protein 6, OSF-1, gas-1, YMP, BTG2, pre-B cell stimulating factor homolog (SDFI a), peripheral benzodiazepine receptor, and cellular ligand of annexin II (p1 1).
• Figure 9 provides the GenBank identification number for each of the human genes corresponding to the differentially expressed genes discovered through microarray analysis, the size of the cDNA of each gene, the coding sequence (CDS), the number of amino acids in the encoded human protein, whether or not the human protein has a signal sequence, and whether or not the human protein is a tra ⁇ smembrane protein.
• the identified differentially expressed genes may in turn be used to design specific oligonucleotide probes and primers.
• the term "primer” as used here includes any nucleic acid capable of priming template-dependent synthesis of a nascent nucleic acid.
• the nucleic acid may be able to hybridize a template, but not be extended for synthesis of nascent nucleic acid that is complementary to the template.
• the term "template” may refer to a nucleic acid that is used in the creation of a complementary nucleic acid strand to the "template" strand.
• the template may be either RNA or DNA, and the complementary strand may also be RNA or DNA.
• the complementary strand may comprise all or part of the complementary sequence to the template, or may include mutations so that it is not an exact, complementary strand to the template. Strands that are not exactly complementary to the template strand may hybridize specifically to the template strand in detection assays described here, as well as other assays known in the art, and such complementary strands that can be used in detection assays are part of the invention.
• the term “amplification” may refer to any method or technique known in the art or described herein for duplicating or increasing the number of copies or amount of a target nucleic acid or its complement.
• the term “amplicon” refers to the target sequence for amplification, or that part of a target sequence that is amplified, or the amplification products of the target sequence being amplified. In certain other embodiments, an “amplicon” may include the sequence of probes or primers used in amplification.
• probes and primers may include repetitive stretches of adenine nucleotides (poly-A tails) normally attached at the ends of the RNA for the identified differentially expressed gene.
• probes and primers may be specifically designed to not include these or other segments from the identified genes, as one of ordinary skill in the art may deem certain segments more suitable for use in the detection methods disclosed.
• sequences that correspond to exon regions of the gene may be used in most cases.
• One skilled in the art may select segments from the published exon sequences, or may assemble them into a reconstructed mRNA sequence that does not contain intronic sequences.
• one skilled in the art may select or assemble segments from any of the identified gene sequences into other useful forms, such as coding segment reconstructions of mRNA sequences from published genomic sequences of the identified differentially expressed genes, as part of the present invention.
• Such assembled sequences would be useful in designing probes and primers, as well as providing coding segments for protein translation and for detection, diagnosis, and prognosis embodiments of the invention described herein.
• Primers can be designed to amplify transcribed portions of the differentially expressed genes of the present invention that would include any length of nucleotide segment of the transcribed sequences, up to and including the full length of each gene. It is preferred that the amplified segments of identified genes be an amplicon of at least about 50 to about 500 base pairs in length. It is more preferred that the amplified segments of identified genes be an amplicon of at least about 100 to about 400 base pairs in length, or no longer in length than the amplified segment used to normalize the quantity of message being amplified in the detection assays described herein. Such assays include RNA diagnosticing methods, however, differential expression may be detected by other means, and all such methods would fall within the scope of the present invention.
• the predicted size of the gene segment calculated by the location of the primers relative to the transcribed sequence, would be used to determine if the detected amplification product is indeed the gene being amplified. Sequencing the amplified or detected band that matches the expected size of the amplification product and comparison of the band's sequence to the known or disclosed sequence of the gene would confirm that the correct gene is being amplified and detected
• the identified differentially expressed genes may also be used to identify and isolate full length gene sequences, including regulatory elements for gene expression, from genomic human DNA libraries.
• the cDNA sequences or portions thereof, identified in the present disclosure may be used as hybridization probes to screen genomic human DNA libraries by conventional techniques. Once partial genomic clones have been identified,
• chromosomal walking may isolate full-length genes (also called “overlap hybridization”). See Chinault et al., Gene 5:111-26 (1979).
• a partial genomic clone has been isolated using a cDNA hybridization probe, nonrepetitive segments at or near the ends of the partial genomic clone may be used as hybridization probes in further genomic library screening, ultimately allowing isolation of entire gene sequences for the disease, specifically cardiac, kidney or inflammatory disease, state genes of interest, it will be recognized that full length genes may be obtained using small ESTs via technology currently available and described in this disclosure (Sambrook et al., supra; Chinault et al., supra). Sequences identified and isolated by such means may be useful in the detection of disease genes using the detection and diagnostic methods described herein, and are part of the invention
• the identified genes may be used to identify and isolate cDNA sequences
• the sequences, or portions thereof, identified in the present disclosure may be used as hybridization probes to screen human cDNA libraries by conventional techniques. Comparison of cloned cDNA sequences with known human or animal cDNA or genomic sequences may be performed using computer programs and databases known in the art. Figure 5 provides a detailed comparison of the sequence similarity between the twelve representative genes of the present invention and their human counterparts.
• the nucleotide sequences of clones derived from the models of disease disclosed herein were matched to known human genes among the GenBank database.
• Any method suitable for detecting protein protein interactions can be employed for identifying interactive gene products by identifying interactions between gene products and the differentially expressed genes of the present invention.
• An interactive gene can be differentially expressed and, therefore, can have the characteristics of a target or diagnostic gene.
• Differentially expressed gene products can be cellular or extracellular proteins. Those gene products that interact with such known gene products represent interactive gene products and the genes that encode them represent interactive genes
• an interactive gene product can be used, in conjunction with standard techniques, to identify its corresponding interactive gene. For example, at least a portion of the ammo acid sequence of the interactive gene product can be ascertained using techniques well known to those of skill in the art, such as the Edman degradation technique (see, e g , Creighton, Proteins: Structures and Molecular Principles, W. H. Freeman & Co.
• ammo acid sequence obtained can be used as a guide for the generation of oligonucleotide mixtures that can be used to screen for interactive gene sequences Screening can be accomplished, for example, by standard hybridization or PCR techniques. Techniques for the generation of oligonucleotide mixtures and the screening are well known. See, e.g., Ausubel et al., supra, and Innis et al., supra
• methods can be employed which result in the simultaneous identification of interactive genes that encode the protein interacting with a protein involved in a disease, specifically cardiac, kidney or inflammatory disease.
• methods include, for example, probing expression libraries with a labeled protein known or suggested to be involved in a disease, using this protein in a manner similar to the well known technique of antibody probing of ⁇ gtll libraries.
• yeast two hybrid system One method that detects protein interactions in vivo, the yeast two hybrid system, is described in detail for illustration only and not by way of limitation. One version of this system has been described (Chien et al., Proc. Natl Acad. Sci. USA 88: 9578 82 (1991)) and is commercially available from Clontech (Palo Alto, CA).
• plasmids are constructed that encode two hybrid proteins: the first hybrid protein consists of the DNA binding domain of a transcription factor (e.g., activation protein) fused to a known protein, in this case, a protein known to be involved in a disease, specifically cardiac, kidney or inflammatory disease; the second hybrid protein consists of the transcription factor's activation domain fused to an unknown protein that is encoded by a cDNA which has been recombmed into this plasmid as part of a cDNA library.
• the plasmids are transformed into a strain of the yeast Saccharomyces cerevisiae that contains a reporter gene (e.g., lacZ) whose expression is regulated by the transcription factor's binding site.
• a transcription factor e.g., activation protein
• Either hybrid protein alone cannot activate transcription of the reporter gene
• the DNA binding hybrid protein cannot activate transcription because it does not provide the activation domain function and the activation domain hybrid protein cannot activate transcription because it lacks the domain required for binding to its target site (e.g., it cannot localize to the transcription activator protein's binding site).
• Interaction between the DNA binding hybrid protein and the library encoded protein reconstitutes the functional transcription factor and results in expression of the reporter gene, which is detected by an assay for the reporter gene product
• the two hybrid system or related methodology can be used to screen activation domain libraries for proteins that interact with a known "bait" gene product.
• gene products known to be involved in a disease can be used as the bait gene products
• Total genomic or cDNA sequences are fused to the DNA encoding an activation domain
• This library and a plasmid encoding a hybrid of the bait gene product fused to the DNA binding domain are co transformed into a yeast reporter strain, and the resulting transformants are screened for those that express the reporter gene
• the bait gene can be cloned into a vector to translationally fuse to the DNA encoding the DNA binding domain of the protein.
• the colonies are purified and the (library) plasmids responsible for reporter gene expression are isolated
• the inserts in the plasmids are sequenced to identify the proteins encoded by the cDNA or fragments into a vector such that they are translationally fused to the activation domain of GAL4 generates the library
• This library can be co transformed aiong with the bait gene-GAL4 fusion plasmid into a yeast strain which contains a lacZ gene whose expression is controlled by a promoter which contains a GALA activation sequence.
• a cDNA encoded protein fused to GAL4 activation domain that interacts with the bait gene product will reconstitute an active GAL4 transcription factor and thereby drive expression of the lacZ gene.
• Colonies expressing lacZ can be detected by their blue color in the presence of X-gal. cDNA containing plasmids from such a blue colony can then be purified and used to produce and isolate the bait gene product interacting protein using techniques routinely practiced in the art.
• an interactive gene Once an interactive gene has been identified and isolated, it can be further characterized, for example, as discussed below.
• X. CHARACTERIZATION OF DIFFERENTIALLY EXPRESSED GENES Differentially expressed genes and interactive genes, as well as genes identified by alternative means, can be further characterized by utilizing methods such as those discussed herein. Analyses such as those described herein yield information regarding the biological function of the identified genes. An assessment of the biological function of the differentially expressed genes, in addition, can lead to their designation as target or diagnostic genes.
• any of the differentially expressed genes whose further characterization indicates that a modulation of the gene's expression or a modulation of the gene product's activity can inhibit or treat a disease, specifically cardiac, kidney or inflammatory disease, can be designated "target genes" as defined above.
• target genes and target gene products along with those discussed below will constitute the focus of the compound discovery strategies discussed below. Further, such target genes, target gene products or modulating compounds can be used as part of disease treatment methods described below.
• any of the differentially expressed genes whose further characterization indicates that such modulations do not positively affect a disease, specifically cardiac, kidney or inflammatory disease, but whose expression pattern contributes to a gene expression "diagnostic" pattern correlative of a disease can be designated a "diagnostic gene.” "Diagnostic patterns" are discussed below.
• Each of the target genes may also function as diagnostic genes, as can all or a portion of the interactive genes.
• the interactive genes may also be characterized according to techniques such as those described herein.
• target genes which yield information indicating that they are differentially expressed and that modulation of the gene's expression or a modulation of the gene product's activity can inhibit a disease, specifically cardiac, kidney or inflammatory disease, or ameliorate a disease associated symptom can also be designated target genes.
• target genes and target gene products may constitute the focus of the compound discovery strategies and treatment methods described below.
• the characterization of one or more of the interactive genes can reveal a lack of differential expression, yet evidence that modulation of the gene's activity or expression can nonetheless ameliorate symptoms of a disease, specifically cardiac, kidney or inflammatory disease. In such cases, these genes and gene products may also be considered a focus of the compound discovery strategies and treatment methods of the present invention Where an interactive gene's characterization indicates that modulation of gene expression or gene product activity cannot retard or treat the disease but is differentially expressed and contributes to a gene expression diagnostic pattern correlative of a disease or its disorders, such interactive genes can additionally be designated as diagnostic genes. A variety of techniques can be utilized to further characterize the identified genes.
• the nucleotide sequence of the identified genes can be used to further characterize such genes.
• the sequence of the identified genes can reveal homologies to one or more known sequence motifs, which can yield information regarding the biological function of the identified gene product.
• an analysis of the tissue or cell type distribution of the mRNA produced by the identified genes can be conducted, utilizing standard techniques well known to those of skill in the art. Such techniques can include, for example, Northern analyses, microarrrays, RT coupled PCR, and RNase protection techniques. In a preferred embodiment, microarrays are utilized.
• Such analyses provide information as to whether the identified genes are expressed in tissues expected to contribute to a disease, specifically cardiac, kidney or inflammatory disease. These techniques can also provide quantitative information regarding steady state mRNA regulation, yielding data concerning which of the identified genes exhibits a high level of regulation preferably in tissues which can be expected to contribute to a disease state.
• standard in situ hybridization techniques can be utilized to provide information regarding which cells within a given tissue express the identified gene. Specifically, these techniques can provide information regarding the biological function of an identified gene relative to a disease, specifically cardiac, kidney or inflammatory disease, where only a subset of the cells within the tissue is thought to be relevant to the disorder.
• the sequences of the identified genes can be used, utilizing standard techniques, to place the genes onto genetic maps, e.g., mouse (Copeiand et al , Trends in Genetics 7:1 13 18 (1991 )) and human genetic maps (Cohen et al . Nature 266:698 701 (1993)).
• This mapping information can yield information regarding the genes' importance to human disease by identifying genes that map within genetic regions to which known genetic disease disorders map
• In vivo systems can include animal systems that naturally exhibit symptoms of a disease, specifically cardiac kidney or inflammatory disease, or ones engineered to exhibit such symptoms as detailed infra.
• the role of identified gene products can be determined by transfecting cDNAs encoding these gene products into appropriate cells or cell lines, such as, biopsy specimens from patients having undergone surgical treatment, as described above. Further, these systems can include transgenic animals.
• In vitro systems can include cell based systems comprising cell types known or suspected of contributing to a disease, specifically cardiac, kidney or inflammatory disease. Cell types may comprise normal cells or non normal cells containing modifications known to contribute or suspected of contributing to a disease. Such systems are discussed in detail below. Additional procedures to identify and isolate the human homolog of the differentially expressed genes of the present invention when non-human model systems are utilized are also described below.
• the expression of these genes can be modulated within the in vivo or in vitro systems, i.e., either over- or under-expressed, and the subsequent effect on the system then assayed.
• the activity of the product of the identified gene can be modulated by either increasing or decreasing the level of activity in the m vivo ox in vitro system of interest, and its subsequent effect then assayed.
• treatment can include a modulation of gene expression or gene product activity. Characterization procedures such as those described herein can indicate where such modulation should involve an increase or a decrease in the expression or activity of the gene or gene product of interest.
• genes of the present invention can be obtained using cloning methods well known to those skilled in the art, including the use of appropriate probes to detect the genes within an appropriate cDNA or gDNA (genomic DNA) library.
• appropriate probes to detect the genes within an appropriate cDNA or gDNA (genomic DNA) library.
• oligonucleotide probes for the novel genes can be synthesized using techniques well known to those of skill in the art based on the human or animal DNA sequences disclosed in the Figures.
• the probes can be used to screen cDNA libraries prepared from an appropriate cell or cell line in which the gene is transcribed. Genomic DNA libraries can be prepared from any source.
• “differentially expressed gene” i.e., target and diagnostic genes) or “interactive gene” also includes (a) a gene comprising at least one of the DNA sequences disclosed herein; (b) any DNA sequence that encodes the ammo acid sequence encoded by the DNA sequences disclosed herein or contained within the coding region of the gene to which the DNA sequences disclosed here belong; (c) any DNA sequence that hybridizes to the complement of the coding sequences disclosed herein or contained within the coding region of the gene to which the DNA sequences disclosed herein belong under highly stringent conditions, e.g., hybridization to filter-bound DNA in 0.5M NaHP0 4 , 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65°C, and washing in O.lx SSC/0.1 % SDS at 68°C.
• SDS sodium dodecyl sulfate
• the invention also includes nucieic acid molecules, preferably DNA molecules, which hybridize to, and are therefore the complements of, the DNA sequences (a) through (e), in the preceding paragraph.
• hybridization conditions can be highly stringent or less highly stringent, as described above.
• nucleic acid molecules are deoxyoligonucleotides ("oligos")
• highly stringent conditions can refer, e.g., to washing in 6x SSC/0.05% sodium pyrophosphate at 37°C (for 14-base oligos), 48°C (for 17-base oligos), 55°C (for 20-base oligos), and 60°C (for 23-base oligos).
• These nucleic acid molecules can act as target gene antisense molecules, useful, for example, in target gene regulation or as antisense primers in amplification reactions of target, diagnostic, or interactive gene nucleic acid sequences. Further, such sequences can be used as part of ribozyme or triple helix sequences, also useful for target gene regulation. Still further, such molecules can be used as components of diagnostic methods whereby disease, specifically cardiac, kidney or inflammatory disease, disorders can be detected.
• the invention also encompasses (a) DNA vectors that contain any of the foregoing coding sequences or their complements (/.e. f antisense); (b) DNA expression vectors that contain any of the foregoing coding sequences operatively associated with a regulatory element that directs the expression of the coding sequences; and (c) genetically engineered host cells that contain any of the foregoing coding sequences operatively associated with a regulatory element that directs the expression of the coding sequences in the host cell.
• regulatory elements include inducible and non- ⁇ nduc ⁇ ble promoters, enhancers, operators and other elements known to those skilled in the art that drive and regulate expression.
• the invention includes fragments of any of the DNA sequences disclosed herein.
• homologs of these gene sequences can, for example, be present in other species.
• homologs can be identified and can readily be isolated, without undue experimentation, by molecular biological techniques well known in the art. Further, there can exist genes at other genetic loci within the genome that encode proteins, which have extensive homology to one or more domains of such gene products. These genes can also be identified using similar techniques.
• the isolated differentially expressed gene sequence can be labeled and used to screen a cDNA library constructed from mRNA obtained from an organism of interest.
• Hybridization conditions will be of a lower stringency when the cDNA library was derived from an organism different from the type of organism from which the labeled sequence was derived.
• the labeled fragment can be used to screen a genomic library derived from the organism of interest, again, using appropriately stringent conditions.
• Such low stringency conditions will be well known to those of skill in the art, and will vary predictably depending on the specific organisms from which the library and the labeled s ⁇ uences are derived.
• a previously unknown differentially expressed or interactive gene-type sequence can be isolated by performing PCR using two degenerate oligonucleotide primer pools designed on the basis of ammo acid sequences within the gene of interest.
• the template for the reaction can be cDNA obtained by reverse transcription of mRNA prepared from human or non human cell lines or tissue known or suspected to express a differentially expressed or interactive gene allele.
• the PCR product can be subcloned and sequenced to ensure that the amplified sequences represent the sequences of a differentially expressed or interactive gene-iike nucleic acid sequence.
• the PCR fragment can then be used to isolate a full-length cDNA clone by a variety of methods.
• the amplified fragment can be labeled and used to screen a bactenophage cDNA library.
• the labeled fragment can be used to screen a genomic library.
• RNA can be isolated, following standard procedures, from an appropriate cellular or tissue source.
• a reverse transcription reaction can be performed on the RNA using an oligonucleotide primer specific for the most 5' end of the amplified fragment for the priming of first strand synthesis
• the resulting RNA/DNA hybrid can then be "tailed" with guanines using a standard terminal transferase reaction, the hybrid can be digested with RNase H, and second strand synthesis can then be primed with a poly C primer.
• cDNA sequences upstream of the amplified fragment can easily be isolated.
• RNA species can be quantitated by means that do not necessarily require amplification by PCR.
• amplification means may include other amplification techniques, for example, isothermic amplification techniques such as the one developed by Gen-Probe (San Diego, CA) or the ligase chain reaction (LCR).
• isothermic amplification techniques such as the one developed by Gen-Probe (San Diego, CA) or the ligase chain reaction (LCR).
• LCR ligase chain reaction
• the differentially expressed or interactive gene identified is the normal, or wild type gene
• this gene can be used to isolate mutant aileles of the gene. Such isolation is preferable in processes and disorders that are known or suspected to have a genetic basis.
• Mutant aileles can be isolated from individuals either known or suspected to have a genotype contributing to disease, specifically cardiac, kidney or inflammatory disease, symptoms. Mutant aileles and mutant allele products can then be utilized in the therapeutic and diagnostic assay systems described below
• a cDNA of a mutant gene can be isolated, for example, by using PCR.
• the first cDNA strand can be synthesized by hybridizing a oligo-dT oligonucleotide to mRNA isolated from tissue known or suspected of being expressed in an individual putativei ⁇ carrying the mutant allele, and by extending the new strand with reverse transcriptase.
• the second strand of the cDNA can then be synthesized using an oligonucleotide that hybridizes specifically to the 5'- end of the normal gene.
• the product is then amplified via PCR, cloned into a suitable vector, and subjected to DNA sequence analysis through methods known to one skilled in the art.
• the mutat ⁇ on(s) responsible for the loss or alteration of function of the mutant gene product can be ascertained.
• a genomic or cDNA library can be constructed and screened using DNA or RNA, respectively, from a tissue known to express or suspected of expressing the gene of interest in an individual suspected of carrying or known to carry the mutant allele.
• the normal gene or any suitable fragment thereof can then be labeled and used as a probe to identify the corresponding mutant allele in the library.
• Clones containing this gene can then be purified through methods routinely practiced in the art, and subjected to sequence analysis as described above.
• an expression library can be constructed utilizing DNA isolated from or cDNA synthesized from a tissue known to express or suspected of expressing the gene of interest in an individual suspected of carrying or known to carry the mutant allele.
• gene products made by the putatively mutant tissue can be expressed and screened using standard antibody screening techniques in conjunction with antibodies raised against the normal gene product as described below.
• standard antibody screening techniques see, Harlow et a/., eds., Antibodies: A Laboratory Manual, Cold Spring Harbor Press (Cold Spring Harbor, NY, 1988).
• a poiyclonal set of antibodies are likely to cross-react with the mutant gene product.
• Library clones detected via their reaction with such labeled antibodies can be purified and subjected to sequence analysis as described supra.
• Molecular cloning and expression techniques for making biological and synthetic oligonucleotides and nucleic acids are well known in the art. Wide varieties of cloning and expression and in vitro amplification methods suitable for the construction of nucleic acids are well known to persons of skill. Examples of techniques and instructions sufficient to direct persons of skill through many cloning exercises for the expression and purification of biological nucleic acids (DNA and RNA) are found in Berger et al., Guide to Molecular Cloning Techniques, Methods in Enzymology, volume 152, Academic Press, Inc. (San Diego, CA); Sambrook et al., supra; and Ausubel et al., supra.
• Nucleic acids such as tag nucleic acids can be cloned into cells (thereby creating recombinant tagged cells) using standard cloning protocols such as those described in Berger et al., supra; Sambrook et al., supra; and Ausubel et al., supra.
• nucleic acid sequences within the context of the present invention will find utility in a variety of applications in disease, specifically cardiac, kidney or inflammatory disease, detection, diagnosis, prognosis and treatment.
• applications within the scope of the present disclosure comprise amplification of differentially expressed genes using specific primers, detection of genes by hybridization with oligonucleotide probes, incorporation of isolated nucleic acids into vectors, expression of vector incorporated nucleic acids as RNA and protein, and development of immunologic reagents corresponding to gene encoded products.
• the sequences of isolated nucleic acids disclosed herein find utility as hybridization probes or amplification primers These nucieic acids may be used, for example, in diagnostic evaluation of biological samples or employed to clone full length cDNAs or genomic clones corresponding thereto.
• these probes and primers comprise oligonucleotide fragments. Such fragments are of sufficient length to provide specific hybridization to an RNA or DNA sample extracted from tissue and, in a preferred embodiment, may be used within the context of microarrays.
• the sequences typically may be 10 20 nucleotides, but may be longer. Longer sequences, e.g., 40, 50, 100, 500 and even up to full length, are preferred for certain embodiments
• nucleic acid molecules having contiguous stretches of about 10, 15, 17, 20, 30, 40, 50, 60, 75 or 100 or 500 nucleotides of a sequence comprising GenBank Accession numbers X57352, S75725, D13665, X67698, M62402, D90226, L13698, U52101 , U72649, L36034, M36035, and M38591 , corresponding to the human genes 1 -8U (SEQ ID N0:25), prostacyclin stimulating factor (SEQ ID N0:26), osf-2 (SEQ ID N0:27), tissue specific mRNA (SEQ ID N0.28), insuhn-like growth factor binding protein 6 (SEQ ID N0:29), OSF-1 (SEQ ID N0:30), gas 1 (SEQ ID N0.31 ), YMP (SEQ ID N0.32), BTG2 (SEQ ID N0.33), pre B cell stimulating factor homolog (SDFI a) (SEQ ID N0.34), peripheral
• Molecules that are complementary to the above mentioned sequences and that bind to these sequences under high stringency conditions are also contemplated. These probes are useful in a variety of hybridization embodiments, such as Southern and Northern blotting and microarray assays and diagnostics. In some cases, it is contemplated that probes may be used that hybridize to multiple target sequences without compromising their ability to effectively diagnose the disease state.
• multiple probes may be used for hybridization to a single sample.
• the use of a hybridization probe of between 17 and 100 nucleotides in length allows the formation of a duplex molecule that is both stable and selective. Molecules having complementary sequences over stretches greater than 20 bases in length are generally preferred in order to increase stability and selectivity of the hybrid, and thereby improve the quality and Degree of hybrid molecules It is generally preferred to design nucleic acid molecules having stretches of 20 to 30 nucleotides, or even longer.
• Such fragments may be readily prepared by directly synthesizing the fragment by chemical means or by introducing selected sequences into recombinant vectors for recombinant production.
• Differentially expressed or interactive gene products can be produced by synthetic techniques or via recombinant DNA technology using techniques well known in the art. Methods for preparing the differentially expressed or interactive gene polypeptides and peptides of the invention by expressing nucieic acid encoding differentially expressed or interactive gene sequences are described herein. Methods known to those skilled in the art can be used to construct expression vectors containing differentially expressed or interactive gene protein coding sequences and appropriate transc ⁇ ptional/translational control signals. These methods include in vitro recombinant DNA techniques, synthetic techniques and in vivo recombination/genetic recombination.
• RNA capable of encoding differentially expressed or interactive gene protein sequences can be chemically synthesized using, for example, synthesizers. See, for example, the techniques described in Gait, ed., Oligonucleotide Synthesis, IRL Press (Oxford, 1984).
• host-expression vector systems can be utilized to express the differentially expressed or interactive gene coding sequences of the invention.
• Such host-expression systems represent vehicles by which the coding sequences of interest can be produced and subsequently purified, but also represent cells which can, when transformed or transfected with the appropriate nucleotide coding sequences, exhibit the differentially expressed or interactive gene protein of the invention in situ.
• These include but are not limited to microorganisms such as bacteria (e.g., E. coli, B.
• subtilis transformed with recombinant bactenophage DNA, plasmid DNA or cosmid DNA expression vectors containing differentially expressed or interactive gene protein coding sequences; yeast (e.g., Saccharomyces, Pichia) transformed with recombinant yeast expression vectors containing the differentially expressed or interactive gene protein coding sequences; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing the differentially expressed or interactive gene protein coding sequences; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing differentially expressed or interactive gene protein coding sequences; or mammalian cell systems (e.g., COS, CHO, BHK, 293, 3T3) harboring recombinant expression constructs containing promoters derived from the genome of
• Pichia may be utilized.
• a number of expression vectors can be advantageously selected depending upon the use intended for the differentially expressed or interactive gene protein being expressed.
• vectors which direct the expression of high levels of fusion protein products that are readily purified are desirable.
• Such vectors include the £ coli expression vector pUR278 (Rather et al., EMBO J.
• differentially expressed or interactive gene protein coding sequence can be ligated individually into the vector in frame with the lacZ coding region so that a fusion protein is produced and plN vectors (Inouye et al., Nucleic Acids Res. 13:3101-09 (1985); Van Heeke et a/.N. Biol. Chem. 264:5503-09 (1989)).
• pGEX vectors can also be used to express foreign polypeptides as fusion proteins with glutathione 5-transferase (GST).
• fusion proteins are soluble and can easily be purified from lysed cells by adsorption to glutathio ⁇ e-agarose beads followed by elation in the presence of free glutathione.
• the pGEX vectors are designed to include thrombm or factor Xa protease cleavage sites so that the cloned target gene protein can be released from the GST moiety.
• a number of viral-based expression systems can be utilized.
• the differentially expressed or interactive gene coding sequence of interest can be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence.
• This chimeric gene can then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non essential region of the viral genome (e.g., region El or E3) will result in a recombinant virus that is viable and capable of expressing differentially expressed or interactive gene protein in infected hosts (see Logan et al , Proc. Natl.
• Specific initiation signals can also be required for efficient translation of inserted differentially expressed or interactive gene coding sequences. These signals include the ATG initiation codon and adjacent sequences. Where an entire identified gene, including its own initiation codon and adjacent sequences, is inserted into the appropriate expression vector, no additional translations! control signals can be needed. However, in cases where only a portion of the identified coding sequence is inserted, exogenous transiational control signals, including, perhaps, the ATG initiation codon, must be provided. Furthermore, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert.
• exogenous transiational control signals and initiation codons can be of a variety of origins, both natural and synthetic.
• the efficiency of expression can be enhanced, for example, by inclusion of appropriate transcription enhancer elements or transcription terminators (see Bittner et al., Methods in Enzymol. 153:516-44 (1987))
• a host cell strain can be chosen that modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Modifications (e.g., giycosylation) and processing (e.g., cleavage) of protein products can be important for the function of the protein. Different host cells have characteristic and specific mechanisms for the post transiational processing and modification of proteins.
• Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed.
• eukaryotic host cells that possess the cellular machinery for proper processing of the primary transcript, giycosylation, and phosphor ⁇ lation of the gene product can be used.
• mammalian host cells include but are not limited to CHO, VERO, BHK, HeLa, COS, MDCK, 293, 3T3, and W138.
• Cell lines that stably express the differentially expressed or interactive gene protein can be engineered. Rather than using expression vectors which contain viral origins of replication, host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter or enhancer sequences, transcription terminators, and polyadenylation sites) and a selectable marker. Following the introduction of the foreign DNA, engineered cells can be allowed to grow for 1 2 days in an enriched media, and then are switched to a selective media.
• expression control elements e.g., promoter or enhancer sequences, transcription terminators, and polyadenylation sites
• the selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines.
• This method advantageously can be used to engineer cell nes that express the identified gene protein.
• Such engineered cell lines can be particularly useful in screening and evaluation of compounds that affect the endogenous activity of the differen tialiy expressed or interactive gene protein
• fusion protein system allows for the ready purification of non denatured fusion proteins expressed in human cell lines (Janknecht et al., Proc. Natl. Acad. Sci. USA 88:8972 76 (1981 )).
• the gene of interest is subcloned into a vaccinia recombination plasmid such that the gene's open reading frame is translationally fused to an ammo-terminal tag consisting of six histidme residues.
• Extracts from cells infected with recombinant vaccinia virus are loaded onto n ⁇ 2 * nitnioacetic acid-agarose columns and histid e-tagged proteins are selectively eluted with imidazole-contaimng buffers.
• the differentially expressed or interactive gene protein can be labeled, either directly or indirectly, to facilitate detection of a complex formed between the differentially expressed or interactive gene protein and a test substance.
• labeling systems including radioisotopes such as enzyme labeling systems that generate a detectable colorimet ⁇ c signal or light when exposed to substrate, and fluorescent labels.
• fusion proteins that can facilitate labeling, solubility, immobilization or detection.
• Indirect labeling involves the use of a third protein, such as a labeled antibody, which specifically binds to either a differentially expressed or interactive gene product.
• a labeled antibody such as a labeled antibody, which specifically binds to either a differentially expressed or interactive gene product.
• Such antibodies include but are not limited to poiyclonal, monoclonal, chimeric, single chain, Fab fragments and fragments produced by a Fab expression library.
• the differentially expressed genes of the present invention can be expressed in an expression vector in which the gene is operably linked to a native or other promoter.
• the promoter is an eukaryotic promoter for expression in a mammalian cell.
• the transcription regulation sequences typically include a heterologous promoter and optionally an enhancer, which is recognized by the host.
• the selection of an appropriate promoter for example trp, lac, phage promoters, glycolytic enzyme promoters and tRNA promoters, depends on the host selected.
• Commercially available expression vectors can be used. Vectors can include host-recognized replication systems, amplifiabie genes, selectable genes, host sequences useful for insertion into the host genome, and the like.
• the means for introducing the expression construct into a host cell may vary depending upon the particular construction and the target host. Suitable means include fusion, conjugation, transfection, transduction, electroporation or injection, as described in Sambrook et al., supra.
• a wide variety of host cells can be employed for expression of the differentially expressed gene, both prokaryotic and eukaryotic. Suitable host cells include bacteria such as E. coli, yeast (particularly Pichia), filamentous fungi, insect cells, mammalian cells, typically immortalized, e.g., mouse, CHO, human and monkey cell lines and derivatives thereof. Preferred host cells are able to process the differentially expressed gene product to produce an appropriate mature polypeptide. Processing includes giycosylation, ubiquitination, disulfide bond formation, and general post transiational modification.
• the differentially expressed protein may be isolated by conventional means of protein biochemistry and purification to obtain a substantially pure product, i.e., 80, 95 or 99% free of cell component contaminants, as described in Jacoby, Methods in Enzymology, Vol. 104, Academic Press (New York, NY, 1984); Scopes, Protein Purification, Principles and Practice 2nd Edition, Sp ⁇ nger-Verlag, (New York, NY 1987); and Deutscher, ed., Guide to Protein Purification, Methods in Enzymology, Vol. 182 (1990). If the protein is secreted, it can be isolated from the supernatant in which the host cell is grown. If the protein is not secreted, the protein can be isolated from a lysate of the host cells.
• the differentially expressed gene encoding the polypeptide is analyzed to detect putative transmembrane sequences.
• sequences are typically very hydrophobic and are readily detected by the use of sequence analysis software such as Lasergene (DNAstar, Madison, WI).
• sequence analysis software such as Lasergene (DNAstar, Madison, WI).
• the presence of transmembrane sequences is often deleterious when a recombinant protein is synthesized in many expression systems, especially £ coli, as it leads to the production of insoluble aggregates that are difficult to renature into the native conformation of the protein. Deletion of transmembrane sequences typically does not significantly alter the conformation of the remaining protein structure.
• transmembrane sequences embedded within a membrane are inaccessible. Antibodies to these sequences will not prove useful for in vivo or in situ studies. Deletion of transmembrane-encoding sequences from the genes used for expression may be achieved by conventional techniques. For example, restriction enzyme sites may be used to excise the desired gene fragment, or PCR-type amplification may be used to amplify only the desired part of the gene.
• computer sequence analysis is used to determine the location of predicted major antigenic determinant epitopes of the polypeptide.
• Software capable of carrying out this analysis is readily available commercially.
• Such software typically uses conventional algorithms such as the Kyte/Doolittie or Hopp/Woods methods for locating hydrophilic sequences characteristically found on the surface of proteins, which likely act as antigenic determinants.
• polypeptides may be prepared which contain at least the essential features of the antigenic determinant and which may be employed the generation of antisera against the polypeptide.
• Mimgenes or gene fusions encoding these determinants may be constructed and inserted into expression vectors by conventional methods, for example, using PCR cloning methodology.
• synthetic peptides corresponding to the antigenic determinants may be prepared.
• Such peptides are preferably at least six ammo acid residues long, and may contain up to approximately 50 residues, which is the approximate upper length limit of automated peptide synthesis machines, such as those available from PE Applied Biosystems (Foster City, CA).
• Use of such small peptides for vaccination typically requires conjugation of the peptide to an immunogenic carrier protein such as hepatitis B surface antigen, keyhole limpet hemocyamn or bovine serum albumin. Methods for performing this conjugation are well known in the art.
• the expression products of the differentially expressed genes of the present invention may comprise differences of ammo acid sequence. These may, for instance, be minor sequence differences of the polypeptide which arise due to natural variation within the population or they may be homologs found in other species and include native sequence polypeptides and their variants. They also may be sequences which do not occur naturally but which are sufficiently similar that they function similarly or elicit an immune response that cross-reacts with natural forms of the polypeptide. Sequence differences may be prepared by conventional methods of site-directed mutagenesis such as those described above for removing the transmembrane sequence.
• Ammo acid sequence mutants of the polypeptide may be substitutio ⁇ al, msertional or deletion mutants.
• Deletion mutants differentially expressed lack one or more residues of the native protein which are not essential for function or immunogemc activity, and are exemplified by the mutants lacking a transmembrane sequence described above.
• Another common type of deletion mutants is one lacking secretory signal sequences or signal sequences directing a protein to bind to a particular part of a cell.
• An example of the latter sequence is the SH2 domain, which induces protein binding to phosphotyrosme residues.
• Substitutional mutants typically exchange one am o acid for another at one or more sites within the protein and may be designed to modulate one or more properties of the polypeptide, such as stability against proteolytic cleavage. Substitutions preferably are conservative, that is, one ammo acid is replaced with another of similar shape and charge.
• Insertional mutants include fusion proteins such as those used to allow rapid purification of the polypeptide and also may include hybrid proteins containing sequences from other homologous proteins and polypeptides.
• an insertional mutant may include portions of the ammo acid sequence of the polypeptide from one species, together with portions of the homologous polypeptide from another species.
• Other insertional mutants may include those in which additional ammo acids are introduced within the coding sequence of the polypeptide. These typically are smaller insertions than the fusion proteins described above and are introduced, for example, to disrupt a protease cleavage site
• major antigenic determinants of the polypeptide are identified by an empirical approach in which portions of the gene encoding the polypeptide are expressed in a recombinant host, and the resulting proteins tested for their ability to elicit an immune response.
• PCR may be used to prepare a range of peptides lacking successively longer fragments of the C-termi ⁇ us of the protein. The immunoprotective activity of each of these peptides then identifies those fragments or domains of the polypeptide which are essential for this activity. Further studies in which only a small number of ammo acids are removed at each iteration then enables the location of the antigenic determinants of the polypeptide.
• Mimetics are peptide-containmg molecules which mimic elements of protein secondary structure. See, e.g , Johnson etal., Biotechnology and Pharmacy, Chapman and Hall (New York, NY, 1993).
• the underlying rationale behind the use of peptide mimetics is that the peptide backbone of proteins exists chiefly to orient ammo acid side chains in such a way as to facilitate molecular interactions, such as those of antibody and antigen.
• a peptide mimetic is expected to permit molecular interactions similar to the natural molecule.
• peptide mimetic concept has thus far focused on mimetics of ⁇ -turns within proteins, which are known to be highly antigenic.
• Computer-based algorithms as discussed above may predict likely ⁇ turn structure within a polypeptide. Once the component am o acids of the turn are determined, peptide mimetics may be constructed to achieve a similar spatial orientation of the essential elements of the ammo acid side chains.
• Differentially expressed and interactive gene products include those proteins, or portions thereof, encoded by the differentially expressed and interactive gene sequences obtained by the methods of the present invention.
• differentially expressed and interactive gene products can include differentially expressed and interactive gene polypeptides encoded by the differentially expressed and interactive gene sequences contained in the coding regions of the genes to which clones and DNA sequences of the differentially expressed genes of the present invention belong.
• differentially expressed and interactive gene products can include proteins that represent functionally equivalent gene products.
• An equivalent differentially expressed or interactive gene product can contain deletions, additions or substitutions of ammo acid residues within the ammo acid sequence encoded by the differentially expressed or interactive gene sequences described above, but which result in a silent change thus producing a functionally equivalent differentially expressed on interactive gene product.
• Am o acid substitutions can be made based on similarity in polarity, charge, solubility, hydrophobicity, hydrophiiicity, or the amphipatic nature of the residues involved.
• “Functionally equivalent” includes a protein capable exhibiting a substantially similar in vivo activity as the endogenous differentially expressed or interactive gene products encoded by the differentially expressed or interactive gene sequences of the present invention.
• “functionally equivalent” includes peptides capable of interacting with other cellular or extracellular molecules in a manner substantially similar to the way in which the corresponding portion of the endogenous differentially expressed or interactive gene product would.
• the methods of the invention can also be used to detect genetic lesions in a differentially expressed gene of the present invention, thereby determining if a subject with the iesioned gene is at risk for a disorder characterized by differentially expressed gene expression or polypeptide activity, in preferred embodiments, the methods include detecting, in a biological sample from a subject, the presence or absence of a genetic lesion characterized by, for example, an alteration affecting the integrity of a gene encoding an polypeptide or the misexpression of the gene.
• such genetic lesions can be detected by ascertaining the existence of at least one of: a deletion of one or more nucleotides from a gene; an addition of one or more nucleotides to a gene; a substitution of one or more nucleotides of a gene; a chromosomal rearrangement of a gene; an alteration in the level of a messenger RNA transcript of a gene; aberrant modification of a gene, such as of the methylation pattern of the genomic DNA; the presence of a non-wild type splicing pattern of a messenger RNA transcript of a gene; a non-wild type level of a gene protein; allelic loss of a gene; and inappropriate post-transiational modification of a gene protein.
• assay techniques known in the art that can be used for detecting lesions in a gene.
• detection of a lesion may involve the use of a probe/primer in PCR (see, e.g. U.S. Patent Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or, alternatively, in LCR (see, e.g.,
• This method can include the steps of collecting a biological sample from a subject, isolating nucleic acid (e.g., genomic, mRNA or both) from the cells of the sample, contacting the nucleic acid sample with one or more primers which specifically hybridize to an differentially expressed gene under conditions such that hybridization and amplification of the cardiac gene (if present) occurs, and detecting the presence or absence of an amplification product, or detecting the size of the amplification product and comparing the length to a control sample.
• nucleic acid e.g., genomic, mRNA or both
• primers which specifically hybridize to an differentially expressed gene under conditions such that hybridization and amplification of the cardiac gene (if present) occurs
• detecting the presence or absence of an amplification product or detecting the size of the amplification product and comparing the length to a control sample.
• mutations in a differentially expressed gene from a sample can be identified by alterations in restriction enzyme cleavage patterns.
• sample and control DNA is isolated, amplified (optionally), digested with one or more restriction endonucleases, and fragment length sizes are determined by gel electrophoresis and compared. Differences in fragment length sizes between sample and control DNA indicates mutations in the sample DNA.
• sequence specific ⁇ bozymes see U.S. Patent No. 5,498,531 can be used to score for the presence of specific mutations by development or loss of a ribozyme cleavage site.
• any of a variety of sequencing reactions known in the art can be used to directly sequence the differentially expressed gene and detect mutations by comparing the sequence of the sample differentially expressed gene with the corresponding wild-type (normal) sequence.
• Examples of sequencing reactions include those based on tecn ⁇ iques developed by Maxim and Gilbert (Maxim etal., Proc. Natl. Acad. Sci. USA 74:560 (1977)) or Sanger (Sanger, Proc. Natl. Acad. Sci. 74:5463 (1977)).
• a variety of automated sequencing procedures can be utilized when performing the diagnostic assays, including sequencing by mass spectrometry (see, e.g., PCT
• RNA/RNA or RNA/DNA duplexes Other methods for detecting mutations in the differentially expressed gene include methods in which protection from cleavage agents is used to detect mismatched bases in RNA/RNA or RNA/DNA duplexes (Myers et al., Science 230:1242 (1985)), Cotton et al., Proc. Natl. Acad. Sci. USA 85:4397 (1988); Saleeba et al., Meth. Enzymol. 2(17):286-95 (1992)), electrophoretic mobility of mutant and wild type nucleic acid is compared (Onta et al., Proc. Natl. Acad. Sci. USA 86.2766 (1989); Cotton, Mutat. Res. 285:125-44 (1993); and Hayashi, Genet.
• the present invention includes biologically active fragments of the polypeptides, or analogs thereof, including organic molecules, which simulate the interactions of the peptides.
• biologically active fragments include any portion of the full-length polypeptide, which confers a biological function on the differentially expressed gene product, including ligand binding, and antibody binding.
• Ligand binding includes binding by nucleic acids, proteins or polypeptides, small biologically active molecules, or large cellular structures.
• purification and in particular embodiments, the substantial purification, of an encoded protein or peptide.
• the term "purified protein or peptide” includes compositions, isolatable from other components, wherein the protein or peptide is purified to any degree relative to its naturally- obtainable state, i.e., relative to its purity within a cell extract.
• a purified protein or peptide therefore also refers to a protein or peptide, free from the environment in which it may naturally occur.
• Partial purification may be accomplished by using fewer purification steps in combination, or by using different forms of the same general purification scheme.
• a cation-exchange column chromatography performed utilizing an HPLC apparatus generally results in a greater-fold purification than the same technique utilizing a low pressure chromatography system.
• Methods exhibiting a lower degree of relative purification may have advantages in total recovery of protein product, or in maintaining the activity of an expressed protein.
• antibodies are produced that bind with high specificity to the protein products of the differentially expressed genes of the present invention, e.g., all or a portion of the ammo acid sequence of the human genes 1 8U (SEQ ID N0.37), prostacyciin-stimulating factor (SEQ ID N0:38), osf 2 (SEQ ID N0:39),t ⁇ ssue specific mRNA (SEQ ID N0:40), insulin like growth factor binding protein 6 (SEQ ID N0:41 ), OSF 1 (SEQ ID N0.42), gas 1 (SEQ ID N0.43), YMP (SEQ ID N0:44), BTG2 (SEQ ID N0:45), pre-B cell stimulating factor homolog (SDFI a) (SEQ ID N0:46), peripheral benzodiazepine receptor (SEQ ID N0:47), and cellular ligand of annexin II (p11 ) (SEQ ID N0.48), respectively.
• antibodies capable of specifically recognizing one or more differentially expressed or interactive gene epitopes.
• Such antibodies can include, but are not limited to poiyclonal antibodies, monoclonal antibodies (mAbs), humanized or chimeric antibodies, single chain antibodies, Fab fragments, F(ab') 2 fragments, fragments produced by a Fab expression library, anti idiotypic (anti Id) antibodies, and epitope binding fragments of any of the above.
• mAbs monoclonal antibodies
• Such antibodies can be used, for example, in the detection of a diagnostic, target, or interactive gene in a biological sample, or, alternatively, as a method for the inhibition of abnormal target gene activity.
• such antibodies can be utilized as a disease, specifically cardiac, kidney or inflammatory disease, treatment method, or can be used as part of diagnostic techniques whereby patients can be tested for abnormal levels of diagnostic, target, or interactive gene proteins, or for the presence of abnormal forms of such proteins.
• diagnostic techniques such as Kohler et al., Nature 256:495-97 (1975), U.S. Patent No. 4,376,110, Kosbor et al., Immunology Today Ml (1983); Cole et al., Proc. Natl. Acad. Sci. USA 80:2026-30 (1983), Cole et al., Monoclonal Antibodies And Cancer Therapy, Alan R. Uss, Inc. (1985), pp.
• Antibody fragments recognizing specific epitopes can be generated by known techniques.
• such fragments include but are not limited to: the F(ab') 2 fragments which can be produced by pepsin digestion of the antibody molecule and the Fab fragments which can be generated by reducing the disulfide bridges of the F(ab') 2 fragments.
• Fab expression libraries can be constructed (Huse etal.. Science 246:1275-81 (1989)) to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity.
• XX. CELL- AND ANIMAL-BASED MODEL SYSTEMS Described herein are cell- and animal-based systems which represent reliable models for disease, specifically cardiac, kidney or inflammatory disease, disorders.
• the cell- and animal-based model systems can be used to identify differentially expressed genes via the models described above. Such systems can also be used to further characterize differentially expressed and interactive genes, for example, as a target gene. Additionally, such assays can be utilized as part of screening strategies designed to identify compounds, which are capable of preventing or ameliorating symptoms of a disease, specifically cardiac, kidney or inflammatory disease, disorders.
• the animal- and cell-based models can be used to identify drugs, pharmaceuticals, therapies and interventions which can be effective in treating a disease and related disorders.
• such animal models can be used to determine the LD 50 and the ED 50 in animal subjects, and such data can be used to determine the in vivo efficacy of potential disease treatments.
• Animal-based in vivo model systems of a disease can include both non-recombi ⁇ a ⁇ t animals as well as recombinantiy engineered transgenic animals.
• Non-recombinant animal models for a disease, specifically cardiac, kidney or inflammatory disease include, for example, munne models of myocardial infarction, cardiac hypertrophy, and kidney disease as described supra.
• Models based on cardioactive drugs may be generated, for example, by introducing such drugs into syngeneic mice After an appropriate period of time, the diseases that result from these injections of drugs can be detected and the mice used as models.
• the role of identified gene products can be determined by transfecting cDNA encoding such gene products into the appropriate ceil line and analyzing its effect on the cells' ability to induce a disease, specifically cardiac, kidney or inflammatory disease, in animal models such as these.
• the role of the identified gene products may be further analyzed by cultu ⁇ ng cells derived from the diseases which develop in the animal models, introducing these cultured cells into animals, and subsequently measuring the level of identified gene product present in the resulting disease.
• cell line variants are developed which can be useful in analyzing the role of quantitative or qualitative differences in the expression of the identified genes on the cells' ability to induce a disease.
• recombinant animal models exhibiting characteristics or symptoms of a disease, specifically cardiac, kidney or inflammatory disease, can be utilized.
• target gene sequences can be introduced into, and overexpressed in, the genome of the animal of interest, or if endogenous target gene sequences are present, they can either be overexpressed, or alternatively, can be disrupted in order to underexpress or inactivate target gene expression.
• the coding portion of the target gene sequence can be ligated to a regulatory sequence capable of driving gene expression in the animal and cell type of interest. Such regulatory regions are known to those of skill in the art.
• an endogenous target gene sequence can be introduced into the genome of the animal of interest such that the endogenous target gene aileles will be inactivated.
• an engineered sequence comprising at least part of the target gene sequence is utilized and is introduced, via gene targeting, such that the endogenous target sequence is disrupted upon integration of the engineered target gene sequence into the animal's genome.
• Gene targeting is discussed below.
• Animals of any species including, but not limited to, mice, rats, rabbits, guinea pigs, pigs, micro pigs, goats, and non human primates, e.g., baboons, monkeys, and chimpanzees, can be used to generate animal models of a disease, specifically cardiac, kidney or inflammatory disease.
• Any technique known in the art can be used to introduce a target gene transgene into animals to produce the founder lines of transgenic animals. Such techniques include, pronuciear microinjection (Hoppe et al., U.S. Patent No. 4,873,191 (1989)); retrovirus mediated gene transfer into germ lines (Van der Fatten et al., Proc. Natl. Acad. Sci.
• the present invention provides for transgenic animals that carry the transgene in all their cells, as well as
• transgene which carry the transgene in some, but not all their cells, i.e., mosaic animals.
• the transgene can be integrated, either as a single transgene or in co ⁇ catamers, e.g., head-to-head tandems or head-to-ta ⁇ l tandems.
• the transgene can also be selectively introduced into and activated in a particular cell type by following, for example, the teaching of Lasko et al., Proc. Natl. Acad. Sci. USA 89:6232 36 (1992).
• the regulatory sequences required for such a cell-type specific activation depends upon the particular cell type of interest, and will be apparent to those of skill in the art.
• the target gene transgene be integrated into the chromosomal site of the endogenous target gene
• gene targeting is preferred.
• vectors containing some nucleotide sequences homologous to the endogenous target gene of interest are designed for the purpose of integrating, via homologous recombination with chromosomal sequences, into and disrupting the function of the nucleotide sequence of the endogenous target gene.
• the transgene can also be selectively introduced into a particular cell type, thus inactivating the endogenous gene of interest in only that cell type, by following the teaching of Gu et al.
• transgenic animals Once transgenic animals have been generated, the expression of the recombinant target gene and protein can be assayed using standard techniques. Initial screening can be accomplished by Southern blot analysis or PCR techniques to analyze animal tissues to assay whether integration of the transgene has taken place. The level of mRNA expression of the transgene in the tissues of the transgenic animals can also be assessed using techniques which include Northern blot analysis of tissue samples obtained from the animal, in situ hybridization analysis, and RT- coupled PCR. Samples of target gene-expressing tissue can also be evaluated immunocytochemically using antibodies specific for the transgenic product of interest.
• target gene transgenic animals that express target gene mRNA or target gene transgene peptide (detected immunocytochemically, using antibodies directed against target gene product epitopes) at easily detectable levels should then be further evaluated to identify those animals which display disease characteristics or symptoms. Additionally, specific cell types within the transgenic animals can be analyzed for cellular phenot ⁇ pes characteristic of a disease, specifically cardiac, kidney or inflammatory disease. Such cellular phe ⁇ ot ⁇ pes can include, for example, differential gene expression characteristic of cells within a given disease state of interest.
• Such cellular phenot ⁇ p ⁇ s can include an assessment of a particular cell type diagnostic pattern of expression and its comparison to known diagnostic expression profiles of the particular cell type in animals exhibiting a disease, specifically cardiac, kidney or inflammatory disease.
• Such transgenic animals serve as suitable models.
• target gene transgenic founder animals i.e., those animals which express target gene proteins in cells or tissues of interest, and which preferably exhibit disease characteristics
• they can be bred, inbred, outbred, or crossbred to pro ⁇ uce colonies of the particular animal.
• CELL-BASED ASSAYS Cells that contain and express target gene sequences which encode target gene protein, and further, exhibit cellular phenotypes associated with disease, specifically cardiac, kidney or inflammatory disease, disorders, can be utilized to identify compounds that exhibit an ability to prevent, treat or identify a disease, and include the in vitro models described supra
• the diagnostic pattern of gene expression of cells of interest can be analyzed and compared to the normal diagnostic pattern.
• Those compounds which cause cells exhibiting cellular phenotypes of disease, specifically cardiac, kidney or inflammatory disease, disorders to produce a diagnostic pattern more closely resembling a normal diagnostic pattern for the cell of interest can be considered candidates for further testing regarding an ability to ameliorate the symptoms of such diseases.
• Such cells include cardiac myocytes, cardiac fibrobiasts, monoc ⁇ te/macrophages, and kidney epithelial ceils.
• cells which can be used for such assays can also include recombinant, transgenic cell lines.
• the animal models of the present invention can be used to generate cell lines, containing one or more cell types involved in a disease, specifically cardiac, kidney or inflammatory disease, that can be used as cell culture models for these disorders
• techniques which can be used to derive a continuous cell line from transgenic animals see Small et al., Mol. Cell Biol. 5:642-48 (1985).
• cells of a cell type known to be involved in disease can be transfected with sequences capable of increasing or decreasing the amount of target gene expression within the cell.
• target gene sequences can be introduced into, and over expressed in, the genome of the cell of interest, or if endogenous target gene sequences are present, they can either be overexpressed or, be disrupted in order to underexpress or inactivate target gene expression.
• the coding portion of the target gene sequence can be ligated to a regulatory sequence capable of driving gene expression in the cell type of interest.
• regulatory regions are well known to those of skill in the art
• an endogenous target gene sequence For underexpression of an endogenous target gene sequence, such a sequence can be isolated and engineered so that reintroduction into the genome of the cell type of interest disrupts the endogenous target gene aileles.
• the engineered target gene sequence is introduced via gene targeting such that the endogenous target sequence is disrupted upon integration of the engineered target gene sequence into the cell's genome.
• Transf ection of target gene sequence nucieic acids can be accomplished by using standard techniques. See, e.g., Ausubel et al., supra Transfected cells should be evaluated for the presence of the recombinant target gene sequences, for expression and accumulation of target gene mRNA, and for the presence of recombinant target gene protein production. Where a decrease in target gene expression is desired, standard techniques can be used to demonstrate whether a decrease in endogenous target gene expression or in target gene product production is achieved XXI.
• Cells involved in a disease can also be compared to unrelated cells (e.g., fibrobiasts), which have been treated with the compound, such that any generic effects on gene expression that might not be related to the disease or its treatment may be identified.
• unrelated cells e.g., fibrobiasts
• Such generic effects might be manifest, for example, by changes in gene expression that are common to the test cells and the unrelated cells upon treatment with the compound.
• the genes and gene products upon which these compounds act can be identified and used in the assays described videow to identify novel therapeutic compounds for inhibition of a disease and related disorders.
• the present invention provides methods for identifying compounds or agents, which can be used to treat a disease, specifically cardiac, kidney or inflammatory disease, associated with differential gene expression or polypeptide activity.
• These drug screening assays typically include the step of screening a candidate/test compound or agent for the ability to interact with (e.g., bind to) a polypeptide, to modulate the interaction of a polypeptide and a target molecule, or to modulate nucleic acid expression or polypeptide activity.
• candidate/test compounds or agents which have one or more of these abilities can be used as drugs to treat disorders characterized by differential nucieic acid expression or polypeptide activity.
• Candidate/test compounds include, for example, peptides such as soluble peptides, including Ig-tailed fusion peptides and members of random peptide libraries (see, e.g., Lam et al., Nature 354:82-84 (1991 ); Houghten et al., Nature 354:84-86 (1991)) and combinatorial chemistry-derived molecular libraries made of D or L configuration ammo acids; phosphopeptides (e.g., members of random and partially degenerate, directed phosphopeptide libraries, see, e.g., Songyang et al., Ce// 72:767 78 (1993); antibodies (e.g., poiyclonal, monoclonal, humanized, anti idiotypic, chimeric, and single chain antibodies as well as Fab, F(ab') 2 , Fab expression library fragments, and epitope-bindmg fragments of antibodies); and small organic and inorganic molecules (e.g.
• the method involves contacting a biological sample obtained from a subject having a disease, specifically cardiac, kidney or inflammatory disease, with the compound or agent, determining the amount of differentially expressed gene nucleic acid expressed or measuring the activity of the polypeptide in the biological sample, comparing the amount of differentially expressed gene expressed in the biological sample or the measurable differentially expressed gene biological activity in the cell to that of a normal sample.
• An alteration in the amount of differentially expressed gene expression or polypeptide activity in the cell exposed to the compound or agent in comparison to the normal sa pie is indicative of a modulation of differentially expressed gene expression or polypeptide activity.
• Microarrays in a preferred embodiment, are utilized to assess differentiated gene expression.
• the invention aiso pertains to methods for modulating a cell associated activity, e.g., proliferation, differentiation, or cytokine production. Such methods include contacting the cell with an differentially expressed gene modulator such that a cell associated activity is altered relative to a cell associated activity (e.g., the same cell associated activity) of the cell in the absence of the agent.
• the differentially expressed gene modulator can stimulate polypeptide activity or differentially expressed gene expression. Examples of such stimulatory differentially expressed gene modulators include small molecules, active polypeptides, and nucleic acids encoding the differentially expressed gene that have been introduced into the cell.
• the differentially expressed gene modulator can inhibit polypeptide activity or differentially expressed gene expression.
• inhibitory modulators include small molecules, antisense nucleic acid molecules, and antibodies that specifically react with an epitope of the gene or its expression product.
• the ceil is present within a subject, and the agent is administered to the subject.
• nucleic acid molecules, polypeptides, polypeptide homologs, modulators, and antibodies described herein preferably can be used in drug screening assays, diagnostic assays, and methods of treatment.
• the isolated nucleic acid molecules of the invention can be used to express polypeptides (e.g., via a recombinant expression vector in a host cell in gene therapy applications), to detect differentially expressed gene mRNA (e.g., in a biological sample) or a genetic lesion in a gene, and to modulate differentially expressed gene activity.
• differentially expressed gene proteins can be used to screen drugs or compounds which modulate polypeptide activity as well as to treat disorders characterized by insufficient production of polypeptide or production of poiypeptide forms which have decreased activity compared to a normal subject.
• anti differentially expressed gene antibodies of the invention can be used to detect and isolate expressed poiypeptide and modulate differentially expressed disease, specifically cardiac, kidney or inflammatory disease, gene poiypeptide activity.
• the invention provides assays for screening candidate/test compounds, which interact with differentially expressed disease genes.
• the assays may be cell free assays which include the steps of combining a poiypeptide, or a bioactive fragment thereof, and a candidate/test compound, under conditions which allow for interaction of the candidate/test compound to the polypeptide or fragment thereof to form a complex, and detecting the formation of a complex, in which the ability of the candidate compound to interact with the polypeptide or fragment thereof is indicated by the presence of the candidate compound in the complex. Formation of complexes between the polypeptide and the candidate compound can be quantitated, for example, using standard immunoassays.
• the invention provides screening assays to identify candidate/test compounds which modulate the interaction (and most likely differentially expressed gene activity as well) between a polypeptide and a moiecule (target molecule) with which the polypeptide normally interacts.
• target molecules includes proteins in the same signaling path as the poiypeptide, e.g., proteins which may function upstream (including both stimulators and inhibitors of activity) or downstream of the polypeptide in a interactive involving regulation of myocyte ion channels.
• the assays are cell free assays which include the steps of combining polypeptide or a bioactive fragment thereof, differentially expressed gene target molecule (e.g., an differentially expressed gene iigand) and a candidate/test compound under conditions where but for the presence of the candidate compound, the poiypeptide or biologically active portion thereof interacts with the target molecule.
• a complex which includes the polypeptide and the target molecule is then formed or the interaction/reaction of the polypeptide and the target molecule is detected.
• Detection of complex formation can include direct quantitation of the complex by measuring inductive effects of the polypeptide.
• a statistically significant change, such as a decrease, in the interaction of the differentially expressed gene and target molecule (e.g., in the formation of a complex between the differentially expressed gene and the target molecule) in the presence of a candidate compound (relative to what is detected in the absence of the candidate compound) is indicative ot a modulation of the interaction between the poiypeptide and the target molecule.
• Modulation of the formation of complexes between the polypeptide and the target molecule can be quantitated using an immunoassay.
• differentially expressed gene or its target molecule to facilitate separation of complexes from uncomplexed forms of one or both of the proteins, as well as to accommodate automation of the assay.
• Interaction of differentially expressed gene 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 microtiter plates, test tubes, and microcentrifuge tubes.
• a fusion polypeptide can be provided which adds a domain that allows the polypeptide to be bound to a matrix.
• glutathione S transferase/diff erentially expressed gene fusion polypeptides can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, MO) or glutathione denvatized microtiter plates, which are then combined with the cell lysates (e.g 35 S-labeled) and the candidate compound, and the mixture incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads are washed to remove any unbound label, and the matrix immobilized and the radiolabel determined directly, or in the supernatant after the complexes are dissociated.
• glutathione sepharose beads Sigma Chemical, St. Louis, MO
• glutathione denvatized microtiter plates which are then combined with the cell lysates (e.g 35 S-labeled) and the candidate compound, and the mixture incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH).
• the complexes can be dissociated from the matrix, separated by SDS PAGE, and the level of differentially expressed disease, specifically cardiac, kidney or inflammatory disease, gene-binding polypeptide found in the bead fraction quantitated from the gel using standard electrophoretic techniques.
• the invention provides a method for identifying a compound (e.g., a screening assay) capable of use in the treatment of a disorder characterized by (or associated with) differentially expressed disease gene expression or polypeptide activity.
• This method typically includes the step of assaying the ability of the compound or agent to modulate the expression of the differentially expressed gene or the activity of the polypeptide, thereby identifying a compound for treating a disorder characterized by differentially expressed gene expression or polypeptide activity.
• Disorders characterized by differentially expressed gene expression or polypeptide activity are described herein.
• Methods for assaying the ability of the compound or agent to modulate the expression of the differentially expressed gene nucleic acid or activity of the polypeptide are typically cell-based assays.
• ceils sensitive to ligands which transduce signals via an interactive involving differentially expressed gene can be induced to overexpress an polypeptide in the presence and absence of a candidate compound.
• Candidate compounds that produce a statistically significant change in gene-dependent responses can be identified, in one embodiment, expression of the differentially expressed disease gene nucleic acid or activity of a polypeptide is modulated in cells and the effects of candidate compounds on the readout of interest (such as rate of cell proliferation or differentiation) are measured.
• genes which are up- or down- regulated in response to gene-dependent signal cascade, can be assayed, in preferred embodiments, the regulatory regions of such genes, e.g., the 5' flanking promoter and enhancer regions, are operably linked to a detectable marker (such as luciferase) which encodes a gene product that can be readily detected.
• a detectable marker such as luciferase
• Phosphorylation of differentially expressed gene or differentially expressed gene target molecules can also be measured, for example, by immunoblotting.
• modulators of differentially expressed disease gene expression e.g., compounds which can be used to treat a disease, specifically cardiac, kidney or inflammatory disease, or related disorders characterized by differentially expressed gene expression or poiypeptide activity
• a ceil is contacted with a candidate compound and the expression of differentially expressed gene mRNA or polypeptide in the cell is determined
• microarrays are utilized to assess expression levels.
• the level of expression of differentially expressed gene mRNA or polypeptide in the presence of the candidate compound is compared to the level of expression of differentially expressed gene mRNA or polypeptide in the absence of the candidate compound
• the candidate compound can then be identified as a modulator of differentially expressed gene expression based on this comparison and be used to treat a disorder characterized by aberrant differentially expressed gene expression. For example, when expression of differentially expressed gene mRNA or polypeptide is greater (preferably statistically significantly greater) in the presence of the candidate compound than in its absence, the candidate compound may be identified as a stimulator of differentially expressed gene expression.
• the candidate compound when differentially expressed gene expression is less (preferably statistically significantly less) in the presence of the candidate compound than in its absence, the candidate compound may be identified as an inhibitor of differentially expressed gene expression
• the level of differentially expressed gene expression in the cells can be determined by methods described herein for detecting differentially expressed gene mRNA or protein.
• the polypeptides can be used as "bait proteins" in a two-hybrid assay (see, e.g., U.S. Patent No. 5,283,317; Zervos et al., Celllll' lZ 32 (1993); Madura et al., Biol. Chem. 268:12046-54 (1993); Bartel et al., Biotechmques 14:920-24 (1993); Iwabuchi etal., Oncogene 8:1693-96 (1993); and PCT Application WO 94/10300), to identify other proteins, which bind to or interact with differentially expressed gene, ("gene binding proteins" or ' gene bp") and modulate polypeptide activity.
• gene binding proteins are also likely to be involved in the propagation of signals by the polypeptides as, for example, upstream or downstream elements of the differentially expressed gene interaction.
• Compounds identified via assays such as those described herein can be useful, for example, in elaborating the biological function of the target gene product, and for ameliorating symptoms of a disease, specifically cardiac, kidney or inflammatory disease.
• compounds that interact with the target gene product can include ones accentuating or amplifying the activity of the bound target gene protein. Such compounds would bring about an effective increase in the level of target gene activity, thus ameliorating symptoms of the disease disorder or state.
• Another aspect of the invention pertains to methods for identifying compounds or agents for treating a disorder characterized by differential gene expression associated with a disease, specifically cardiac, kidney or inflammatory disease. These methods typically include assaying the ability of the compound or agent to modulate the expression of the differentially expressed gene or the activity of a polypeptide encoded by that gene thereby identifying a compound or agent for treating a disease, specifically cardiac, kidney or inflammatory disease, characterized by differential nucleic acid expression or polypeptide activity.
• the method involves contacting a biological sample, e.g., a cell or tissue sample obtained from a subject, with the compound or agent, determining the amount of the differentially expressed gene expressed or measuring the activity of the polypeptide encoded by that gene in the biological sample, comparing the amount of differentially expressed gene in the biological sample or the measurable biological activity of the encoded polypeptide in the cell to that of a sample from a normal subject.
• a biological sample e.g., a cell or tissue sample obtained from a subject
• the compound or agent determines the amount of the differentially expressed gene expressed or measuring the activity of the polypeptide encoded by that gene in the biological sample, comparing the amount of differentially expressed gene in the biological sample or the measurable biological activity of the encoded polypeptide in the cell to that of a sample from a normal subject.
• An alteration in the amount of the differentially expressed gene expression or poiypeptide activity in the cell exposed to the compound or agent in comparison to the control is indicative of a modulation of the differentially expressed gene
• Compounds identified can be useful, for example, in modulating the activity of normal or mutant target gene products, preferably mutant target gene proteins, can be useful in elaborating the biological function of the target gene product, can be utilized in screens for identifying compounds that disrupt normal target gene interactions, or can in themselves disrupt such interactions.
• the principle of the assays used to identify compounds that bind to the target gene product involves preparing a reaction mixture of the target gene protein and the test compound under conditions and for a time sufficient to allow the two components to interact and bind, thus forming a complex which can be removed or detected in the reaction mixture. These assays can be conducted in a variety of ways.
• one method to conduct such an assay would involve anchoring target gene product or the test substance onto a solid phase and detecting target gene product/test compound complexes anchored on the soiid phase at the end of the reaction.
• the target gene product can be anchored onto a solid surface, and the test compound, which is not anchored, can be labeled either directly or indirectly.
• microtiter plates can conveniently be utilized as the solid phase.
• the anchored component can be immobilized by non-covalent or covalent attachments. No ⁇ -covalent attachment can be accomplished by simply coating the solid surface with a solution of the protein and drying.
• an immobilized antibody preferably a monoclonal antibody, specific for the protein to be immobilized can be used to anchor the protein to the solid surface.
• the surfaces can be prepared in advance and stored.
• the no ⁇ immobiiized component is added to the coated surface containing the anchored component. After the reaction is complete, unreacted components are removed (e.g., by washing) under conditions such that any complexes formed will remain immobilized on the solid surface.
• the detection of complexes anchored on the solid surface can be accomplished in a number of ways. Where the previously immobilized component is pre-labeled, the detection of label immobilized on the surface indicates that complexes were formed.
• an indirect label can be used to detect complexes anchored on the surface, e.g., using a labeled antibody specific for the immobilized component (the antibody, in turn, can be directly labeled or indirectly labeled with a labeled anti Ig antibody).
• a reaction can be conducted in a liquid phase, the reaction products separated from unreacted components, and complexes detected, e.g., using an immobilized antibody specific for target gene or the test compound to anchor any complexes formed in solution, and a labeled antibody specific for the other component of the possible complex to detect anchored complexes.
• ASSAYS FOR CELLULAR PROTEINS THAT INTERACT WITH THE TARGET GENE PRODUCT Any method suitable for detecting protein-protein interactions can be employed for identifying novel target product cellular or extracellular protein interactions. The methods outlined, supra, for the identification of interactive genes can be utilized herein with respect to the identification of proteins, which interact with identified target proteins. In such a case, the target gene serves as the known "bait" gene.
• the target gene products of the invention can, in vivo, interact with one or more cellular or extracellular macromolecuies, such as proteins.
• macromolecuies include nucleic acid molecules and those products identified via methods such as those described above.
• Such cellular and extracellular macromoiecules are referred to herein as "binding partners.”
• binding partners Compounds that disrupt such interactions can be useful in regulating the activity of the target gene product, especially mutant target gene products.
• the assay systems used to identify compounds that interfere with the interaction between the target gene product and its cellular or extracellular binding partner or partners involve preparing a reaction mixture containing the target gene product and the binding partner under conditions and for a time sufficient to allow the two products to interact and bind, thus forming a complex.
• the reaction mixture is prepared in the presence and absence of the test compound.
• the test compound can be initially included in the reaction mixture, or can be added at a time subsequent to the addition of target gene and its cellular or extracellular binding partner. Control reaction mixtures are incubated without the test compound or with a placebo. The formation of any complexes between the target gene product and the cellular or extracellular binding partner is then detected.
• complex formation within reaction mixtures containing the test compound and normal target gene product can also be compared to complex formation within reaction mixtures containing the test compound and mutant target gene product. This comparison can be important in those cases where it is desirable to identify compounds that disrupt interactions of mutant but not normal target gene products.
• the assay for compounds that interfere with the interaction of the target gene products and binding partners can be conducted in a heterogeneous or homogeneous format.
• Heterogeneous assays involve anchoring either the target gene product or the binding partner onto a solid phase and detecting complexes anchored on the solid phase at the end of the reaction, in homogeneous assays, the entire reaction is carried out in a liquid phase. In either approach, the order of addition of reactants can be varied to obtain different information about the compounds being tested.
• Test compounds interfering with the interaction between the target gene products and the binding partners can be identified by conducting the reaction in the presence of the test substance; i.e., by adding the test substance to the reaction mixture prior to or simultaneously with the target gene product and interactive cellular or extracellular binding partner.
• test compounds that disrupt preformed complexes e.g., compounds with higher binding constants that displace one of the components from the complex, can be tested by adding the test compound to the reaction mixture after complexes have been formed.
• the various formats are described briefly below.
• the target gene product or the interactive cellular or extracellular binding partner is anchored onto a solid surface, while the non-anchored species is labeled, either directly or indirectly.
• the anchored species can be immobilized by non-covalent or covalent attachments. Non-covalent attachment can be accomplished simply by coating the solid surface with a solution of the target gene product or binding partner and drying. Alternatively, an immobilized antibody specific for the species to be anchored can be used to anchor the species to the solid surface. The surfaces can be prepared in advance and stored. in order to conduct the assay, the partner of the immobilized species is exposed to the coated surface with or without the test compound.
• any complexes formed will remain immobilized on the solid surface.
• the detection of complexes anchored on the solid surface can be accomplished in a number of ways. Where the non-immobiiized species is pre-labeled, the detection of label immobilized on the surface indicates that complexes were formed. Where the non-immobiiized species is not pre- labeled, an indirect label can be used to detect complexes anchored on the surface; e.g., using a labeled antibody specific for the initially non-immobilized species (the antibody, in turn, can be directly labeled or indirectly labeled with a labeled anti-ig antibody). Depending upon the order of addition of reaction components, test compounds which inhibit complex formation or which disrupt preformed complexes can be detected.
• the reaction can be conducted in a liquid phase in the presence or absence of the test compound, the reaction products separated from unreacted components, and complexes detected; e.g., using an immobilized antibody specific for one of the binding components to anchor any complexes formed in solution, and a labeled antibody specific for the other partner to detect anchored complexes.
• test compounds which inhibit complex or which disrupt preformed complexes can be identified.
• a homogeneous assay can be used.
• a preformed complex of the target gene product and the interactive cellular or extracellular binding partner product is prepared in which either the target gene products or their binding partners are labeled, but the signai generated by the label is quenched due to complex formation (see, e.g., U.S. Patent No. 4,109,496, which utilizes this approach for immunoassays).
• the addition of a test substance that competes with and displaces one of the species from the pre- formed complex will result in the generation of a signal above background. In this way, test substances disrupting target gene product-cellular or extracellular binding partner interaction can be identified.
• the target gene product can be prepared for immobilization using recombinant DNA techniques described supra.
• the target gene coding region can be fused to a glutathione-S- transferase (GST) gene using a fusion vector such as pGEX-5X-1, in such a manner that its binding activity is maintained in the resulting fusion product.
• GST glutathione-S- transferase
• the interactive cellular or extracellular product can be purified and used to raise a monoclonal antibody, using methods routinely practiced in the art and described above.
• the GST Target gene fusion product can be anchored to glutathione-agarose beads.
• the interactive cellular or extracellular binding partner product can then be added in the presence or absence of the test compound in a manner that allows interaction and binding to occur. At the end of the reaction period, unbound material can be washed away, and the labeled monoclonal antibody can be added to the system and allowed to bind to the complexed components.
• the interaction between the target gene product and the interactive cellular or extracellular binding partner can be detected by measuring the amount of radioactivity that remains associated with the glutathione-agarose beads. A successful inhibition of the interaction by the test compound will result in a decrease in measured radioactivity.
• the GST-target gene fusion product and the interactive cellular or extracellular binding partner product can be mixed together in liquid in the absence of the solid glutathione-agarose beads.
• test compound can be added either during or after the binding partners are allowed to interact. This mixture can then be added to the glutathione-agarose beads and unbound material is washed away. Again the extent of inhibition of the binding partner interaction can be detected by adding the labeled antibody and measuring the radioactivity associated with the beads. in another embodiment of the invention, these same techniques can be employed using peptide fragments that correspond to the binding domains of the target gene product and the interactive cellular or extracellular binding partner (in case where the binding partner is a product), in place of one or both of the full length products. Any number of methods routinely practiced in the art can be used to identify and isolate the protein's binding site.
• mutage ⁇ esis of one of the genes encoding one of the products and screening for disruption of binding in a co-immu ⁇ oprecipitation assay.
• Compensating mutations in the gene encoding the second species in the complex can be selected. Sequence analysis of the genes encoding the respective products will reveal the mutations that correspond to the region of the product involved in interactive binding.
• one product can be anchored to a solid surface, and allowed to interact with and bind to its labeled binding partner, which has been treated with a proteol ⁇ tic enzyme, such as trypsin.
• a short labeled peptide comprising the binding domain can remain associated with the solid material, which can be isolated and identified by am o acid sequencing. Also, once the gene coding for the cellular or extracellular binding partner product is obtained, short gene segments can be engineered to express peptide fragments of the product, which can then be tested for binding activity and purified or synthesized.
• XXV. ASSAYS FOR AMELIORATION OF DISEASE SYMPTOMS Any of the binding compounds, including compounds such as those identified in the foregoing assay systems, can be tested for the ability to prevent or ameliorate symptoms of a disease, specifically cardiac, kidney or inflammatory disease. Cell based and animal model-based assays for the identification of compounds exhibiting an ability to prevent or ameliorate disease symptoms are described herein.
• cell based in vitro systems such as those described above can be used to identify compounds that can act to ameliorate symptoms of a disease.
• such cell systems can be exposed to a compound suspected to exhibit an ability to ameliorate a disease or its symptoms, at a sufficient concentration and for a time sufficient to elicit such an amelioration in the exposed cells. After exposure, the cells are examined to determine whether one or more disease disorder phenotypes has been altered to resemble a more normal or more wild- type disease phenotype.
• animal-based in vivo models can be used to identify compounds capable of ameliorating symptoms of a disease, specifically cardiac, kidney or inflammatory disease.
• Such animal models can be used as test substrates for the identification of drugs, pharmaceuticals, therapies, and interventions which can be effective in treating a disease, specifically cardiac, kidney or inflammatory disease, and related disorders.
• animal models can be exposed to a compound suspected to exhibit an ability to ameliorate a disease or its symptoms at a sufficient concentration and for a time sufficient to elicit such amelioration in the exposed animals. The response of the animals to the exposure can be monitored by assessing the reversal of disorders associated with a disease.
• any treatments that reverse any aspect of symptoms of a disease should be considered as candidates for human therapeutic intervention in the treatment of a disease.
• Dosages of test agents can be determined by deriving dose-response curves, as discussed below.
• gene expression patterns can be utilized to assess the ability of a compound to ameliorate symptoms of a disease, specifically cardiac, kidney or inflammatory disease. For example, diagnostic gene expression or a diagnostic pattern can then be used in such an assessment. Diagnostic gene expression and diagnostic patterns are described below.
• Diagnostic patterns can be characterized for known disease states within the cell- or animal-based model systems. Subsequently, these known diagnostic patterns can be compared to ascertain the effect a test compound has to modify such diagnostic patterns and to cause the pattern to more closely resemble that of a more desirable diagnostic pattern. For example, administration of a compound can cause the diagnostic pattern of a disease, specifically cardiac, kidney or inflammatory disease, model system to more closely resemble a control, normal system. Administration of a compound can alternatively cause the diagnostic pattern of a control system to begin to mimic a disease state. XXVI.
• MONITORING OF EFFECTS DURING CLINICAL TRIALS Monitoring the influence of compounds in a disease, specifically cardiac, kidney or inflammatory disease, can be applied not only in basic drug screening, but also in clinical trials. In such clinical trials, the expression of a panel of genes that have been discovered by the methods of the present invention can be used as a indicator of the disease, specifically cardiac, kidney or inflammatory disease, state of a particular cell.
• peripheral blood can be isolated, and RNA prepared and analyzed by microarray as described supra.
• the levels of expression of the diagnostic genes can be quantified by microarray or RT-PCR, or alternatively by measuring the amount of protein produced.
• the diagnostic profiles can serve as putative biomarkers indicative of the disease.
• a protocol for suitable drugs can be developed based on the gene expression potential of the subject cardiac cells.
• biological samples can be periodically obtained from a treated subject for measurement of gene expression so that the efficacy of a drug can be measured by monitoring the degree of restored expression of the gene.
• the present invention relates to methods and compositions that can be used to ameliorate symptoms of a disease, specifically cardiac, kidney or inflammatory disease, and its related disorders by target gene modulation.
• Target gene modulation can be of a positive or negative nature, depending on the specific situation involved, but each modulatory event preferably yields a net result in which disease, specifically cardiac, kidney or inflammatory disease, symptoms are ameliorated.
• “Negative modulation,” as used herein, refers to a reduction in the level or activity of target gene product relative to the level or activity of the target gene product in the absence of the modulatory treatment.
• “Positive modulation,” as used herein, refers to an increase in the level or activity of target gene product relative to the level or activity of target gene product in the absence of modulatory treatment.
• Another aspect of the invention pertains to methods for treating a subject, e.g., a human, having a disease or disorder characterized by (or associated with) differentially expressed gene expression or polypeptide activity. These methods include the step of administering a differentially expressed gene modulator to the subject such that treatment occurs.
• the terms "treating” or “treatment” include the reduction or alleviation of at least one adverse effect or symptom of a disorder or disease, e.g., a disorder or disease characterized by or associated with differentially expressed gene expression.
• a differentially expressed gene modulator is a molecule, which can modulate differentially expressed gene expression or polypeptide activity.
• a differentially expressed gene modulator can modulate, e.g., upregulate (activate) or downregulate (suppress), differentially expressed gene expression.
• a differentially expressed gene modulator can modulate (e.g., stimulate or inhibit) expression product or polypeptide activity. If it is desirable to treat a disease, specifically cardiac, kidney or inflammatory disease, associated with differentially expressed gene expression or polypeptide activity by inhibiting differentially expressed gene expression, an differentially expressed gene modulator can be an antisense molecule.
• antisense molecules which can be used to inhibit differentially expressed gene expression include antisense molecules which are complementary to a portion of the 5' untranslated region of the coding sequence of the gene (e.g., SEQ ID N0:25) which also includes the start codon and antisense molecules which are complementary to a portion of the 3' untranslated region of the gene
• a differentially expressed gene modulator that inhibits differentially expressed gene expression can also be a small molecule or other drug, e g., a small molecule or drug identified using the screening assays described herein, which inhibits differentially expressed gene expression.
• a differentially expressed gene modulator can be, for example, a nucleic acid molecule encoding differentially expressed gene (e.g., a nucleic acid molecule comprising a nucleotide sequence homologous to the nucleotide sequence of SEQ ID N0:25) or a small molecule (e.g., a peptide) or drug identified using the screening assays described herein, which stimulates differentially expressed gene expression.
• the modulator can be the differentially expressed gene or expression product itself.
• an differentially expressed gene modulator can be an anti differentially expressed gene antibody or a small molecule or other drug, eg., a small molecule or drug identified using the screening assays described herein, which inhibits polypeptide activity
• an differentially expressed gene modulator can be an active polypeptide or portion thereof, including the differentially expressed polypeptide itself (e.g., an polypeptide or portion thereof having an ammo acid sequence which is homologous to the ammo acid sequence of SEQ ID N0:37 (or a portion thereof)) or a small molecule or other drug, e.g., a small molecule or drug identified using the screening assays described herein, which stimulates polypeptide activity.
• cell associated activity refers to a normal or abnormal activity or function of a cell. Examples of cell associated activities include proliferation, migration, differentiation, production or secretion of molecules such as proteins, and cell survival.
• the cell mav be a cardiac cell of the heart, e.g., a cardiac myocyte.
• altered relates to a change, e.g., an increase or decrease, of a cell associated activity.
• the agent stimulates polypeptide activity or nucleic acid expression.
• stimulatory agents include an active gene protein, a nucleic acid molecule encoding differentially expressed gene that has been introduced into the cell, and a modulatory agent which stimulates polypeptide activity or differentially expressed gene expression and which is identified using the drug screening assays described herein.
• the agent inhibits polypeptide activity or differentially expressed gene expression.
• inhibitory agents include an antisense differentially expressed gene nucleic acid molecule, an anti differentially expressed gene antibody, and a modulatory agent which inhibits polypeptide activity or differentially expressed gene expression and which is identified using the drug screening assays described herein.
• modulatory methods can be performed in vitro (e.g., by cultunng the cell with the agent) or, alternatively, in vivo (e.g., by administering the agent to a subject).
• the modulatory methods are performed in vivo, i.e., the cell is present within a subject and the subject has a disorder or disease characterized by or associated with abnormal or aberrant polypeptide activity or differentially expressed gene expression.
• a disease can be brought about, at least in part, by an abnormal level of differentially expressed gene product, or by the presence of a gene product exhibiting abnormal activity. As such, the reduction in the level or activity of such gene products would bring about the amelioration of disease symptoms. Negative modulatory techniques for the reduction of target gene expression levels or target gene product activity levels are discussed below.
• a disease specifically cardiac, kidney or inflammatory disease
• a disease can be brought about, at least in part, by the absence or reduction of the level of gene expression, or a reduction in the level of a gene product's activity.
• an increase in the level of gene expression or the activity of such gene products would bring about the amelioration of a disease, specifically cardiac, kidney or inflammatory disease, symptoms.
• diagnostic genes discovered by the methods of the present invention are observed to be down-regulated in the disease state (e.g., BTG-2).
• a positive modulatory technique that increases such gene expression in cells within a disease state should, therefore, act to ameliorate the symptoms of such a state.
• the gene product may exhibit suppressor features, it is possible that a positive modulatory technique could ameliorate symptoms of many disease events.
• Positive modulatory techniques for increasing the target gene expression levels or target gene product activity levels are discussed below.
• treatment of disease can be brought about by techniques that serve to inhibit the expression or activity of target gene products.
• compounds within the context of the present invention that exhibit negative modulatory activity can be used in accordance with the invention to prevent or ameliorate symptoms of a disease, specifically cardiac, kidney or inflammatory disease.
• Such molecules can include, but are not limited to peptides, phosphopeptides, small organic or inorganic molecules, or antibodies (including, for example, poiyclonal, monoclonal, humanized, anti-idiotypic, chimeric or single chain antibodies, and Fab, F(ab') 2 and Fab expression library fragments, and epitope binding fragments thereof).
• Negative modulatory techniques involving antibody administration are described below, as well as techniques for the determination and administration of such compounds.
• antisense and ribozyme molecules which inhibit expression of the target gene can also be used in accordance with the invention to reduce the level of target gene expression, thus effectively reducing the level of target gene activity.
• triple helix molecules can be utilized in reducing the level of target gene activity.
• the invention also pertains to methods for modulating a cell associated activity. Such methods include contacting the cell with a modulator of the differentially expressed gene such that a cell associated activity is altered relative to a cell associated activity (e.g., the same cell associated activity) of the cell in the absence of the agent.
• the differentially expressed gene modulator can stimulate polypeptide activity or nucleic acid expression related to the differentially expressed gene.
• stimulatory modulators include small molecules, active polypeptides encoded by the differentially expressed gene, and nucleic acids encoding the differentially expressed gene that have been introduced into the cell.
• the modulator can inhibit the polypeptide activity of the differentially expressed gene or nucleic acid expression.
• inhibitory modulators include small molecules, antisense nucieic acid molecules, and antibodies that specifically react with an epitope of the differentially expressed gene product
• the cell is present within a subject and the agent is administered to the subject.
• the nucleic acid molecules, polypeptides, polypeptide homologs, modulators, and antibodies described herein preferably may be used in drug screening assays, diagnostic assays, and methods of treatment.
• a differentially expressed gene poiypeptide of the invention has one or more of the activities described herein and can thus be used, for example, to modulate a function in a cell involved in a disease, specifically cardiac, kidney or inflammatory disease.
• the isolated nucieic acid molecules of the invention can be used to express polypeptide encoded by a differentially expressed gene of the present invention (e.g., via a recombinant expression vector in a host cell in gene therapy applications), to detect mRNA of the differentially expressed gene (e.g., in a biological sample) or a genetic lesion in a differentially expressed gene, or to modulate activity of the differentially expressed gene or polypeptides encoded thereby, as described further below.
• polypeptides encoded by the differentially expressed genes of the present invention can be used to screen drugs or compounds which modulate polypeptide activity related to the differentially expressed gene as well as to treat disorders characterized by insufficient production of polypeptide encoded by the differentially expressed gene or production of polypeptide forms which have decreased activity compared to wild type forms of the polypeptides encoded by the differentially expressed genes of the present invention
• antibodies to the polypeptides encoded by the differentially expressed genes of the present invention can be used to detect and isolate such polypeptides and modulate polypeptide activity.
• XXIX NEGATIVE MODULATORY ANTISENSE, RIBOZYME AND TRIPLE HELIX APPROACHES
• antisense ribozyme
• triple helix molecules Such molecules can be designed to reduce or inhibit either normal or, if appropriate, mutant target gene activity. Techniques for the production and use of such molecules are well known to those of skill in the art.
• Anti sense RNA and DNA molecules act to directly block the translation of mRNA by hybridizing to targeted mRNA and preventing protein translation.
• antisense DNA oligodeoxyribonucleotides derived from the translation initiation site, eg., between the 10 and + 10 regions of the target gene nucleotide sequence of interest, are preferred.
• Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA (Rossi, Current Biology 4:469 71 (1994))
• the mechanism of ribozyme action involves sequence specific hybridization of the ribozyme molecule to complementary target RNA, followed by an endonucleolytic cleavage.
• the composition of ribozyme molecules must include one or more sequences complementary to the target gene mRNA and must include the well known catalytic sequence responsible for mRNA cleavage. For this sequence, see U.S. Patent No. 5,093,246, which is incorporated by reference herein in its entirety.
• engineered hammerhead motif ribozyme molecules that specifically and efficiently catalyze endonucleolytic cleavage of RNA sequences encoding target gene proteins.
• ribozyme cleavage sites within any potential RNA target are initially identified by scanning the molecule of interest for ribozyme cleavage sites which include the following sequences, GUA, GUU and GUC. Once identified, short RNA sequences of between 15 and 20 ribonucieotides corresponding to the region of the target gene containing the cleavage site can be evaluated for predicted structural features, such as secondary structure, that can render the oligonucleotide sequence unsuitable. The suitability of candidate sequences can also be evaluated by testing their accessibility to hybridization with complementary oligonucleotides, using nbonuclease protection assays.
• Nucleic acid molecules preferably used in triplex helix formation for the inhibition of transcription are single stranded and composed of deox ⁇ nucleotides.
• the base composition of these oligonucleotides must be designed to promote triple helix formation via Hoogsteen base pairing rules, which generally require sizeable stretches of either purines or pyrimidmes to be present on one strand of a duplex
• Nucleotide sequences can be pyrimidme based, which will result in TAT and CGC + triplets across the three associated strands of the resulting triple helix
• the py ⁇ midine- rich molecules provide base complementanl ⁇ to a purine rich region of a single strand of the duplex in a parallel orientation to that strand
• nucleic acid molecules can be chosen that are purine rich, for example, contain a stretch of G residues.
• Switchback molecules are synthesized in an alternating 5' 3', 3' 5' manner, such that they base pair with first one strand of a duplex and then the other, eliminating the necessity for a sizeable stretch of either purines or pyrimidmes to be present on one strand of a duplex.
• nucleic acid molecules that encode and express target gene polypeptides exhibiting normal target gene activity can be introduced into cells via gene therapy methods, such as those described below, that do not contain sequences susceptible to the antisense, ribozyme, or triple helix treatments utilized.
• target gene encodes an extracellular protein
• Antisense RNA and DNA, ribozyme and triple helix molecules of the invention can be prepared by any method known in the art for the synthesis of DNA and RNA molecules. These include techniques for chemically synthesizing oligodeoxyribonucleotides and oligo ⁇ bonucleotides well known in the art such as solid phase phosphoramidite chemical synthesis. Alternatively, RNA molecules can be generated by in vitro and in vivo transcription of DNA sequences encoding the antisense RNA molecule. Such DNA sequences can be incorporated into a variety of vectors that incorporate suitable RNA polymerase promoters such as the T7 or SP6 polymerase promoters.
• antisense cDNA constructs that synthesize antisense RNA constitutively or mducibly, depending on the promoter used, can be introduced stably into cell lines.
• Another class of antisense molecules the phosphorothioates, has a sulfur in the oligonucleotide backbone instead of an oxygen atom and is a DNA analog capable of use in the present invention.
• modification at the 2'-pos ⁇ t ⁇ on of the sugar by creating methoxyethyl, ammopropyl, and fluorine conjugates has dramatic effects on stability and target-binding efficiency. Nonspecific effects due to their association with proteins and to the base sequence of the oligonucleotide can limit or even alter the expected antisense effects.
• PNAs peptide nucieic acids
• PNAs have a peptide-like backbone instead of the normal sugar and phosphate groups of DNA.
• PNA may be used to turn on specific genes by binding to a promoter region of a gene to initiate RNA transcription. Chimeric molecules of PNA and DNA may also be considered.
• the DNA portion will allow enzymes attacking DNA-RNA hybrids to cut the RNA part of the complex into pieces (leading to dissociation of the drug molecule, which can then be reused), whereas the PNA portion will contribute stability and selectivity.
• Genetic drugs can also be directed at the gene itself.
• the first chemical approach to target double stranded DNA has been to use oligonucleotides to bind in the major groove of DNA and form a specific local triple helix. Tests of blocking transcription of the HIV genes nef and pol in cell cultures was performed by using oligonucleotides linked to intercalators. Some PNA sequences bind to double-stranded DNA by an invasion mechanism: two PNA molecules form a triplex structure with the complementary DNA target sequence, whereas the other strand of the DNA duplex is displaced into a single stranded loop.
• PNA-DNA complexes are extremely stable Minor-groove binding polyamides that contain combinations of three different aromatic ammo acids, which pair and uniquely rec ognize each of the four Watson-Crick base pairs may also be used.
• Hairpin polyamides bind specifically to predetermined DNA sequences with the affinity and specificity of protein transcription factors. Cells are believed to be permeable to these polyamides, a property that may be related to the fact that they are significantly smaller than oligonucleotide analogs used in antisense approaches.
• These synthetic DNA binding ligands can enter the nucleus and inhibit expression of specific genes by blocking promoter-specific transcription factors.
• the arrays of immobilized DNA fragments may also be used for genetic diagnostics.
• a microarray containing multiple forms of a mutated gene or genes can be probed with a labeled mixture of a subject DNA, which will preferentially interact with oniy one of the immobilized versions of the gene.
• Arrays of immobilized DNA fragments can also be used in DNA probe diagnostics.
• identity of a differentially expressed gene of the present invention can be established unambiguously by hybridizing a sample of a subject's DNA to an array comprising known differentially expressed DNA.
• Other molecules of genetic interest, such as cDNAs and RNAs can be immobilized on the array or alternately used as the labeled probe mixture that is applied to the array.
• Antibodies can be generated which are both specific for target gene product and which reduce target gene product activity. Such antibodies may, therefore, by administered in instances whereby negative modulatory techniques are appropriate for the treatment of a disease, specifically cardiac, kidney or inflammatory disease. Anti bodies can be generated using standard techniques against the expressed proteins themselves or against peptides corresponding to portions of the proteins.
• the antibodies include poiyclonal, monoclonal, Fab fragments, single chain antibodies, chimeric antibodies.
• the target gene protein to which the antibody is directed is mtracellular and whole antibodies are used, internalizing antibodies can be preferred.
• lipofectm or liposomes can be used to deliver the antibody or a fragment of the Fab region that binds to the target gene epitope into cells. Where fragments of the antibody are used, the smallest inhibitory fragment that binds to the target protein's binding domain is preferred.
• peptides having an ammo acid sequence corresponding to the domain of the variable region of the antibody that binds to the target gene protein can be used.
• Such peptides can be synthesized chemically or produced via recombinant DNA technology using methods well known in the art (see, e.g., Creighton, supra; and Sambrook et al., supra)
• single chain neutralizing antibodies that bind to mtracellular target gene product epitopes can also be administered.
• Such single chain antibodies can be administered, for example, by expressing nucleotide sequences encoding single-chain antibodies within the target cell population by utilizing, techniques such as those described in Marasco etal. (Marasco etal., Proc. Natl. Acad. Sci. USA 90:7889 93 (1993)).
• any of the administration techniques described below that are appropriate for peptide can be utilized to effectively administer inhibitory target gene antibodies to their site of action.
• Such molecules can include, but are not limited to, peptides, phosphopeptides, small organic or inorganic molecules, or antibodies (including, for example, poiyclonal, monoclonal, humanized, anti idiot ⁇ pic, chimeric or single chain antibodies, and Fab, F(ab') 2 and Fab expression library fragments, and epitope-bmdmg fragments thereof.
• a target gene protein at a level sufficient to ameliorate a disease, specifically cardiac, kidney or inflammatory disease, can be administered to a patient. Any of the techniques discussed below can be utilized for such administration. One of skill in the art will readily know how to determine the concentration of effective, non-toxic doses of the normal target gene protein, using known techniques.
• administered compound is a peptide
• DNA sequences encoding the peptide compound can, alternatively, be directly administered to a patient exhibiting disease symptoms, at a concentration sufficient to generate the production of an amount of target gene product adequate to ameliorate the disease symptoms. Any of the techniques described below, which achieve mtracellular administration can be utilized for the administration of such DNA molecules.
• the DNA molecules can be produced by known recombinant techniques.
• the DNA molecules encoding such peptides can be taken up and expressed by any cell type, so long as a sufficient circulating concentration of peptide results for the elicitation of a reduction in disease, specifically cardiac, kidney or inflammatory disease, symptoms.
• the DNA molecules encoding such peptides may be taken up and expressed by cells involved in the disease at a sufficient level to bring about the reduction of disease symptoms. Any technique that selectively serves to administer DNA molecules to a cell involved in a disease is preferred for the DNA molecules encoding intracellularly acting peptides.
• patients can be treated for symptoms of a disease, specifically cardiac, kidney or inflammatory disease, by gene replacement therapy.
• One or more copies of a normal target gene or a portion of the gene that directs the production of a normal target gene protein with target gene function can be inserted into cells, using vectors which include, adenovirus, adeno-associated virus, and retrovirus vectors, in addition to other particles that introduce DNA into cells, such as liposomes.
• Techniques such as those described above can be utilized for the introduction of normal target gene sequences into human cells. in instances wherein the target gene encodes an extracellular, secreted gene product, such gene replacement techniques may be accomplished either in vivo or in vitro.
• target gene sequences can be introduced into autologous cells.
• Cells expressing the target gene sequence of interest can then be reintroduced, preferably by intravenous administration, into the patient such that there results an amelioration of disease symptoms.
• the gene replacement involves a gene encoding a product which acts intracellularly, it is preferred that gene replacement is accomplished in vivo.
• the cell type in which the gene replacement must occur is the cell type involved in a disease, specifically cardiac, kidney or inflammatory disease, such techniques must successfully target such cells.
• a down-regulated differentially expressed gene of the present invention as an example (e.g., BTG2)
• an increase in expression may serve to ameliorate disease, specifically cardiac, kidney, or inflammatory disease, symptoms. Therefore, any positive modulatory agent which increases the gene product or gene product activity to a level that is sufficient to ameliorate cardiac disease symptoms represents a successful therapeutic treatment.
• XXXII PHARMACEUTICAL PREPARATIONS AND METHODS OF ADMINISTRATION
• the identified compounds that inhibit target gene expression, synthesis or activity can be administered to a patient at therapeuticall ⁇ effective doses to prevent, treat or ameliorate a disease, specifically cardiac, kidney or inflammatory disease, or its symptoms.
• a therapeuticall ⁇ effective dose refers to that amount of the compound sufficient to result in treatment or amelioration of symptoms of a disease, specifically cardiac, kidney or inflammatory disease.
• Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD 50 (the dose lethal to 50% of the population) and the ED 50 (the dose therapeutically effective in 50% of the population).
• the dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD 60 /ED 50 .
• Compounds exhibiting large therapeutic indices are preferred. While compounds that exhibit toxic side effects can be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.
• Data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans.
• the dosage of such compounds lies preferably within a range of circulating concentrations that include the ED 50 with little or no toxicity.
• the dosage can vary within this range depending upon the dosage form employed and the route of administration utilized.
• the therapeutically effective dose can be estimated initially from cell culture assays.
• a dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC 50 (i.e., the concentration of the test compound, which achieves a half-maximal inhibition of symptoms) as determined in cell culture.
• IC 50 i.e., the concentration of the test compound, which achieves a half-maximal inhibition of symptoms
• levels in plasma can be measured, for example, by high performance liquid chromatography.
• compositions for use in accordance with the present invention can be formulated in conventional manner using one or more physiologically acceptable carriers or excipients.
• the compounds and their physiologically acceptable salts and solvates can be formulated for administration by inhalation or insufflation (either through the mouth or the nose) or oral, buccal, parenteral or rectal administration.
• the pharmaceutical compositions can take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinised maize starch, polyvinylp ⁇ rrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystallme cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate. talc or silica); dismtegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium iauryi sulfate).
• binding agents e.g., pregelatinised maize starch, polyvinylp ⁇ rrolidone or hydroxypropyl methylcellulose
• fillers e.g., lactose, microcrystallme cellulose or calcium hydrogen phosphate
• lubricants e.g., magnesium stearate. talc or
• Liquid preparations for oral administration can take the form of, for example, solutions, syrups or suspensions, or they can be presented as a dry product for constitution with water or other suitable vehicle before use.
• Such liquid preparations can be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives (e.g., methyl or propyl p-hydroxybenzoates or sorbic acid).
• suspending agents e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats
• emulsifying agents e.g., lecithin or acacia
• non-aqueous vehicles e.g., almond oil, oily esters, ethy
• compositions can also contain buffer salts, flavoring, coloring and sweetening agents as appropriate.
• Preparations for oral administration can be suitably formulated to give controlled release of the active compound.
• buccal administration the compositions can take the form of tablets or lozenges formulated in conventional manner.
• the compounds for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dicfilorodifluoromethane, trichlorofiuoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
• a suitable propellant e.g., dicfilorodifluoromethane, trichlorofiuoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
• the dosage unit can be determined by providing a valve to deliver a metered amount.
• Capsules and cartridges of e.g., gelatin for use in an inhaler or insufflator can be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
• the compounds can be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion.
• Formulations for injection can be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative.
• the compositions can take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and can contain formulator ⁇ agents such as suspending, stabilizing or dispersing agents
• the active ingredient can be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen free water, before use.
• the compounds can also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glyce ⁇ des.
• the compounds can also be formulated as a depot preparation. Such long acting formulations can be administered by implantation (for example, subcutaneously or intramuscularly) or by intramuscular injection.
• the compounds can be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
• compositions can, if desired, be presented in a pack or dispenser device that can contain one or more unit dosage forms containing the active ingredient.
• the pack can for example comprise metal or plastic foil, such as a blister pack.
• the pack or dispenser device can be accompanied by instructions for administration XXXIII.
• DIAGNOSIS OF A DISEASE A variety of methods can be employed for the diagnosis of a disease, specifically cardiac, kidney or inflammatory disease, and of disorders involving such diseases. Such methods can, for example, utilize reagents such as diagnostic gene nucleotide sequences and antibodies directed against differentially expressed and interactive gene peptides. Specifically, such reagents can be used, for the detection of the presence of target gene mutations, or the detection of either over or under expression of target gene mRNA.
• the methods described herein can be performed by utilizing pre packaged diagnostic kits comprising at least one specific diagnostic gene nucleic acid or anti-diagnostic gene antibody reagent described herein, which can be conveniently used, e.g., in clinical settings, to diagnose patients exhibiting symptoms of a disease, specifically cardiac, kidney or inflammatory diseases.
• Microarrays as disclosed preferably may be used.
• a microarray comprising one or more cDNA clones representing differentially expressed genes is hybridized with total cDNA from a subject to monitor differentially expressed gene expression for research or diagnostic purposes.
• such microarrays may comprise sequences specific for differentially expressed disease, specifically cardiac, kidney or inflammatory disease, genes, preferably chosen from one or more of the group including nucleic acids (and portions thereof) corresponding to the sequences specified in GenBank Accession numbers X57352, S75725, D13665, X67698, M62402, D90226, L13698, U52101 , U72649, L36034, M36035, and M38591 , as well as nucleic acids (and portions thereof) encoding the human genes 1 8U (SEQ ID N0:37), prostacyclin stimulating factor (SEQ ID N0:38), osf-2 (SEQ ID N0:39), tissue specific mRNA (SEQ ID N0:40), insulin-like growth factor binding protein 6 (SEQ ID N0:41), OSF-1 (SEQ ID N0:42), gas-1 (SEQ ID N0:43), YMP (SEQ ID N0:44), BTG2 (SEQ ID N0:45
• DNA or RNA from the cell type or tissue to be analyzed can easily be isolated using procedures known to those in the art. Diagnostic procedures can also be performed directly upon tissue sections (fixed or frozen) of subject tissue obtained from biopsies or resections, such that no nucieic acid purification is necessary. Nucleic acid reagents such as those described above can be used as probes or primers for such in situ procedures (see, e.g., Nuovo, PCR in situ hybridization: Protocols and Applications, Raven Press (New York, 1992)).
• Diagnostic gene nucleotide sequences can, be used in hybridization or amplification assays of biological samples to detect gene structures and expression associated with a disease, specifically cardiac, kidney or inflammatory disease.
• assays can include microarray analyses, Southern or Northern analyses, single stranded conformational polymorphism analyses, in situ hybridization assays, and polymerase chain reaction analyses.
• analyses can reveal both quantitative aspects of the expression pattern of the diagnostic gene, and qualitative aspects of the diagnostic gene expression or gene composition. That is, such techniques can include, for example, point mutations, insertions, deletions, chromosomal rearrangements, or activation or inactivation of gene expression.
• Preferred diagnostic methods for the detection of diagnostic gene-specific nucleic acid molecules can involve, for example, microarray analysis via contacting and incubating nucleic acids derived from the cell type or tissue being analyzed, with microarrays under conditions favorable for the specific hybridization of these reagents to their complementary sequences within the nucleic acid molecule or interest.
• Alternative diagnostic methods for the detection of diagnostic gene specific nucleic acid molecules can involve their amplification, e.g., by PCR (the experimental embodiment set forth in U.S. Patent No. 4,683,202), ligase chain reaction (Barany, Proc. Natl. Acad. Sci. USA 88:189-93 (1991)), self sustained sequence replication (Guatelli et al., supra), transcriptional amplification system (Kwoh et al., supra), Q-Beta Replicase (Lizardi et al., Bio/Technology 6:1 197 (1988)), or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well known to those of skill in the art. These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low numbers.
• diagnostic profiles can also be assessed in such detection schemes. Diagnostic profiles can be generated, by using microarrays, differential display procedures, Northern analysis or RT-PCR. XXXV. DETECTION OF TARGET GENE PEPTIDES
• Antibodies directed against normal or mutant diagnostic gene peptides can also be used in disease, specifically cardiac, kidney or inflammatory disease, diagnostics and prognostics as described above.
• diagnostic methods can be used to detect abnormalities in the level of diagnostic gene protein expression, or abnormalities in the structure or tissue, cellular, or subcellular location of diagnosticing gene protein.
• Structural differences can include, for example, differences in the size, electronegativity, or a ⁇ tigemcit ⁇ of the mutant diagnostic gene protein relative to the normal diagnostic gene protein
• Protein from the tissue or cell type to be analyzed can be isolated using techniques known to those of skill in the art. The protein isolation methods employed herein can, be such as those described in (Harlow et al., supra), which is incorporated herein by reference in its entirety.
• diagnostic methods for the detection of normal or mutant diagnostic gene peptide molecules can involve, immunoassays wherein diagnostic gene peptides are detected by their interaction with an anti diagnostic gene specific peptide antibody
• diagnostic gene peptides are detected by their interaction with an anti diagnostic gene specific peptide antibody
• antibodies, or fragments of antibodies useful in the present invention can be used to quantitatively or qualitatively detect the presence of normal or mutant diagnostic gene peptides. This can be accomplished, for example, by immunofluorescence techniques employing a fluorescently labeled antibody coupled with light microscopic, flow cytometnc, or fluorimet ⁇ c detection.
• the antibodies (or fragments thereof) useful in the present invention can, additionally be employed histologically, as in immunofluorescence or immunoelectron microscopy, for in situ detection of target gene peptides.
• In situ detection can be accomplished by obtaining a biological sample from a patient, and applying thereto a labeled antibody of the present invention.
• the antibody (or fragment) is preferably applied by overlaying the labeled antibody
• Immunoassays for normal or mutant diagnostic gene peptides may typically comprise incubating a biological sample, such as a biological fluid, a tissue extract, freshly harvested cells, or cells which have been incubated in tissue culture, in the presence of a detectably labeled antibody capable of identifying diagnostic gene peptides, and detecting the bound antibody by any of a number of techniques well known in the art.
• a biological sample such as a biological fluid, a tissue extract, freshly harvested cells, or cells which have been incubated in tissue culture
• the biological sample can be brought in contact with and immobilized onto a solid phase support or carrier such as nitrocellulose, or other solid support which is capable of immobilizing cells, cell particles or soluble proteins.
• a solid phase support or carrier such as nitrocellulose, or other solid support which is capable of immobilizing cells, cell particles or soluble proteins.
• the support can then be washed with suitable buffers followed by treatment with the detectably labeled diagnostic gene specific antibody.
• the solid phase support can then be washed with the buffer a second time to remove unbound antibody.
• the amount of bound label on solid support can then be detected by conventional means.
• ETA enzyme immunoassay
• ELISA Enzyme Linked Immunosorbent Assay
• the enzyme which is bound to the antibody, will react with an appropriate substrate, preferably a chromogenic substrate, in such a manner as to produce a chemical moiety that can be detected by spectrophotometric fluorimet ⁇ c or by visual means. Detection can also be accomplished using any of a variety of other immunoassays.
• RIA radioimmunoassay
• the radioactive isotope can be detected by such means as the use of a gamma counter or a scintillation counter or by autoradiography.
• XXXVI USE OF DIAGNOSTIC GENES AS SURROGATE MARKERS IN CLINICAL TRIALS
• the expression pattern of the diagnostic genes of the invention may be utilized as surrogate markers to monitor clinical human trials of drugs being tested for their efficacy as disease treatments, specifically for cardiac, kidney or inflammatory disease, or may additionally be used to monitor patients undergoing clinical evaluation for the treatment of such disease.
• the effect of the compound on the diagnostic gene expression normally displayed in connection with a disorder involving a disease, specifically cardiac, kidney or inflammatory disease, can be used to evaluate the efficacy of the compound as a treatment for such a disorder. Additionally, diagnostic gene expression can be used to monitor patients undergoing clinical evaluation for the treatment of the disorder.
• the diagnostic gene expression and diagnostic pattern can be used to monitor clinical trials of drugs in human patients, indeed, the influence of modulating agents on a disease can be measured by performing microarray analysis of mRNA obtained from biological samples of patients undergoing clinical tests.
• XXXVII TREATMENT OF PATIENTS SUFFERING FROM A DISEASE, SPECIFICALLY CARDIAC, KIDNEY OR INFLAMMATORY DISEASE
• Another aspect of the present invention pertains to methods for treating a subject having a disease, specifically cardiac, kidney or inflammatory disease, characterized by (or associated with) differential gene expression or polypeptide activity. These methods include the step of administering a modulator of the differential nucieic acid expression or polypeptide activity to the subject such that treatment occurs.
• a modulator of the differential nucieic acid expression or polypeptide activity to the subject such that treatment occurs.
• differential activity or expression interferes with the normal system function.
• a modulator includes a molecule, which can modulate nucleic acid expression or polypeptide activity.
• a modulator can modulate, e.g., upregulate (activate) or downregulate (suppress), nucleic acid expression.
• a modulator can modulate (e.g., stimulate or inhibit) polypeptide activity.
• a modulator can be an antisense molecule, e.g., a ribozyme, as described herein.
• antisense molecules which can be used to inhibit nucleic acid expression include antisense molecules which are complementary to a portion of the 5' untranslated region of the cDNA encoding the genes and polypeptides of the present invention, e.g., SEQ ID N0:25, which also includes the start codon and antisense molecules which are complementary to a portion of the 3' untranslated region of the cDNA.
• a modulator that inhibits nucleic acid expression can also be a small molecule or other drug, e.g., a small molecule or drug identified using the screening assays described herein, which inhibits nucieic acid expression.
• a modulator can be, for example, a nucleic acid molecule encoding the differentially expressed polypeptide (e.g., a nucleic acid molecule comprising a nucleotide sequence homologous to the nucleotide sequence encoding the polypeptide) or a small molecule or other drug, e.g., a small molecule (peptide) or drug identified using the screening assays described herein, which stimulates nucleic acid expression.
• a nucleic acid molecule encoding the differentially expressed polypeptide e.g., a nucleic acid molecule comprising a nucleotide sequence homologous to the nucleotide sequence encoding the polypeptide
• a small molecule or other drug e.g., a small molecule (peptide) or drug identified using the screening assays described herein, which stimulates nucleic acid expression.
• a modulator can be an anti-polypeptide antibody or a small molecule or other drug, e.g., a small molecule or drug identified using the screening assays described herein, which inhibits polypeptide activity.
• a modulator can be an active polypeptide or portion thereof (e.g., a polypeptide or portion thereof having an amino acid sequence which is homologous to the amino acid sequence of the polypeptide or a portion thereof) or a small molecule or other drug, e.g., a small molecule or drug identified using the screening assays described herein, which stimulates polypeptide activity.
• a subject having a disease can be treated according to the present invention by administering to the subject a polypeptide or portion thereof modulating the differentially expressed nucleic acid expression and or polypeptide activity such that treatment occurs.
• a cell associated activity refers to a normal or abnormal activity or function of a cell.
• examples of cell associated activities include proliferation, migration, differentiation, production or secretion of molecules, such as proteins, and cell survival, in a preferred embodiment, the cardiac cell is a myocyte.
• altered refers to a change, e.g., an increase or decrease, in a cell associated activity.
• the agent stimulates polypeptide activity or nucleic acid expression
• stimulatory agents include an active protein, a nucleic acid molecule encoding polypeptide that has been introduced into the cell, and a modulatory agent which stimulates polypeptide activity or nucleic acid expression and which may be identified using the drug screening assays described herein.
• the agent inhibits polypeptide activity or nucleic acid expression.
• inhibitory agents include an antisense nucleic acid molecule, an anti polypeptide antibody, and a modulatory agent which inhibits polypeptide activity or nucleic acid expression and which is identified using the drug screening assays described herein.
• modulatory methods can be performed in vitro (e.g., by cultu ⁇ ng the cell with the agent), or alternatively in vivo (e.g., by administering the agent to a subject).
• the modulatory methods are performed in vivo, / , the cell is present within a subject and the subject has a disease, specifically cardiac, kidney or inflammatory disease, characterized by or associated with differential polypeptide activity or nucleic acid expression.
• a nucleic acid molecule, a polypeptide, a modulator, or a compound used in the methods of treatment can be incorporated into an appropriate pharmaceutical composition described herein and administered to the subject through a route, which allows the molecule, polypeptide, modulator, or compound to perform its intended function. Examples of routes of administration are also described herein.
• Test patients can be administered compounds suspected of disease, specifically cardiac, kidney or inflammatory disease, modulating activity.
• Control patients can be given a placebo Cardiac cell biopsies or peripheral blood can be drawn from each patient after a determined period of treatment and RNA can be isolated as described supra for analysis.
• XXXVIII is also described herein.
• kits for detecting the presence of differentially expressed genes of the present invention in a biological sample can comprise a labeled or labelable compound or agent capable of detecting polypeptide or mRNA in a biological sample, means for determining the amount of differentially expressed gene in the sample, and means for comparing the amount of differentially expressed gene in the sample with a standard
• the kit may preferably comprise a microarray comprising one or more oligonucleotides complementary to reference DNA or RNA sequences encoding the differentially expressed genes of the present invention obtained from tissue from a normal subject and tissue from a subject exhibiting a disease, specifically cardiac, kidney or inflammatory disease
• a biological sample is obtained from the subject, particularly tissue or blood, from which cDNA probes are made and hybridized on a microarray to create fluoromet ⁇ c, colorimet ⁇ c or such identifying emissions
• the compound or agent can be packaged in a suitable container
• the kit can further comprise instructions for using the kit
• kits comprising at least one oligonucleotide of the present invention.
• the kits contain one or more pairs of allele-specific oligonucleotides hybridizing to different forms of a polymorphism.
• the allele specific oligonucleotides are provided immobilized to a substrate.
• Optional additional components of the kit can include, restriction enzymes, reverse transcriptase or polymerase, the substrate nucleoside t ⁇ phosphates, means used to label (e.g., an avidm-enzyme conjugate and enzyme substrate and chromogen if the label is biotin), and the appropriate buffers for reverse transcription, PCR, or hybridization reactions.
• the kit may also contain instructions for carrying out the methods.
• the kit may comprise one or more antibodies that bind with high specificity to the protein products of the differentially expressed genes of the present invention, e.g., all or a portion of the ammo acid sequence of the human genes 1 8U (SEQ ID N0:37), prostacyciin-stimulating factor (SEQ ID N0:38), osf-2 (SEQ ID N0.39),t ⁇ ssue specific mRNA (SEQ ID N0:40), insuhn-like growth factor binding protein 6 (SEQ ID N0:41), OSF 1 (SEQ ID N0:42), gas 1 (SEQ ID N0:43), YMP (SEQ ID N0:44), BTG2 (SEQ ID N0:45), pre B cell stimulating factor homolog (SDFI a) (SEQ ID N0:46), peripheral benzodiazepine receptor (SEQ ID N0:47), and cellular ligand of annexin II (pi 1 ) (SEQ ID N0:
• kits This generally comprises preselected primers for specific genes. Also included may be enzymes suitable for amplifying nucleic acids including various polymerases, deoxynucleotides and buffers to provide the necessary reaction mixture for amplification.
• kits generally comprise, in suitable means, distinct containers for each individual reagent and enzyme as well as for each gene primer pair Preferred pairs of primers for amplifying nucleic acids are selected to amplify the sequences specified in GenBank Accession numbers X57352, S75725, D13665, X67698, M62402, D90226, L13698, U52101, U72649, L36034, M36035, and M38591, encoding the human genes 1 8U (SEQ ID N0:37), prostacyciin-stimulating factor (SEQ ID N0:38), osf 2 (SEQ ID N0:39), tissue specific mRNA (SEQ ID N0:40), insulin- like growth factor binding protein 6 (SEQ ID N0:41), OSF 1 (SEQ ID N0:42), gas-1 (SEQ ID N0:43), YMP (SEQ ID N0:44), BTG2 (SEQ ID NO 45), pre B cell stimulating factor homolog (SDFI a) (SEQ ID N
• such microarrays may comprise sequences specific for differentially expressed disease, specifically cardiac, kidney or inflammatory disease, genes, preferably chosen from a group including nucleic acids, or portions thereof, corresponding to the sequences specified in GenBank Accession numbers X57352, S75725, D13665, X67698, M62402, D90226, L13698, U52101 , U72649, L36034, M36035, and M38591 , encoding the human genes 1 8U (SEQ ID N0:37), prostacyclin stimulating factor (SEQ ID N0:38), osf-2 (SEQ ID N0:39), tissue specific mRNA (SEQ ID N0:40), insu n-like growth factor binding protein 6 (SEQ ID N0:41), OSF-1 (SEQ ID N0:42), gas-1 (SEQ ID N0:43), YMP (SEQ ID N0:44), BTG2 (SEQ ID N0:45), pre-B cell stimulating factor homolog (SD
• Neonatal rat ventricular cardiom ⁇ ocytes were isolated from one or two days old rat pups using the following reagents and isolation procedure: Reagents:
• Dissociation buffer CBFHH (Calcium- and Bicarbonate-Free Hanks with Hepes), pH 7.5
• Cryopreserved human peripheral blood mononuclear cells were purchased from Clonetics Corp. and maintained in short term culture in lymphocyte growth media. Experimental incubations utilized 2x10 5 cells/well/ 100 ⁇ l in a 96 well format.
• Cryopreserved human cardiac fibrobiasts obtained from a single adult male donor (60 years) were purchased from Clonetics Corp. and maintained in short term culture in fibroblast growth media.
• Experimental incubations utilized 4x10 3 cells/well/100 ⁇ l in a 96 well format.
• Cells were treated with known stimuli in quadruplet over a time course.
• the known stimuli reflect the therapeutic areas of inflammation and fibrosis.
• Cell Type Stimuli Cell Type Stimuli
• Real time Quantitative PCR uses a fluorogenic 5' nuclease assay performed with the TaqMan® PCR Reagents designed and optimized for use with a 7700 Sequence Detector. Using this assay, one can detect and monitor target gene sequences.
• a fluorogenic probe consisting of an oligonucleotide with both a reporter and a quencher dye attached, anneals specifically between the forward and reverse primers.
• the reporter dye is separated from the quencher dye, and a sequence-specific signal is generated.
• the genes covered include those genes identified by high density differential display as described hereinbefore, thus allowing us to examine these gene expressions in tissue culture (and human samples) in cells that have been treated with a number of known pathological stimuli pertaining the inflammation or fibrosis. Additionally, a number of assays have been developed which reflect the dieases phenotype themselves. These assays are used to determine whether treatment of a cell type with a recombinantly expressed protein will induce a phenotype which relates to inflammation and/or fibrosis.
• RNA isolated from neonatal rat cardiac myocytes treated with stimuli known to induce a hypertrophic response was analyzed for the expression of OSF 2. The results are illustrated in Figure 10. Following 24 hours of incubation cardiotroph (CT-1) and TGF- ⁇ were shown to induce OSF-2 by eight fold compared the untreated cells. Other hypertrophic stimuli (IL-1 ⁇ , TNF- ⁇ and Angll) also induced OSF-2 by three-fold above control. This is the first demonstration of regulation of OSF-2 in a tissue culture setting in response to hypertrophic stimuli.
• IGFBP7 IGFBP7
• NPC-30753 p38 ⁇ inhibitor
• Rat neonatal myocytes were treated with 0, 0.2 or 1 ⁇ g/ml OSF-1 for 0.5, 1, 2 and 24 hours.
• Total RNA was isolated and assayed for ANP and GAPDH transcript levels.
• the data shows a two-fold up-regulat ⁇ on of ANP by OSF-1 at 0.5 hours treated with 1 ⁇ g/ml OSF-1, indicating a possible hypertrophy promoting activity of OSF- 1.
• IGFBP-6 insuhn-like growth factor binding protein 6

Landscapes

• Chemical & Material Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Health & Medical Sciences (AREA)
• Organic Chemistry (AREA)
• Wood Science & Technology (AREA)
• Analytical Chemistry (AREA)
• Zoology (AREA)
• Genetics & Genomics (AREA)
• Engineering & Computer Science (AREA)
• Pathology (AREA)
• Immunology (AREA)
• Microbiology (AREA)
• Molecular Biology (AREA)
• Biotechnology (AREA)
• Biophysics (AREA)
• Physics & Mathematics (AREA)
• Biochemistry (AREA)
• Bioinformatics & Cheminformatics (AREA)
• General Engineering & Computer Science (AREA)
• General Health & Medical Sciences (AREA)
• Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
• Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Priority Applications (1)

Applications Claiming Priority (3)

Related Child Applications (1)

Publications (1)

Family

ID=22347087

Family Applications (1)

Country Status (3)

Families Citing this family (47)

Family Cites Families (3)

• 1999
  • 1999-12-15 AU AU20553/00A patent/AU2055300A/en not_active Abandoned
  • 1999-12-15 EP EP99964277A patent/EP1140137A2/de not_active Ceased
  • 1999-12-15 WO PCT/US1999/029941 patent/WO2000035473A2/en not_active Application Discontinuation

Non-Patent Citations (1)

Also Published As

Similar Documents

Legal Events