JP2002516664A - CNRE binding factors and uses thereof - Google Patents

Abstract

  (57) [Summary] The present invention relates to nucleic acids encoding CNRE-binding polypeptides, including fragments of CNRE-binding polypeptides and biologically functional variants thereof. The present invention also relates to therapeutic and diagnostic agents, including the proteins and genes described above, and agents that bind to the proteins and genes described above.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001] government support this study, by Grant No. R37 HL-35610 from the National Institutes of Health, received a partially funds. Accordingly, the United States government may have certain rights in the invention.

RELATED APPLICATIONS This application is filed under 35 USC §119 (e) with the title "NRE BINDING FACTORS AND USES THERE."
US Provisional Patent Application Ser. No. 60 / 082,997, filed Apr. 24, 1998, entitled "OF".
Claim priority from issue. The contents of the application identified above are expressly incorporated herein.

FIELD OF THE INVENTION [0003] The present invention relates to nucleic acid and encoding polypeptides of the regulatory element binding factor (REB), as well as diagnostic methods and treatments associated with medical disorders associated with such genes and polypeptides, including hypertension and cancer. About the law.

[0004] Background Hypertension of the invention is, perhaps, is the most important public health in developed countries of the problem. Hypertension is common, asymptomatic, easily detectable, and often leads to fatal complications if left untreated. Although the pathophysiology of elevated arterial pressure is well understood, in 90-95% of cases, the etiology (and thus potentially prevention and treatment) is still largely unknown. As a result, in most cases, hypertension is treated non-specifically, resulting in numerous insignificant side effects and relatively high (approximately 50%) noncompliance.

[0005] Patients with arterial hypertension and whose cause cannot be defined are said to have primary, essential, or idiopathic hypertension. Approximately 15% of patients with essential hypertension have elevated plasma renin activity levels above the normal range. Therefore, it has been suggested that plasma renin plays an important role in the pathogenesis of elevated arterial pressure in these patients.

[0006] Renin is a proteolytic enzyme (aspartate proteinase), which is produced in and stored in granules of paraglomerular cells surrounding the glomerular import arterioles in the kidney. Renin acts basic substrate, the angiotensinogen (circulating alpha ₂ globulin created in the liver) to form decapeptide, angiotensin I. Angiotensin I is then enzymatically converted to the octapeptide, angiotensin II, by a converting enzyme present in many tissues, especially in the pulmonary vascular endothelium. Angiotensin II is a potent vasopressor and exerts its effects by directly affecting arteriolar smooth muscle. In addition, angiotensin II stimulates aldosterone production by the globular glands of the adrenal cortex. Currently, some of the most common drug treatments for hypertension target this renin-angiotensin pathway. Examples of such classes of drugs include angiotensin converting enzyme (ACE) inhibitors. These drugs inhibit the enzyme that converts angiotensin I to angiotensin II. Although effective in treating hypertension, the drug and other types of anti-hypertensive drugs are often associated with several side effects.

[0007] Studies in mice have shown that renin exhibits tissue-specific expression. In mice, renin is encoded at two loci (Ren1 ^d and Ren2 ^d ) and is expressed in a variety of extrarenal tissues, such as the submandibular gland (SMG), adrenal gland, heart, testis and ovary.

[0008] Tissue-specific gene expression relies on the specific interaction of a cis DNA sequence with a nuclear transactivation protein. Certain trans-acting factors are ubiquitous, while others exhibit transient, tissue, and / or cell-specific expression. Other researchers have shown that the interaction of negative nuclear regulatory elements (NREs or silencers) with specific nuclear trans-acting proteins plays an important role in tissue-specific regulation of gene expression (Williams, TM Et al., Science, 1991, 254: 1791-1794; Laden, JM, Annu Re
v Immun, 1993, 11: 539-570). Further complications arise when multiple trans-acting factors, especially those that exert the opposite effect, can bind to the same cis element. In this case, competition from the same element can play an important role in gene regulation.

[0009] Because renin is a rate-limiting enzyme that controls the formation of angiotensin II and the initiation of the renin-angiotensin system cascade, factors that control renin expression may be very useful in reducing hypertension Will be proved.

[0010] We have previously established the presence of a CNRE element in the Ren1 ^d gene (Nak
amura, N. et al., Proc Natl Acad Sci USA, 1989, 86: 56-59). This CNRE element was later characterized as a 3030 base pair DNA fragment (Barrett, G. et al., Proc N
atl Acad Sci USA, 1992, 89: 885-889). Such CNRE motifs are also found in promoter regions found in various genes including human renin, collagen II, T cell receptor and c-myc.

There is a need to improve the course of hypertension and other cardiovascular diseases. There is also a need to identify genes responsible for hypertension, cardiovascular disease, cancer and other disorders.

It is an object of the present invention to desirably provide compounds that affect hypertension and the course of cancer. Another object of the present invention is to provide a therapeutic method for treating diseases or disorders involving CNRE and CNRE binding factors.

Yet another object of the present invention is to provide diagnostic methods and research tools related to hypertension, cardiovascular disease, and other disorders. These and other purposes are:
It will be described in more detail below.

SUMMARY OF THE INVENTION The present invention relates to CNRE binding factor polypeptides (CNREBs), nucleic acids encoding such polypeptides, and fragments and biologically functional variants thereof. The present invention also provides expression vectors containing these molecules and host cells transfected with these molecules. The invention also relates to compositions for therapeutic and diagnostic uses, as well as therapeutic and diagnostic methods related to the aforementioned polypeptides and nucleic acids. Also included in the present invention are agents that bind to the aforementioned CNREB polypeptides and nucleic acids.

[0015] In one aspect, the invention involves cloning a cDNA encoding a CNRE binding transcription factor. CNRE-binding transcription factors are generally present in the human, mouse and rat renin genes, the human c-myc gene, type II collagen, and the 5 'flanking region (also known as the promoter) of the T cell receptor. SEQ ID NO: 21, SEQ ID NO: 22,
Or binds to any of the sequences set forth in SEQ ID NO: 23 and regulates the expression of these genes.

According to one aspect of the present invention there is provided an isolated nucleic acid molecule encoding a CNREB-2 polypeptide, and comprising: (a) the nucleic acid of SEQ ID NO: 7, the nucleic acid of SEQ ID NO: 17, and A nucleic acid molecule that hybridizes under highly stringent conditions to a molecule selected from the group consisting of nucleic acids of SEQ ID NO: 19, and that encodes a CNREB-2 polypeptide; (b) each CNREB- (A) deletions, additions and substitutions of (a) encoding the polypeptide; (c) in the codon sequence due to the degeneracy of the genetic code;
a) a nucleic acid molecule different from that of (a) or (b); and (d) the complement of (a), (b) or (c). In certain embodiments, the isolated nucleic acid molecule is SEQ ID NO: 17
including. In some embodiments, the isolated nucleic acid molecule is a human cDNA or SEQ ID NO:
It contains the gene corresponding to 17. An isolated nucleic acid molecule also includes a molecule that encodes a polypeptide of SEQ ID NO: 18 and that has CNREB-2 activity. Preferably, the isolated nucleic acid molecule consists of the nucleotide sequence of SEQ ID NO: 17.

The invention provides in another aspect: (a) a unique nucleic acid molecule selected from the group consisting of a nucleic acid of SEQ ID NO: 7, a nucleic acid of SEQ ID NO: 17, and a nucleic acid of SEQ ID NO: 19 fragment,
(B) the complement of (a), wherein the unique fragment is: (1) SEQ ID NO: 10, SEQ ID NO
: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ I
The sequence described in D NO: 16, the complement of (2) (1), and (3) (1) and (2)
Or a sequence of contiguous nucleotides that is not identical to any sequence selected from the group consisting of:

In one embodiment, the sequence of the flanking nucleotides is (1) at least two flanking nucleotides that are not identical to the group of sequences;
3 adjacent nucleotides, (3) at least 4 adjacent nucleotides that are not identical to the sequence group, (4) at least 5 adjacent nucleotides that are not identical to the sequence group,
(5) at least six adjacent nucleotides that are not identical to the sequence group; and (6) at least seven adjacent nucleotides that are not identical to the sequence group.

In another embodiment, the fragment comprises at least: 8 nucleotides, 10 nucleotides, 1
2 nucleotides, 14 nucleotides, 16 nucleotides, 18 nucleotides, 20 nucleotides, 22 nucleotides, 24 nucleotides, 26 nucleotides, 28 nucleotides,
It has a size selected from the group consisting of 30 nucleotides, 40 nucleotides, 50 nucleotides, 75 nucleotides, 100 nucleotides, 200 nucleotides, 1000 nucleotides and all integer lengths therebetween.

According to another aspect, the invention provides an expression vector, comprising the nucleic acid molecule described above, and a host cell transformed or transfected with such an expression vector.

In accordance with another aspect of the present invention, there is provided an isolated polypeptide. The isolated polypeptide is encoded by an isolated nucleic acid molecule of the invention, as described above. In some embodiments, the isolated polypeptide is encoded by the nucleic acid of SEQ ID NO: 7, resulting in a polypeptide having the sequence of SEQ ID NO: 8, having CNREB-2 activity. In other embodiments, the isolated polypeptide is encoded by the nucleic acid of SEQ ID NO: 19, and has CNREB-2 activity.
This results in a polypeptide having the sequence of ID NO: 20. In a preferred embodiment, the isolated polypeptide is encoded by the nucleic acid of SEQ ID NO: 17, and has CNREB-2 activity.
This results in a polypeptide having the sequence of D NO: 18. In a further embodiment, the isolated polypeptide is of sufficient length to exhibit sequence uniqueness within the human genome and
It may be a fragment or variant of the foregoing, which binds to a polypeptide having REB-2 activity, provided that the fragment comprises SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12,
Includes sequences of contiguous amino acids that are not identical to any sequence encoded by the nucleic acid sequence as set forth in SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16. In another aspect, an immunogenic fragment of the above-described polypeptide molecule is provided.

According to another aspect of the present invention there is provided an isolated binding polypeptide which selectively binds to a polypeptide encoded by an isolated nucleic acid molecule of the present invention as described above. Preferably, the isolated binding polypeptide selectively binds to a polypeptide comprising the sequence of SEQ ID NO: 18, or a fragment thereof. In a preferred embodiment, the isolated binding polypeptide comprises an antibody and an antibody fragment (eg, an antibody fragment comprising a Fab, F (ab) ₂ , Fd and a CDR3 region that selectively binds a CNREB-2 polypeptide). In certain embodiments,
The antibodies are human antibodies.

According to another aspect of the present invention, there is provided a method for isolating a nucleic acid molecule encoding a polypeptide having CNREB-2 activity. The method comprises: (a) SEQ ID NO: 17
Or a fragment of at least eight contiguous nucleotides thereof; (b) under stringent screening conditions, as a probe for obtaining a nucleic acid molecule encoding a polypeptide that is a candidate having CNREB-2 activity; Using the nucleic acid molecule of (a); (c) expressing the isolated nucleic acid molecule obtained in (b) in a host cell to produce the polypeptide; and (d) expressing the expressed polypeptide CNREB-2 The presence of such an activity, which measures the activity, is accompanied by an indication that the isolated nucleic acid encodes a polypeptide having CNREB-2 activity.

According to another aspect of the present invention, there is provided a method for reducing renin expression in a mammalian cell expressing renin. The method comprises providing a mammalian cell with an effective amount of CNREB- to reduce renin expression in the mammalian cell.
1) contacting with an inhibitor. In some embodiments, the mammalian cells are C
Contacting with a NREB-1 inhibitor occurs in vitro or in vivo. CNREB-1 inhibitors inhibit antisense CNREB-1 nucleic acids, dominant negative CNREB-1 nucleic acids, dominant negative CNREB-1 polypeptides, anti-CNREB-1 antibodies, anti-CNREB-1 antibody fragments, and CNREB-1 activity Includes polypeptides, and functional fragments or variants thereof. In certain embodiments, the CNREB-1 inhibitor is a nucleic acid. In a preferred embodiment, the nucleic acid is the nucleic acid of SEQ ID NO: 1, the nucleic acid of SEQ ID NO: 3, the nucleic acid of SEQ ID NO: 5
And a CNREB-1 nucleic acid selected from the group consisting of the nucleic acid of SEQ ID NO: 6 and the antisense nucleic acid or the dominant negative nucleic acid. In some embodiments, the CNREB-1 inhibitor is not an adenylate cyclase inhibitor. However, any of the foregoing embodiments may further comprise co-administration of an adenylate cyclase inhibitor in an amount effective to reduce renin expression in mammalian cells.

[0025] In accordance with yet another aspect of the present invention, there is provided a method for reducing CNREB-1 activity in a patient. The method comprises administering to a patient in need of such treatment an amount of a CNREB-1 inhibitor effective to reduce CNREB-1 activity in the patient. In some embodiments, the amount is less than the normal baseline level.
1 Sufficient to reduce activity. In certain embodiments of the aforementioned method, the patient has an increased risk of hypertension, has hypertension, has an increased risk of myocardial infarction, has an increased risk of ischemic stroke, has an ischemic stroke. Experienced renal disease, nephropathy, congestive heart failure, coronary artery disease, optic disc swelling,
And increased risk of abnormalities selected from the group consisting of stenosis of the aorta, or progressive renal disease, nephropathy, congestive heart failure, coronary artery disease, optic disc swelling,
And an abnormality selected from the group consisting of aortic constriction. In certain embodiments, the CNREB-1 inhibitor is administered acutely. In other embodiments, the agent is administered C
NREB-1 inhibitor. In a further embodiment, the method further comprises co-administering an agent other than the CNREB-1 inhibitor. Preferred non-CNREB-1 inhibitors include diuretics, anti-adrenergic agents, vasodilators, calcium entry blockers, and angiotensin converting enzyme inhibitors in amounts effective to reduce CNREB-1 activity in the patient. It is. Any of the foregoing embodiments may further comprise co-administration of an adenylate cyclase inhibitor in an amount effective to reduce CNREB-1 activity in the patient.

According to another aspect of the present invention, there is provided a method for increasing CNREB-1 activity in a mammalian cell. The method involves contacting a mammalian cell with an effective amount of a CNREB-1 activator to increase CNREB-1 activity in the mammalian cell. In some embodiments, contacting the mammalian cell with the CNREB-1 activator may occur in vitro or in vivo. In certain embodiments, C
NREB-1 activators are nucleic acids. In a preferred embodiment, the nucleic acid has SEQ ID NO: 1.
, The nucleic acid of SEQ ID NO: 3, the nucleic acid of SEQ ID NO: 5, and the nucleic acid of SEQ ID NO: 6. In a further embodiment, the CNREB-1 activator is a polypeptide. In a preferred embodiment, the CNREB-1 activator comprises SEQ ID NO: 1
A nucleic acid of SEQ ID NO: 3, a nucleic acid of SEQ ID NO: 5 and a polypeptide that is an expression product of a nucleic acid selected from the group consisting of a nucleic acid of SEQ ID NO: 6, or a functional fragment or variant thereof It is.

In accordance with another aspect of the present invention, there is provided a method for increasing CNREB-1 activity in a patient. The method can be used in patients in need of such treatment to treat CNRE in patients.
Administering a CNREB-1 activator in an amount effective to increase B-1 activity.
In some embodiments, the amount is sufficient to increase CNREB-1 activity above normal baseline levels. In certain embodiments, the patient has a condition involving hypertension, impotence, spinal cord injury, toxic shock syndrome or blood transfusion. In a further embodiment, the CNREB-1 activator is a nucleic acid. In a preferred embodiment, the nucleic acid is a nucleic acid of SEQ ID NO: 1, a nucleic acid of SEQ ID NO: 3, a nucleic acid of SEQ ID NO: 5 and a nucleic acid of SEQ ID NO: 5.
D NO: 6 is a nucleic acid selected from the group consisting of nucleic acids. In a further embodiment, the CN
REB-1 activators are polypeptides. In a preferred embodiment, the CNREB-1 activator comprises the nucleic acid of SEQ ID NO: 1, the nucleic acid of SEQ ID NO: 3, the nucleic acid of SEQ ID NO: 5 and the nucleic acid of SEQ ID NO: 5.
The polypeptide is an expression product of a nucleic acid selected from the group consisting of NO: 6 nucleic acids, or a functional fragment or variant thereof. In some embodiments, CNREB-1
The activator is administered acutely. In certain embodiments, the CNREB-1 activator is administered prophylactically. In a further embodiment, the method further comprises co-administering an agent other than the CNREB-1 activator. Preferred non-CNREB-1 activators include fluorocortisone, potassium tablets, vasopressin analogs, somatostatin analogs, beta blockers, sympathomimetic agents, dopamine antagonists, venous constrictors (veno
constrictor), and adenylate cyclase activator.

According to another aspect of the present invention, there is provided a method for measuring the level of CNREB-1 expression in a patient. The method includes: (a) obtaining a test sample from an individual; (b) measuring the level of CNREB-1 expression in the test sample; and (c) comparing the measured level of CNREB-1 expression to a control; It involves determining the level of CNREB-1 expression in the patient. In some embodiments, the expression of CNREB-1 in step (b) is CNREB-1 mRNA expression. In certain embodiments, the expression of CNREB-1 in (b) is CNREB-1 polypeptide expression. In a further aspect, the test sample is a tissue or a biological fluid. For methods of measuring CNREB-1 mRNA expression, preferred assays include nucleic acid amplification and hybridization assays,
For example, Northern blotting is included. For methods of measuring CNREB-1 polypeptide expression, a preferred assay involves using a monoclonal antiserum to CNREB-1 or a polyclonal antiserum to CNREB-1. In a further embodiment, an increase in CNREB-1 expression levels as compared to a control indicates that the patient is susceptible to developing a renin-angiotensin system-mediated disorder. In certain embodiments, the renin-angiotensin system mediated disorder is an increased risk of hypertension,
Selected from the group consisting of hypertension, increased risk of myocardial infarction, increased risk of ischemic stroke, ischemic stroke, progressive renal disease, nephropathy, congestive heart failure, coronary artery disease, optic disc swelling, and aortic constriction Or an abnormality selected from the group consisting of progressive kidney disease, nephropathy, congestive heart failure, coronary artery disease, optic disc swelling, and aortic constriction.

According to a further aspect of the present invention there is provided a method for determining a susceptibility of a patient to develop a renin-angiotensin system mediated disorder. The method includes: (a) characterizing the CNREB-1 nucleic acid sequence in a test sample, wherein the test sample is obtained from a patient; (b) replacing the CNREB-1 nucleic acid sequence in the test sample with the control sample CNREB- An alteration or match in the CNREB-1 nucleic acid sequence in the test sample, relative to the nucleic acid sequence, where compared to the CNREB-1 nucleic acid sequence in the control sample, indicates that the patient develops a renin-angiotensin system-mediated disorder , With that. In some embodiments, the observed alteration is evident when the CNREB-1 nucleic acid sequence in the test sample is compared to the wild-type CNREB-1 nucleic acid sequence in a control sample. In other embodiments, the observed match is when the CNREB-1 nucleic acid sequence in the test sample is compared to the mutant CNREB-1 nucleic acid sequence in the control sample.
it is obvious. In a further embodiment, CNREB-1 mRNA molecules are compared. In a further embodiment, the modification of the CNREB-1 mRNA comprises modifying the mRNA from the test sample with the nucleic acid of SEQ ID NO: 1, the nucleic acid of SEQ ID NO: 3, the nucleic acid of SEQ ID NO: 5 and the nucleic acid of SEQ ID NO: 6. Is detected by hybridizing to a CNREB-1 nucleic acid selected from the group consisting of:
In some embodiments, the CNREB-1 cDNA sequences are compared, and the comparison comprises a CNREB-
1 Performed by hybridizing a cDNA probe to genomic DNA isolated from a test sample. In certain embodiments, the method further comprises: (a) subjecting the genomic DNA isolated from the patient test sample to Southern hybridization using a CNREB-1 cDNA probe; and (b) (i) the patient test sample And (ii) comparing the hybridization of the CNREB-1 cDNA probe to a control sample. In any of the forgoing embodiments, the CNRE
The B-1 cDNA probe detects restriction fragment length polymorphism. In a further embodiment, a CNRE
Comparing the B-1 nucleic acid sequences, wherein the comparison is performed by determining the sequence of at least a portion of the CNREB-1 cDNA in the test sample using a polymerase chain reaction;
Deviations in the CNREB-1 cDNA determined from those of the wild-type CNREB-1 nucleic acid sequence set forth in NO: 1 indicate a patient's susceptibility to develop a renin-angiotensin system-mediated disorder. The renin-angiotensin system-mediated disorder is as described above.

[0030] In other embodiments, the modification of the CNREB-1 nucleic acid sequence is accomplished by modifying the molecule (a) the CNREB-1 cDNA or mRNA isolated from the test sample, and (b) the human wild-type CNREB-1 nucleic acid sequence. Where complementary nucleic acid probes hybridize to each other and form a duplex, they are detected by identifying a mismatch between molecules (a) and (b). In some embodiments, the CNREB-1 nucleic acid sequences are compared and the CNREB-1 nucleic acid sequence is modified by: (a) amplifying the CNREB-1 cDNA sequence in a test sample; and (b)
The amplified CNREB-1 cDNA sequence is detected by hybridizing to a nucleic acid probe containing the CNREB-1 sequence.

In a further embodiment, the CNREB-1 nucleic acid molecules are compared and the modification of the CNREB-1 nucleic acid sequence is performed by molecular cloning and cloning of the CNREB-1 gene in a test sample.
It is detected by sequencing all or part of the NREB-1 gene.

[0032] In any of the forgoing embodiments, detecting alterations in the CNREB-1 nucleic acid sequence comprises screening for deletion, point, and / or insertion mutations. The test sample may be a tissue, a tissue biopsy and / or a biological fluid. In a preferred embodiment, the test sample comprises a brain, heart, breast, colon, bladder, uterus, prostate, stomach, testis, ovary, pancreas, pituitary, adrenal gland, thyroid, salivary gland, mammary gland, kidney, liver,
It may be obtained from a patient's tissues, including intestine, spleen, thymus, bone marrow, trachea, and lung. In a further preferred embodiment, the test sample is amniotic fluid, aqueous humor, bile, blood, bronchoalveolar lavage, bronchial fluid, cerebrospinal fluid, follicular fluid, gingival crevicular fluid, middle ear fluid, peritoneal fluid, pleural fluid, prostate fluid, It may be obtained from a patient's biological fluids, including saliva, semen, serum, sweat, synovial fluid, tears, culture supernatant, and urine.

According to another aspect of the present invention, there is provided a method for regulating c-myc expression in a cell. The method involves contacting a cell expressing c-myc in a cell with an amount of a CNREB-1 activity modulator effective to modulate c-myc expression in the cell. In some embodiments, the mammalian cell expressing c-myc is CNREB-1
Contacting with an activity modulator can occur in vitro or in vivo. In certain embodiments, the CNREB-1 activity modulator is a nucleic acid. In a further embodiment, C
NREB-1 activity modulators are polypeptides.

According to another aspect of the present invention, there is provided a method for regulating type II collagen expression in a cell. The method involves contacting a cell expressing type II collagen in a cell with an amount of a CNREB-1 activity modulator that is effective to modulate type II collagen expression in the cell. Preferred conditions and CNREB-1 activity modulators include c
-as described above in regulating myc expression.

According to a further aspect of the present invention there is provided a method for modulating T cell receptor expression in a cell. The method involves contacting a cell expressing a T cell receptor in a cell with an amount of a CNREB-1 activity modulator that is effective to modulate T cell receptor expression in the cell. Preferred conditions and CNREB-1 activity modulators include c-myc
As described above in the regulation of expression.

[0036] In some aspects of the invention, a pharmaceutical composition is also provided. The pharmaceutical compositions of the present invention comprise a pharmaceutically effective amount of a CNREB-1 inhibitor to inhibit CNREB-1 activity, and a pharmaceutically acceptable carrier. In certain embodiments, the pharmaceutical composition further comprises a pharmaceutically effective amount of an adenylate cyclase inhibitor to inhibit CNREB-1 activity. In any of the forgoing embodiments, the CNREB-1 inhibitor is a nucleic acid. In a preferred embodiment, the nucleic acid is a CNREB- selected from the group consisting of a nucleic acid of SEQ ID NO: 1, a nucleic acid of SEQ ID NO: 3, a nucleic acid of SEQ ID NO: 5, and a nucleic acid of SEQ ID NO: 6.
One nucleic acid is an antisense nucleic acid or a dominant negative nucleic acid.

[0037] In another aspect of the invention, methods are provided for screening for CNREB fragments and CNREB variants that bind to CNRE. In another aspect of the present invention, there are provided methods for screening functional fragments of CNREB and functional mutants of CNREB that alter renin expression. In yet another aspect of the invention,
Methods are provided for screening for molecules that prevent the binding of native CNREB to CNRE in a cell, in vitro or in vivo. Assays for screening for such molecules are described in the detailed description below. The present invention also relates to CNREB-binding that binds to CNRE and can be regulated by preventing or promoting the expression of renin, c-myc, type II collagen, and T cell receptors.
Also included are products identified by the above-described screening methods, including functional fragments and functional variants of CNREB-2 polypeptide.

According to yet another aspect of the present invention there is provided the use of the foregoing molecules and compositions in the preparation of a medicament, particularly for the treatment of the aforementioned diseases. The invention also includes all functional variants and equivalents of the molecules described above.

[0039] These and other objects of the invention will be described in further detail in connection with the detailed description of the invention. BRIEF DESCRIPTION OF SEQUENCES SEQ ID NO: 1 is the nucleotide sequence of mouse CNREB-1 cDNA.

[0040] SEQ ID NO: 2 is the predicted amino acid sequence of the translation product of mouse CNREB-1 cDNA. SEQ ID NO: 3 is the nucleotide sequence of the largest open reading frame of mouse CNREB-1 cDNA of SEQ ID NO: 1, which encodes the polypeptide of SEQ ID NO: 2.

[0041] SEQ ID NO: 4 is the nucleotide sequence of EST AA415858. SEQ ID NO: 5 is the nucleotide sequence of GenBank accession number U11685. SEQ ID NO: 6 is the nucleotide sequence of GenBank accession number U22662.

[0042] SEQ ID NO: 7 is the nucleotide sequence of mouse CNREB-2 cDNA. SEQ ID NO: 8 is the predicted amino acid sequence of the translation product of mouse CNREB-2 cDNA. SEQ ID NO: 9 is the nucleotide sequence of the largest open reading frame of mouse CNREB-2 cDNA of SEQ ID NO: 7, which encodes the polypeptide of SEQ ID NO: 8.

[0043] SEQ ID NO: 10 is the nucleotide sequence of EST AA756195. SEQ ID NO: 11 is the nucleotide sequence of EST AA619904. SEQ ID NO: 12 is the nucleotide sequence of EST W63814.

[0044] SEQ ID NO: 13 is the nucleotide sequence of EST AA014739. SEQ ID NO: 14 is the nucleotide sequence of EST W64822. SEQ ID NO: 15 is the nucleotide sequence of EST C78804.

[0045] SEQ ID NO: 16 is the nucleotide sequence of EST C80984. SEQ ID NO: 17 is the nucleotide sequence of mouse CNREB-2S cDNA (short isomer).

SEQ ID NO: 18 is the predicted amino acid sequence of the translation product of mouse CNREB-2S cDNA (short isomer). SEQ ID NO: 19 is the nucleotide sequence of mouse CNREB-2L cDNA (long isomer).

[0047] SEQ ID NO: 20 is the predicted amino acid sequence of the translation product of mouse CNREB-2L cDNA (long isomer). SEQ ID NO: 21 is the nucleotide sequence of mouse CNRE.

[0048] SEQ ID NO: 22 is the nucleotide sequence of rat CNRE. SEQ ID NO: 23 is the nucleotide sequence of human CNRE. DETAILED DESCRIPTION OF THE INVENTION One embodiment of the present invention involves the cloning of a cDNA encoding a CNRE-binding transcription factor, wherein the CNRE-binding transcription factor comprises the human, mouse and rat renin genes (Bu
rt, DW. et al., J. Biol. Chem., 1989, 264, 7357-62), the human c-myc gene (Hay, N.
Et al., Genes Dev., 1989, 3, 293-303), which are commonly present in the 5'-flanking regions of many genes, including type II collagen and T cell receptors (while also known as promoters). , SEQ ID NO: 21, SEQ ID NO: 22 or SEQ ID NO:
It binds to any sequence shown in 23 and regulates the expression of these genes.

According to the present invention, CNREB-2 is set forth in SEQ ID NO: 18 and defined in the claims,
Also, under stringent conditions, SEQ ID NO: 7, SEQ ID NO: 17 and / or SEQ ID NO: 17
A novel CNREB transcription factor having an amino acid sequence encoded by a sequence that hybridizes to ID NO: 19. CNREB-2 binds to the 5'-flanking region of the renin gene and regulates the expression of the renin gene. The sequence of the mouse gene was changed to SEQ ID N
O: 7, SEQ ID NO: 17 or any one of the sequences SEQ ID NO: 19, the predicted amino acid sequence of the gene protein product is shown in SEQ ID NO: 8, SEQ ID NO: 19, respectively.
Shown as NO: 18 and SEQ ID NO: 20. Preferably, CNREB-2 is a polypeptide encoded by the nucleic acid of SEQ ID NO: 17, having the predicted amino acid sequence of SEQ ID NO: 18. This indicates a short isoform (CNREB-2S). This short isoform has two other
It shows stronger binding to the renin promoter CNRE when compared to the two isoforms.
Sequence analysis by comparison to nucleic acid and protein databases indicates that CNREB-2 has significant homology to any other known gene or protein. Sequence analysis also indicates that CNREB-2 is a novel zinc finger transcription factor.

Also according to the present invention, CNREB-1 is another CNRE-binding transcription factor,
Coded by EQ ID NO: 1. CNREB-1 also binds to the 5′-flanking region of the renin gene and regulates the expression of the renin gene. The sequence of the mouse gene is shown in SEQ ID NO: 1, and the predicted amino acid sequence of the gene protein product is shown in SEQ ID NO: 2. Sequence analysis by comparison to nucleic acid and protein databases shows that CNREB-1 has high homology to rattus norvegicus orphan receptor RLD-1 (SEQ ID NO: 5, 93% homology at nucleotide level)
And has high homology to human nuclear orphan receptor LXR-α (SEQ ID NO: 6, 89% homology to nucleotide level).

[0051] In one aspect, the invention includes isolated nucleic acids and polypeptides. As used herein, the term "isolated" with respect to nucleic acids refers to (i) amplified in vitro, for example, by the polymerase chain reaction (PCR); (ii) recombinantly produced by cloning; iii) purified by cleavage and gel separation; or (iv) synthesized, for example, by chemical synthesis. An isolated nucleic acid is a nucleic acid that can be readily engineered by recombinant DNA techniques well known in the art. Thus, nucleotide sequences contained within a vector for which the 5 'and 3' restriction sites are known or for which a polymerase chain reaction (PCR) primer sequence is disclosed are considered to be isolated. However, a nucleic acid sequence that is present in its natural state in its natural host is not. The isolated nucleic acid can be, but need not be, substantially purified. For example, nucleic acids isolated in cloning or expression vectors are not pure in that they contain only a small amount (%) of the material that remains in the cell. However, such a nucleic acid is "isolated" as the term is used herein because it can be readily engineered by standard techniques known to those skilled in the art.

As used herein, the term “isolated” with respect to a polypeptide is separated from its natural environment in a sufficiently pure form so that it can be engineered or used in the invention. Can be used as any polypeptide of interest. Thus, "isolated" is used to (i) raise and / or isolate an antibody, (ii) as a reagent in an assay, or (iii) for sequencing. Means that it is pure enough to be

In general, polypeptides of the invention having CNREB activity (and nucleic acids encoding them) can be determined in a variety of different ways. For direct measurement, CNREB
The protein or the complex of CNREB protein can be combined with any CNRE sequence (SEQ ID NO: 21, SEQ ID NO: 22 or SEQ ID NO: 22) under conditions that allow binding of CNRE to the putative CNREB protein.
ID NO: 23), preferably SEQ ID NO: 21. In binding experiments, CNREB: CNRE binding can be assayed directly or indirectly, such as in competition experiments using the putative CNRE as a competitor nucleic acid for a known CNREB: CNRE binding pair. Generally, such conditions are known to those skilled in the art of protein-nucleic acid binding and can be accomplished using routine experimentation. Certain preferred binding conditions are described in more detail in the Examples below. CNREB activity can also be measured more indirectly, such as by mRNA levels or by a phenotypic response to the presence of CNREB. One phenotypic measurement, as described in more detail below,
Detecting renin levels in cells (or in fluids that come into contact with cells).

[0054] CNREB proteins that bind to the CNRE motif include CNREB-1 and CNREB-2. In cells, other CNREB proteins are thought to bind to the CNRE motif. As shown below, in cells transfected with an expression vector encoding the CNREB protein, as a result, the expression of genes having the CNRE motif in their 5'-flanking regions is regulated. In some embodiments, CNREB
The proteins bind to the CNRE motif and increase the expression of genes with the CNRE motif in their 5'-flanking regions. CNREB activity also includes CNREB polypeptides that bind to a CNRE motif.

[0055] Homologs and alleles of the CNREB nucleic acids of the invention can be identified by conventional techniques. Accordingly, an embodiment of the present invention are those nucleic acid sequences that encode CNREB polypeptides and hybridize under stringent conditions to nucleic acid molecules consisting of the coding region of the sequences identified herein. As used herein, the term "stringent conditions" refers to parameters that are well known in the art.
Nucleic acid hybridization parameters can be found in references that summarize such methods, eg, Molecular Cloning: A Laboratory Manual, J. Sambrook et al., Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1
989, or edited by Current Protocols in Molecular Biology, FM Ausubel et al., Jo
hn Wiley & Sons, Inc., New York. More specifically, the stringent conditions used herein include, for example, a hybridization buffer (3.5 × SSC, 0.02% Ficoll, 0.02% polyvinylpyrrolidone, 0.
This refers to hybridization at 65 ° C. in 02% bovine serum albumin, 2.5 mM NaH ₂ PO ₄ (pH 7), 0.5% SDS, 2 mM EDTA). SSC is 0.15 M sodium chloride / 0.15
M sodium citrate, pH 7; SDS is sodium dodecyl sulfate; EDTA
Is ethylenediaminetetraacetic acid. After hybridization, the membrane for transferring DNA was washed at 2 × SSC at room temperature, and further washed at 0.1 × SSC / 0.1 × SDS at 65 ° C.
Wash at ° C.

There are other conditions, reagents, etc. that can be used and result in similar stringency. The expert will be familiar with such conditions.
Therefore, they are not described here. However, those skilled in the art will appreciate that the
It will be appreciated that conditions could be tailored in a manner that allows unambiguous identification of homologs and alleles of the REB-2 nucleic acid. Also, one of skill in the art would screen cells and libraries expressing such molecules, and then routinely isolate,
Familiarity with the methodology for subsequently isolating and sequencing related nucleic acid molecules.

In general, homologs and alleles typically have at least 40% nucleotide identity and / or at least 50% amino acid identity to any CNREB-2 isoform (SEQ ID NO: 17 and SEQ ID NO: ID NO: 18), and in some cases, at least 50% nucleotide identity and / or at least 65% amino acid identity (
SEQ ID NO: 19 and SEQ ID NO: 20, respectively), and in other cases,
It will have at least 60% nucleotide identity and / or at least 75% amino acid identity (SEQ ID NO: 7 and SEQ ID NO: 8, respectively). Watson-Crick complement of the foregoing nucleic acids is also encompassed by the present invention. Homology can be obtained from a variety of NCB1 (Be Be
The calculations can be made using publicly available software tools developed by Thesda, Maryland). Exemplary tools include http: //wwww.ncbi.nlm.nih
Includes the BLAST system available at .gov. MacVetor sequence analysis software (
Pairwise and ClustalW alignment (BLO
SUM30 matrix setting) as well as Kyte-Doolittle hydropathic analysis. Watson-Crick complement of the foregoing nucleic acids is also encompassed by the present invention.

For screening for CNREB-related genes, such as homologues and alleles of CNREB-2, Southern blots can be performed using radioactive probes, using the conditions described above.
It can be carried out. After washing the membrane to finally transfer DNA, the membrane is
The radioactive signal can be detected by placing it on a line film or a phosphoimager plate.

The expression of the CNREB-1 and CNREB-2 genes is abundant in certain human tissues and, given the teachings herein of the full-length CNREB cDNA clone, the human cDNA corresponding to the mouse CNREB gene Other mammalian sequences, such as clones, can be isolated from cDNA libraries prepared from one or more tissues rich in CNREB expression using standard colony hybridization techniques.

[0060] The invention also includes degenerate nucleic acids that include those alternative codons that are present in natural substances. For example, serine residues are encoded by codons TCA, AGT, TCC, TCG, TCT and AGC. Each of the six codons is equivalent for the purpose of encoding a serine residue. Thus, any serine-encoding nucleotide triplet can be used directly on a protein synthesizer, in vitro or in vivo, to incorporate a serine residue into the elongating CNREB-2 polypeptide. Similarly, nucleotide sequence triplets encoding other amino acid residues include, but are not limited to: CCA, CCC, CCG and CCT (proline codon); CGA, CGC, CGG, CGT, AGA and AGG (arginine codon) A); ACA, ACC, ACG and ACT (threonine codon); A
AC and AAT (asparagine codon); and ATA, ATC and ATT (isoleucine codon). Other amino acid residues can be encoded by more than one nucleotide sequence as well. Thus, the present invention also includes degenerate nucleic acids that differ in codon sequence from biologically isolated nucleic acids due to the degeneracy of the genetic code.

The present invention also relates to isolated fragments of the unique CNREB-2 isoforms (SEQ ID NO: 17 and SEQ ID NO: 18, respectively; SEQ ID NO: 19 and SEQ ID NO: 18, respectively).
20; SEQ ID NO: 7 and SEQ ID NO: 8, respectively, or their complements. A unique fragment is one fragment that is a "signature" for a larger nucleic acid. For example, a unique fragment is long enough to ensure that its exact sequence is not found in the human genome (and human allele) molecule outside the CNREB-2 nucleic acid as defined above. One skilled in the art would not apply more than routine methods to ascertain whether a fragment is unique within the human genome. However, the unique fragment contains (1) SEQ I
D NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID
NO: 15 or the sequence set forth in SEQ ID NO: 16, (2) the complement of (1), and (3) (1)
And (2), or any other sequence already published as of the filing date of the present application: except for a fragment that is completed from the nucleotide sequence of any sequence selected from the sequence group consisting of:

Fragments that become complete from the sequences set forth in the foregoing sequence group are those that do not contain any nucleotides that are unique to the sequences of the present invention. Thus, a unique fragment must include a nucleotide sequence other than the exact sequence of GenBank and / or a sequence within the foregoing sequence group, or a fragment thereof. The difference may be an addition, deletion or substitution of GenBank, or may be a completely different sequence from the GenBank sequence.

The unique fragments can be used as probes in Southern and Northern blot assays to identify such nucleic acids or can be used in amplification assays such as those used in PCR. As known to those skilled in the art, large probes, such as 200, 250, 300 or more nucleotides, are preferred for certain applications, such as Southern and Northern blots, while smaller fragments are more suitable for PCR. Would be preferred for such applications. Also,
The unique fragment can be used to generate a fusion protein, as shown in the examples, to generate antibodies or to determine the binding of polypeptide fragments, or to make immunoassay components. Similarly, using unique fragments, for example, CNs useful in immunoassays or therapeutic applications
Non-fusion fragments of a REB-2 polypeptide can also be made. In addition, the unique fragments can be used as antisense molecules to inhibit expression of CNREB-2 nucleic acids and polypeptides, particularly for therapeutic purposes as described in more detail below.

As will be appreciated by those skilled in the art, the size of a unique fragment will depend on its conservation in the genetic code. Thus, some regions of the CNREB-2 molecule (nucleic acids and polypeptides, as described herein and in the claims) and their complements require longer segments to be unique But otherwise typically 12-32 nucleotides (e.g.,
12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,
Only short segments (30, 31, and 32 bases in length) will be required. In fact, SEQ ID NO: 7 starting at nucleotide 1 and ending at nucleotide 3,847, or SEQ ID NO: 9 starting at nucleotide 1 and ending at nucleotide 1,288, or SEQ ID NO: starting at nucleotide 1 and ending at nucleotide 2,336 : 17, or any segment of the region of SEQ ID NO: 19, beginning at nucleotide 1 and ending at nucleotide 2,651, or its complement, ie, 12 or more nucleotides in length (SEQ ID NOs: 10, 11,
12, 13, 14, 15, and 16) will be unique.

The claims state that a unique fragment excludes sequences that are identical to a particular nucleic acid, or is identical to only that fragment. The claims do not intend to cover such molecules which are within the prior art. Thus, nucleic acids consisting solely of these sequences, or consisting solely of fragments of these sequences,
It is believed to be within the prior art. However, nucleic acids comprising any portion of the novel sequences of the present invention are intended to be encompassed by the claims, including sequences comprising the novel sequences and adjacent portions of the prior art sequences. Such sequences have unexpected properties, as described herein. In one embodiment, the unique fragment does not include any portion of the excluded prior art sequence. Those of skill in the art are familiar with how to select unique sequences, typically based on the ability of the unique fragment to selectively discriminate sequences of interest from non-CNREB-2 nucleic acids. Comparison of the sequence of the fragment with those on known databases is typically all that is required, but in vitro definitive hybridization and sequencing analyzes can also be performed.

As noted above, the invention encompasses antisense oligonucleotides that selectively bind to a nucleic acid encoding a CNREB polypeptide to reduce CNREB activity, particularly CNREB-1 and CNREB activity. This is virtually desirable for any medical condition where it is desirable to reduce CNREB activity, including certain renin-angiotensin mediated disorders. For example, the use of antisense CNREB molecules can slow or reduce the growth of cancer cells that express c-myc in vivo. When using antisense preparations of the present invention, slow intravenous administration is preferred.

As used herein, the term “antisense oligonucleotide” or “antisense” refers to a DNA that contains a particular gene or a DNA that contains an mRNA transcript of that gene that hybridizes under physiological conditions. Refers to an oligonucleotide that is an oligoribonucleotide, oligodeoxyribonucleotide, modified oligoribonucleotide, or modified oligodeoxyribonucleotide; and CNREBy
Inhibits the transcript of the gene and / or the translation of the mRNA. Antisense molecules, upon hybridization with the target gene or its transcript,
It is designed to inhibit the transcription or translation of the target gene. One of skill in the art will appreciate that the exact length of the antisense oligonucleotide and its degree of complementarity with its target will depend on the specific target selected, including the sequence of the target and the particular base containing the sequence. You will recognize that The antisense oligonucleotide selectively binds to the target under physiological conditions, i.e., so that it hybridizes substantially more with the target sequence under physiological conditions than any other sequence in the target cell. It is desirable to build and arrange. Any CNREB cDNA disclosed herein
Based on the sequence, or based on the allele or homologous genomic and / or cDNA sequence, one of skill in the art can readily select and synthesize any of a number of suitable antisense molecules for use in accordance with the present invention. . To be sufficiently selective and effective in inhibiting, such antisense oligonucleotides must contain at least 10, more preferably at least 15 contiguous bases that are complementary to the target, but in some cases In some cases, modified oligonucleotides as short as 7 bases in length have been used successfully as antisense oligonucleotides (Wagner
Nat Med., 1 (11), 1116-1118, 1995). Most preferably, the antisense oligonucleotide comprises a complementary sequence of 20-30 bases. Although oligonucleotides that are antisense to any region of the gene or mRNA transcript can be selected, in a preferred embodiment, the antisense oligonucleotide is an N-nucleotide such as a translation initiation, transcription initiation or promoter site. Matches with terminal or 5 'upstream site. In addition, the 3'-untranslated region can be targeted by an antisense oligonucleotide. Targeting to mRNA splicing sites is also used in the art, but is less preferred if alternative mRNA splicing occurs. Furthermore, it is not possible to predict antisense, preferably mRNA secondary structure (
See, e.g., Sainio et al., Cell Mol. Neurobiol., 14 (5), 439-457, 1994), and sites where proteins are not expected to bind.
Finally, although the present invention discloses the CNREB cDNA sequence, those skilled in the art can readily derive genomic DNA corresponding to this sequence, both CNREB-1 and CNREB-2. Therefore, the present invention also relates to SEQ ID NO: 1, SEQ ID NO: 17, SEQ ID NO: 1
Antisense oligonucleotides that are complementary to genomic DNA corresponding to SEQ ID NO: 9 or SEQ ID NO: 7 are provided. Similarly, alleles or homologous CNREB-1 and CNREB-2 cDN
Antisense to A as well as genomic DNAs can be obtained without undue experimentation.

[0068] In one set of embodiments, the antisense oligonucleotides of the invention can consist of "natural" deoxyribonucleotides, ribonucleotides, or any combination thereof. That is, the 5 'end of one natural nucleotide and the 3' end of another natural nucleotide can be covalently linked within the natural system through a phosphodiester intranucleoside linkage. These oligonucleotides can be prepared by art-recognized methods that can be performed manually, or by automated synthesizers. They can also be made by recombination with a vector.

[0069] In a preferred embodiment, however, the antisense oligonucleotides of the present invention can also include "modified" oligonucleotides. That is, oligonucleotides are modified in a number of ways that do not inhibit them from hybridizing to their target, but enhance their stability or targeting, or otherwise enhance their therapeutic effect. can do.

As used herein, the term “modified oligonucleotide” refers to (1
And / or) at least two of the nucleotides are covalently linked through a synthetic intranucleoside linkage (ie, a linkage other than a phosphodiester linkage between the 5 'end of one nucleotide and the 3' end of another nucleotide) and / or (2) Usually refers to an oligonucleotide in which a chemical group not associated with a nucleic acid is covalently bonded to the oligonucleotide. Preferred synthetic intranucleoside linkages are phosphorothioates,
Alkyl phosphonates, phosphorodithioates, phosphates, alkylphoronothioates, phosphoramidates, carbamates, carbonates, phosphate triesters, acetamidodates, carboxymethyl esters and peptides.

The term “modified (modified) oligonucleotide” also encompasses oligonucleotides having covalently modified bases and / or sugars. For example, modified oligonucleotides include oligonucleotides having a sugar skeleton covalently linked to a low molecular weight organic group that is different from the 3 'hydroxyl group and different from the 5' phosphate group. Thus, the modified oligonucleotide can include a 2'-O-alkylated ribose group. Further, the modified oligonucleotide can include a sugar such as arabinose instead of ribose. Thus, according to the present invention, a pharmaceutical preparation comprising a modified antisense molecule that is complementary to and capable of hybridizing under physiological conditions with a nucleic acid encoding a CNREB (1 and 2) polypeptide is pharmaceutically acceptable. Planned, with possible carriers. Antisense oligonucleotides can also be administered as part of a pharmaceutical composition. As such, the pharmaceutical composition comprises:
Antisense oligonucleotides can be included in combination with any standard physiologically and / or pharmaceutically acceptable carriers known in the art. The composition must be sterile and must contain a therapeutically effective amount of the antisense oligonucleotide in a unit or weight suitable for administration to a patient. The term "pharmaceutically acceptable" means a non-toxic substance that does not interfere with the effect of the biological activity of the active ingredient. The term “physiologically acceptable” refers to a non-toxic substance that is compatible with a biological system, such as a cell, cell culture, tissue, or organism. The characteristics of the carrier will depend on the route of administration. Physiologically and pharmaceutically acceptable carriers include diluents, fillers, salts, buffers, stabilizers, solubilizers and other materials well known in the art.

The present invention also relates to CNREB proteins and fragments and variants thereof (varia
nt), as well as host cells containing those expression vectors. Virtually any cell, prokaryotic or eukaryotic that can be transformed with heterologous DNA or RNA and that can be grown or maintained in culture for use in the practice of the invention be able to. Examples are described in E.I. bacterial cells such as E. coli and mammalian cells such as mice, hamsters, pigs, goats, primates and the like. They can be of a wide variety of tissues, including mast cells, fibroblasts, oocytes and lymphocytes, and can be primary cells or cell lines. Particular examples include CHO cells and COS cells. Cell-free transcription systems can also be used instead of cells.

As used herein, “vector” refers to any of a number of nucleic acids into which a desired sequence can be inserted by restriction and ligation, for transport between different genetic environments, or for expression in a host cell. Can be. RNA vectors are also available, but the vector is typically DNA. Vectors include, but are not limited to, plasmids, phagemids, and viral genomes. A cloning vector is capable of replicating in a host cell, and
The vector is cut in a determinable manner and the desired DNA sequence is ligated so that the resulting novel recombinant vector retains its ability to replicate in its host cell.
It is further characterized by the above endonuclease restriction sites. In the case of plasmids, replication of the desired sequence may occur many times, such as increasing the copy number of the plasmid in the host bacterium, or may occur only once, before the host multiplies by mitosis. In the case of phage, replication can occur actively during the lysis phase and can be immobile during the lysogenization phase. An expression vector is one into which a desired DNA sequence can be inserted by restriction enzyme treatment and ligation, so that it can be operably connected to control sequences and expressed as an RNA transcript. The vector may further comprise one or more marker sequences suitable for use in identifying cells that have been or have not been transformed or transfected by the vector. Markers include, for example, genes encoding proteins that increase or decrease either resistance or sensitivity to antibiotics or other compounds, enzymes whose activity can be detected by standard assays known in the art (eg, Genes encoding [beta] -galactosidase or alkaline phosphatase) and visually affect the phenotype of transformed or transfected cells, hosts, colonies, plaques (e.g., green fluorescent protein)
) Including genes. Preferred vectors are those that are capable of autonomous replication and are capable of expressing the structural gene products present in the DNA segments to which they are operatively connected.

As used herein, coding and control sequences are “operable” when they are covalently linked such that expression or transcription of the coding sequence occurs under the influence or control of the control sequences. It is said to be "connected."
If it is desired that the coding sequence be translated into a functional protein, 5
'The introduction of a promoter in the control sequence causes transcription of the coding sequence,
And the nature of the linkage between the two DNA sequences is (1) the consequence of inducing frameshift mutation; (2) interference by the activity of the promoter region to direct transcription of the coding sequence; or (3) translation into protein. Two DNA sequences are said to be operably linked when they are not interfering with the ability of the corresponding RNA transcript to be performed. Therefore, a promoter region is operably connected to a coding sequence if the promoter region allows for the efficient transcription of that DNA sequence so that the resulting transcription can be translated into the desired protein or polypeptide. It is thought that it is.

The exact nature of the regulatory sequences required for gene expression may vary between species or cell types, but generally, as necessary, in turn dictates the initiation of transcription and translation. It includes related 5 'non-transcribed and 5' non-translated sequences, such as TATA box, capping sequence, CAAT sequence and the like. The control sequence may also desirably include an enhancer sequence or an upstream activator sequence. The vectors of the present invention may optionally include a 5 'leader or signal sequence. The selection and design of an appropriate vector is within the skill and best of skill in the art.

[0076] Expression vectors containing all necessary elements for expression are commercially available and known to those of skill in the art. Sambrook et al., Molecular Cloning: A Labor
See atory Manual, 2nd edition, published by Cold Spring Harbor Laboratory, 1989. Cells are genetically processed by introduction into heterologous DNA (RNA cells) encoding a CNREB-2 polypeptide or fragment or a variant thereof.

A system for mRNA expression in mammalian cells comprises a pRc / CMV (Invitrogen, Inc.), which includes a selectable marker such as a gene that confers G418 resistance and a human cytomegalovirus (CMV) enhancer-promoter sequence. Carlsbad, available from CA). In addition, preferred for expression in primate or canine cell lines is the pCEP4 vector (Invitrogen, Carlsbad, CA), which facilitates the maintenance of the plasmid as a multicopy extrachromosomal element, the Epstein-Barr virus (EBV ) Including the replication origin. Another expression vector is the pEF-BOS plasmid, which contains the promoter of the polypeptide elongation factor 1α, which effectively stimulates transcription in vitro. Mishizuma and Na
Plasmids designed by gata (Nuc. Acids Res. 18: 5322, 1990) and their use in transfection experiments are disclosed, for example, by Demoulin (Mol. Cell. Biol. 16: 4710-4716, 1996). ). Another preferred expression vector is the adenovirus designed by Stratford-Perricaudet, which has drawbacks for the E1 and E3 proteins (J. Clin. Invest. 90: 6
26-630, 1992). The use of adenovirus as an Adeno.P1A recombinant in mice subcutaneously for anti-P1A immunization has been disclosed by Warnier et al. (Int. J. Cancer 67: 303-310, 1996).

The present invention also includes so-called expression kits, which allow a technician to prepare the desired expression vector (s). Such an expression kit comprises at least a separate part of each of the coding sequences described above. If desired, other components may be added in the arrangements already mentioned.

The present invention is directed to the use of host cells and cell lines (either prokaryotic (eg, E. coli) or eukaryotic (eg, CHO cells, COS cells, yeast expression systems, and recombinant baculovirus expression in insect cells). It will also be appreciated that this includes the use of the CNREB cDNA sequence containing the expression vector described above to transfect). Particularly useful are mammalian cells such as mice, hamsters, pigs, goats, primates, and the like. Specific examples include dendritic cells, U293 cells, peripheral blood lymphocytes, bone marrow stem cells,
Contains embryonic stem cells. The present invention also provides for the construction of CNREB (particularly novel CNREB-2) gene "knockouts" in cells and animals that supply material for the study of certain embodiments of CNREB activity (including CNREB-2 activity). enable.

The present invention also provides an isolated polypeptide, which is provided in SEQ ID NO: 18
, SEQ ID NO: 20 and / or SEQ ID NO: 8, and unique fragments thereof. Such polypeptides may be used, for example, for stimulation of cell growth, effects on renin expression, detection of genes flanked by CNRE, alone or as fusion proteins to obtain antibodies, components of immunoassays, etc. Useful as

A unique fragment of a CNREB polypeptide generally has the composition and characteristics of a unique fragment as discussed above in relation to nucleic acids. As will be appreciated by those skilled in the art, the size of a unique fragment will depend on factors such as whether the fragment constitutes part of a conserved protein domain. Therefore, some regions of SEQ ID NOs: 18, 20, and / or 8 are unique, while others are thought to require only short segments, typically between 5 and 12 amino acids. It is believed that some require longer segments (e.g., including, in order, each integer up to 434, 539 and / or 763 amino acids in length, and 5, 6, 7, 8, 9, 10, 11, and 12 amino acids in length). Or greater). In fact, any segment of SEQ ID NO: 18, 20, and / or 8 that is 9 or more amino acids in length is encoded by the nucleic acid of SEQ ID NO: 10, 11, 12, 13, 14, 15, and 16 Except for those having the same identity as the polypeptide). As will be appreciated by those skilled in the art, the size of a unique fragment will depend on factors such as whether the fragment constitutes part of a conserved protein domain. One of skill in the art is familiar with methods for the selection of unique amino acid sequences, typically based on the ability of unique fragments, and can distinguish key sequences from non-family members in a selectable manner. Typically, all that requires a comparison of the sequence of the fragment to a sequence on a known database.

[0082] A unique fragment of a polypeptide is preferably such a fragment that retains the distinct functional capabilities of the polypeptide. The functional activity that can be maintained in a unique fragment of a polypeptide is the interaction with an antibody, the interaction with other polypeptides or their fragments, and in one of the important embodiments of the invention, CNRE Includes binding to the motif. In general, the CNRE binding properties of a putative CNREB polypeptide fragment can be determined by experiments in which the CNRE is contacted with the putative CNREB binding fragment under conditions that allow binding of the CNRE to the CNREB binding fragment. Binding experiments were performed using the known CNRE-CNREB
Binding analysis can be performed directly or indirectly by a competition experiment using a putative CNREB binding fragment as a competitor polypeptide for the binding pair. These conditions are generally known to those skilled in the art of protein-nucleic acid binding and can be performed as routine experiments. As mentioned above, such binding (due to interference by binding, such as in competitive binding experiments or tests for predominant negative polypeptides), seeks for changes in phenotype, such as expression of renin, etc. And can be analyzed indirectly.

[0083] The present invention includes variants of the CNREB polypeptides described above. C as used herein
A “variant” of a NREB polypeptide is a variant of the amino acid sequence of the original CNREB polypeptide.
A polypeptide containing the above modification. Modifications of the CNREB polypeptide are typically made to the nucleic acid encoding the CNREB polypeptide, and may include deletions, point mutations, truncations, amino acid substitutions, and the addition of amino acids or non-amino acid portions. Alternatively, modifications can be made directly to the polypeptide, for example, by elimination or addition of a linker molecule, addition of a detectable moiety such as biotin, addition of a fatty acid, and the like. Modifications also include fusion proteins made up of part or all of the CNREB amino acid sequence.

Variants may include CNREB polypeptides that have been specifically modified to render the characteristics of the polypeptide independent of its physiological activity. For example, cysteine residues can be substituted or deleted to undesired disulfide bonds. Similarly, by altering certain amino acids to eliminate proteolysis by proteases in the expression system, expression of CNREB polypeptide can be enhanced (eg, in yeast expression systems in which KEX2 protease activity is present). 2 base amino acid residues).

In important embodiments, mutations in the nucleic acid encoding the CNREB polypeptide preserve the amino acid reading frame of the coding sequence and preferably hybridize to the hairpin such that it can be detrimental to the expression of the mutant polypeptide. Do not create regions in nucleic acids that are likely to form secondary structures such as loops and loops. They also preserve, enhance or eliminate the binding of CNREB polypeptide to CNRE, depending on the desired use.

[0086] Mutations can be made by amino acid substitutions or by random mutagenesis of a selected site in a nucleic acid encoding the polypeptide. The variant polypeptide is then expressed and tested for one or more activities to determine that the mutation results in a variant polypeptide having the desired properties.
Additional mutations can be made to create variants (or non-mutant CNREB polypeptides) that are silent for the amino acid sequence of the polypeptide, but provide preferred codons for translation in a particular host. Preferred codons for translation of the nucleic acid, for example in E. coli, are well known to those skilled in the art. Still other mutations can be made to create cDNA clones that enhance expression of the non-coding sequence of the CNREB gene or the polypeptide.

One skilled in the art can make conservative amino acid substitutions in CNREB polypeptides,
It will be appreciated that functionally equivalent variants of the above-described polypeptides can be provided, i.e., variants that retain the functional capacity of CNREB-1 and / or CNREB-2 polypeptides. As used herein, "conservative amino acid substitution" refers to an amino acid substitution that does not alter the specific charge or size characteristics of the protein in which the amino acid substitution occurred. Methods for altering polypeptide sequences known to those of skill in the art, such as references accumulating such methods, e.g., Molecular Cloning: A
Laboratory Manual (J. Sambrook, et al., Eds., Second Edition, Cold Spri
ng Harbor Laboratory Press, Cold Spring Harbor, New York, 1989) or Cu
rrent Protocols in Molecular Biology (FM Ausubel, et al., eds., John W
Mutants can be prepared according to methods such as those found in iley & Sons, Inc., New York). Exemplary CNREB-2 polypeptide functional equivalent variants include conservative amino acid substitutions of SEQ ID NO: 18. Conservative amino acid substitutions include the following groups: (a) M, I, L, V; (b) F, Y, W; (e) K, R, H; (d) A, G; (e) S ,
T; (f) Q, N; and (g) E, D;

Thus, a variant of a CNREB polypeptide that is functionally equivalent (eg, a native CNREB
CNREB variants that retain the function of the polypeptide) are contemplated by the present invention. Substitution of conservative or non-conservative amino acids in the amino acid sequence of a CNREB polypeptide to create a functionally active variant of the CNREB polypeptide is typically accomplished by CN
This is accomplished by altering the nucleic acid encoding the REB polypeptide. Such substitutions can be made in various ways known to those skilled in the art. For example, amino acid substitutions can be
R-specific mutagenesis and site-directed mutagenesis (by the method of Kunkel et al .; Kunkel,
Proc. Na Acad Sci. USA 82: 488-492, 1985), or by chemical synthesis of a gene encoding a CNREB polypeptide. The activity of a functionally active fragment of a CNREB polypeptide can be tested as follows: the nucleic acid encoding the modified CNREB polypeptide is cloned into a bacterial or mammalian expression vector and the vector is then transformed into a suitable host cell. To express the modified CNREB polypeptide and test the functional ability of the CNREB polypeptide as disclosed herein.

As used herein, a functional variant of a nucleic acid means a nucleic acid that encodes a functional variant of a CNREB polypeptide. As disclosed herein, the invention has a number of uses, some of which are described elsewhere herein. The present invention allows for the isolation of CNREB polypeptides. Various methodologies known to those skilled in the art can be used to obtain an isolated CNREB molecule. The polypeptide may be purified from cells that naturally produce the polypeptide by chromatography or immunological recognition. Alternatively, the expression vector may be introduced into cells to produce the polypeptide. According to another method, mRNA transcripts are microinjected or otherwise introduced into cells to produce the encoded polypeptide. Translation of CNREB mRNA in a cell-free extract, eg, a reticulocyte lysate system, may be utilized for polypeptide production. One skilled in the art can readily perform known isolation procedures on CNREB polypeptides. Such isolation methods include immunochromatography, HP
Examples include, but are not limited to, LC, size exclusion chromatography, ion exchange chromatography, immunoaffinity chromatography, and the like. The same applies to fragments or mutants.

The present invention also includes isolated peptides and non-peptide binders (which may be, for example, antibodies or fragments capable of specifically binding to CNREB polypeptide). Examples of the antibody include a polyclonal or monoclonal antibody prepared according to a conventional method.

Importantly, it is well known to those skilled in the art that only a small part of the antibody (paratope) is involved in binding the antibody to its epitope (Clark,
WR (1986) The Experimental Foundations of Modem Immuno Pay Wiley & Son
s, Inc., New York; Roitt, 1. (1991) Essential Immunology, 7th Ed., Black
well Scientific: See Publications, Oxford, etc.). pFc 'and Fc regions
For example, it is an effector of the complement cascade, but is not involved in antigen binding. Antibodies whose pFc 'region is enzymatically cleaved or created without the pFc' region are called F (ab ') ₂ fragments, but retain both antigen binding sites with the intact antibody. Similarly, an antibody whose Fc region has been enzymatically cleaved or created without an Fc region is called a Fab fragment, but retains any of the antigen binding sites for an intact antibody molecule. Furthermore, Fab fragments consist of a covalently linked antibody light chain and a portion of an antibody heavy chain called Fd. The Fd fragment is
It is a major determinant of antibody specificity (one Fd fragment can bind up to 10 different types of light chains without altering antibody specificity), and Fd fragments can be isolated Retains the ability to bind to the epitope.

As is well known to those skilled in the art, within the antigen binding site of an antibody, complementarity determining regions (CDRs) that directly interact with the epitope of the antigen, and a framework that maintains the three-dimensional structure of the paratope Regions (FRs) exist (see Clark, 1986; Roitt, 1991)
. In both the heavy chain Fd fragment and the light chain of IgG, four framework regions (from FRI to CDR3) separated by three complementarity determining regions (CDR1 to CDR3), respectively.
FR4) exists. CDRs (especially CDR3 regions, more particularly heavy chain CDR3s) are deeply involved in antibody specificity.

[0093] It is well established to those skilled in the art that the non-CDR region of a mammalian antibody can be modified by a similar or heterospecific antibody while retaining the epitope specificity of the original antibody. It may be replaced by a region. This is a "humanization" in which non-human CDRs are covalently linked to human FRs and / or Fc / pFc 'regions to create functional antibodies.
It is most apparent in the development or use of antibodies. For example, PCT International Publication WO92 / 04
No. 381 discloses the production and use of a humanized mouse RSV antibody in which at least a part of the mouse FR region has been replaced with a human-derived FR region. Such antibodies (including those of intact antibodies that have the ability to bind antigen) are often referred to as "chimeric" antibodies.

Thus, as will be apparent to those skilled in the art, the present invention provides F (ab ′) ₂ , Fab, Fv and
Fd fragments; chimeric antibodies in which the Fe and / or FR and / or CDRI and / or CDR2 and / or light chain CDR3 regions have been replaced by homologous human or non-human sequences; FR and / or CDR1 and / or CDR2 and / or A chimeric F (ab ') ₂ fragment antibody in which the light chain CDR3 region has been replaced by homologous human or non-human sequences; FR and / or
A chimeric Fab fragment antibody in which the CDR1 and / or CDR2 and / or L chain CDR3 regions are replaced with homologous human or non-human sequences; and FR and / or CDR
It is intended to provide a chimeric Fd fragment antibody in which the I and / or CDR2 region has been replaced with a homologous human or non-human sequence. The present invention also includes so-called single-chain antibodies.

Thus, the present invention provides a method for specifically binding to a CNREB polypeptide,
It includes polypeptides of various sizes and types that interact with the binding of the REB polypeptide or its CNRE binding partner. Such polypeptides may be obtained from sources other than antibody technology. For example, such polypeptide binders may be supplied in solution in fixed form by a degenerate peptide library that can be readily prepared as a bacterial flagellar peptide display library or a phage display library. Combinatorial libraries can also be synthesized from peptides containing one or more amino acids. Libraries can also be synthesized from peptide and non-peptide synthetic components.

[0096] Phage display is particularly useful for identifying binding peptides useful according to the invention. Briefly, a phage library is prepared (eg, m13, fd,
Alternatively, using lambda phage), inserts of 4 to about 80 amino acid residues are displayed by conventional techniques. Such an insertion may, for example, represent a fully degenerate or bias array. The phage can then be selected for having a CNREB-2 polypeptide or an insert that is a complex of CNREB-2 with a binding partner. In this method, reselection of phage that binds to the CNREB-2 polypeptide or complex can be repeated for several cycles. By repeating, phage having a specific sequence can be concentrated. DNA sequence analysis can identify the sequence of the expressed polypeptide. The minimal linear portion of the sequence that binds to the CNREB-2 polypeptide or complex can be determined. This method can be repeated using a bias library that includes an insert that includes some or all of the minimal linear portion plus one or more additional degenerate residues upstream or downstream thereof. Yeast two hybrid screening methods may be used to identify polypeptides that bind to CNREB-2 polypeptide. Therefore, the CNREB-2 polypeptide of the present invention, a fragment thereof, or a complex of CNREB-2 and a binding partner can be converted to a peptide library such as a phage display library, a chemical substance, a peptide and a naturally occurring substance such as a carbohydrate or lipid. Peptide binding partners of the CNREB-2 polypeptides of the invention are identified and selected for use in screening combinatorial libraries of compounds present and for use in computer model programs. It will be apparent to those skilled in the art that such molecules can be used for screening assays, purification protocols, interacting directly with CNREB-2 function, and for other purposes.

[0097] The CNREB polypeptide or a fragment thereof can also be used to isolate its native binding partner. Isolation of the binding partner can be performed according to well-known methods. For example, after attaching the isolated CNREB-2 polypeptide to a substrate, the CNREB-2
A solution suspected of containing the -2 binding partner may be applied to the substrate. If a CNREB-2 polypeptide binding partner is present in the solution, it will bind to the substrate to which the CNREB-2 polypeptide is bound. Then, the binding partner will be isolated. Other proteins that are binding partners of CNREB-2 can be isolated in a similar manner without undue experimentation.

According to one aspect of the present invention there is provided a "dominant negative" polypeptide derived from CNREB-1 and CNREB-2 polypeptides. A dominant-negative polypeptide is an inactive variant of a protein that interacts with a cellular mechanism to displace the active protein from interacting with the cellular mechanism or to compete with the active protein, thereby producing an active protein. It reduces the effects of proteins. For example, a dominant-negative receptor that binds to a ligand but does not transmit a signal through its binding reaction can reduce the onset of the biological effect of the ligand. Similarly, a dominant negative catalytically inactive kinase that normally interacts with a target protein but does not phosphorylate the target protein may reduce phosphorylation of the target protein on cellular signals. Similarly, dominant-negative transcription factors that bind to the promoter region of the gene regulatory region but do not increase gene transcription may reduce the effects of normal transcription factors by occupying the promoter binding site without increasing transcription. it can.

The end result of the expression of a dominant negative polypeptide in a cell is a diminished function of the active protein. One of skill in the art can assess the potential of a dominant negative variant of a protein and create one or more dominant negative variant polypeptides using standard mutagenesis techniques. For example,
US Patent No. 5,580,723 and Sambrook et al., Molecular Cloning: A Lab
oratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, 198
See 9 One of skill in the art can then test the mutated polypeptides for a diminished activity and / or retention of such activity. Other similar methods for creating and testing dominant negative variants of a protein will be apparent to those skilled in the art.

The isolation of CNREB-2 cDNA and the newly identified use of CNREB-1 cDNA also allows one of ordinary skill in the art to diagnose disorders characterized by aberrant expression of CNREB. These methods include determining the expression of the CNREB gene and / or CNREB polypeptide derived therefrom. In the case of genes, such a determination can be made by any standard nucleic acid determination method, including the replication chain reaction, or analysis with labeled hybridization probes, as exemplified below. In the case of polypeptides, such a determination can be made, for example, using an antibody that binds a secreted CNREB protein.
It can be performed by any standard immunological assay.

As used herein, a subject is a human, non-human primate, cow, horse, pig, sheep, goat, dog, cat or rodent. In all embodiments, human CNREB molecules and human subjects are preferred.

The present invention also encompasses determining the likelihood of a subject suffering from a renin-angiothecin system mediated disorder. Such obstacles are known to those skilled in the art. The renin-angiotensin system is involved in the control of hemodynamics, as well as in the balance of water and electrolytes. Factors that reduce blood volume, renal perfusion pressure, or Na ⁺ concentration in plasma tend to activate the system, while factors that increase these variables tend to suppress the function of the system. is there. In determining the likelihood of each individual for a renin-angiothecin system-mediated disorder, the level of CNREB expression in each individual is obtained. The level of CNREB expression in each individual can be obtained by a number of art-recognized methods. If CNREB activity is abnormal, the disorder is present, or there is a high (or low) likelihood of becoming a disorder.

Typically, CNREB expression levels can be determined by first obtaining a test sample from a subject. The test sample can be a tissue or a biological fluid. Tissues include kidney and skin, and biological fluids include blood and urine. Both invasive and non-invasive techniques can be used to obtain such samples, and these techniques are well described in the art. At the molecular level, both PCR and Northern blots are used to determine the level of CNREB mRNA by art-recognized protocols found in the products of the invention described above, as well as references that compile such methods. Can be used. At the protein level, CNREB expression can be determined by using polyclonal or monoclonal anti-CNREB sera in combination with standard immunological assays. A preferred method will compare the measured level of CNREB expression in the test sample with a control. The controls are
It can include a known amount of a nucleic acid probe, a CNREB epitope (eg, a CNREB expression product), or a similar test sample of a subject with control or “normal” levels of CNREB expression. The control may be of any type recognized in the art, for example, a reagent to be tested in parallel with the test liquid, or a predetermined value such as color or number. The control population preferably includes only individuals of the same age group as the individual being tested (eg, within 5-10 years). The predetermined value also refers to a range such as the following, e.g., to equally divide the population used to obtain CNREB expression levels into, for example, a low risk group and a high risk group to be used as a control. (Or an unequal split) or a quadrant (the lowest quarter is the lowest risk population and the highest quarter is the highest risk population). You may. Thus, the predetermined value may depend on the particular population selected. For example, an apparently healthy population (having no detectable disease and no history of disease) is distinguished from a population of members who have previously had a renin-angiotensin system disorder by a CNREB activity level. It may have a different "normal" range. Thus, the predetermined value selected is
The category to which the individual belongs may be considered. Appropriate ranges and categories can be selected without exceeding the routine experimentation of a person skilled in the art.

In one aspect, a method for determining the likelihood of a subject afflicted with a renin-angiothecin system mediated disorder comprises: (a) CNRE in a test sample
Characterizing the B-1 nucleic acid sequence, wherein the test sample is obtained from a subject; (b
) Comparing the CNREB-1 nucleic acid sequence of the test sample with the CNREB-1 nucleic acid sequence of the control sample, wherein the CNREB-1 nucleic acid sequence in the test sample is compared to the CNREB-1 nucleic acid sequence of the control sample The change or agreement made is an indication of the subject's susceptibility to a renin-angiothecin system-mediated disorder. In some embodiments, the changes observed are apparent when the CNREB-1 nucleic acid sequence in the test sample is compared to the wild-type CNREB-1 nucleic acid sequence in a control sample. In other embodiments, the observed match is evident when the CNREB-1 nucleic acid sequence in the test sample is compared to the mutant CNREB-1 nucleic acid sequence in a control sample. In a further embodiment, CNREB-1 mRNA molecules are compared. In yet another embodiment, the change in CNREB-1 mRNA is the mRNA of the test sample, SEQ ID NO:
1, nucleic acid of SEQ ID NO: 3, nucleic acid of SEQ ID NO: 5, and nucleic acid of SEQ ID NO: 6. In certain embodiments, the CNREB-1 cDNA sequences are compared, and the comparison is made by hybridization of a CNREB-1 cDNA probe to genomic DNA isolated from a test sample. In some embodiments, the method further comprises: (a) subjecting the genomic DNA isolated from the test sample of interest to Southern hybridization with a CNREB-1 cDNA probe; and (b) (i) the CNREB-1 cDNA Comparing the hybridization of the probe to the test sample of interest, and (ii) the hybridization of the CNREB-1 cDNA probe to the control sample. In any of the foregoing embodiments, the CNREB-1 cDNA probe detects restriction fragment length polymorphisms. In yet another embodiment, the CNREB-1 nucleic acids are compared, and the comparison is performed by using a replication chain reaction to sequence at least a portion of the CNREB-1 cDNA in the test sample. Here, in the determined CNREB-1 cDNA, the wild type shown in SEQ ID NO: 1
Deviation of the CNREB-1 nucleic acid sequence from the sequence is an indicator of the subject's potential morbidity for a renin-angiothecin system-mediated disorder. Renin-angiothecin system-mediated disorders include those described above.

In another embodiment, the alteration of the CNREB-1 nucleic acid sequence comprises the steps of: (a) a CNREB-1 cDNA or CNREB-1 mRNA isolated from a test sample; and (b) a human wild-type CNREB-1 nucleic acid. Detected by identifying a mismatch between the nucleic acid probe, complementary to the sequence, wherein the molecules (a) and (b) hybridize to each other to form a duplex. In some embodiments, the CNREB-1 nucleic acid sequences are compared and the CNREB-1
The alteration of the nucleic acid sequence can be accomplished by the following steps: (a) amplify the CNREB-1 cDNA sequence in the test sample, and (b) hybridize the amplified CNREB-1 cDNA sequence to a nucleic acid probe containing the CNREB-1 sequence. Let the soy be detected.

In yet another embodiment, the CNREB-1 nucleic acid sequences are compared, wherein the CNREB-1 nucleic acid sequence alteration comprises molecular cloning of the CNREB-1 gene in a test sample, and the cloned CNREB-1 gene Is determined by sequencing all or part of

[0107] In any of the above embodiments, detecting a change in the CNREB-1 nucleic acid sequence comprises a deletion mutation, a site mutation, and / or an insertion mutation. The test sample may be a tissue, a tissue biopsy, and / or a biological fluid. In a preferred embodiment, the test sample is brain, heart, breast, colon, bladder, uterus, prostate, stomach, testis, ovary, pancreas, pituitary, adrenal gland, thyroid,
It may be obtained from tissues of the subject including salivary glands, mammary gland, kidney, liver, intestine, spleen, thymus, bone marrow, trachea and lung. In a further preferred embodiment, the test sample comprises amniotic fluid, aqueous humor, bile,
Blood, bronchoalveolar lavage fluid, bronchial fluid, cerebrospinal fluid, follicular fluid, intergingival fluid, middle ear fluid, peritoneal fluid, pleural fluid, prostate fluid, saliva, semen, serum, sweat, synovial fluid fluid), tears, culture supernatants and urine.

The “change” observed is that the CNREB-1 nucleic acid sequence in the test sample is compared to a control sequence in a control sample, where the control sequence is a wild-type CNREB-1 nucleic acid of the invention. (Eg, SEQ ID NO: 1 or a human homolog), and that the test sample CNREB-1 sequence differs from the control sample CNREB-1 sequence. Alternatively, CNREB in the test sample
-1 nucleic acid sequence is compared to a control sequence in a control sample, wherein the control sequence is a mutated CNREB-1 nucleic acid and that the test sample CNREB-1 sequence is identical to the control sample CNREB-1 sequence May be found, the observed "match" has been detected. Appropriate differences and / or matches in the sequence can be determined without departing from the routine experimentation of those skilled in the art.

The present invention also provides novel kits that can be used to measure levels of a nucleic acid of the invention, an expression product of the invention, or an anti-CNREB antibody. For nucleic acid detection, CN
A primer pair for amplifying a REB nucleic acid may be included. A preferred kit can include a known amount of a nucleic acid probe, a CNREB epitope (such as a CNREB expression product), or a control, such as an anti-CNREB antibody, and instructions or other printed material. The reagent may be packaged in a container and / or coated on the wells in a predetermined amount, and the kit may comprise a labeled immunological reagent (eg, a labeled anti-IgG antibody). ) May be included. One kit is CNREB
Protein-coated, packaged polystyrene microtiter plates, as well as containers containing labeled anti-human IgG antibodies. The wells of the plate are contacted, for example, with serum, washed, and contacted with an anti-IgG antibody. Then, the label is detected.

The present invention further includes a method for treating a control with a renin-angiotensin system mediated condition. Such conditions include cardiovascular disorders associated with atherosclerotic diseases. "Cardiovascular disorders associated with atherosclerotic disease" include hypertension, myocardial infarction, stroke, angina and peripheral arterial vascular disease.

[0111] Hypertension or high blood pressure refers to a condition in which arterial blood pressure is continuously rising above a specified level and is associated with an increased risk of experiencing a major cardiovascular failure. Is believed to be. Hypertension is treated by reducing arterial pressure to levels below those designated as hypertension.

[0112] Congestive heart failure is associated with defective heart pumps. This can be due to many different situations. Heart failure exists when the heart does not pump in proportion to the demands of metabolizing tissue, or only from elevated filling pressures.

Myocardial infarction involves interruption of blood flow to the heart tissue, with consequent death of part of the heart muscle. The treatment of myocardial infarction may be for patients who have already experienced myocardial infarction or may be prophylactic treatment. In the prophylactic case, treatment is for subjects whose risk of myocardial infarction is abnormally high. The criteria for such individuals are well known to those skilled in the art, and are described in Harrison's Principles of Experimental Medicine, 13th E.
See dition, McGraw-Hill, Inc.

Ischemic stroke (ischemic cerebral infarction) is an acute neurological disorder caused by impaired blood flow, including blood vessels in the brain. Ischemic attacks fall into two broad categories: thrombotic and embolic.

[0115] In particular, the treatment methods of the present invention may reduce brain damage following ischemic stroke. Brain damage reduction may be measured by determining the reduction in infarct size in a treated individual as compared to a control group. Similarly, functional tests that measure neurological deficits provide further evidence of a reduction in brain damage in treated subjects as compared to controls. Thus, in various accepted models of brain injury following an attack, a definite effect can be measured in treated patients versus control subjects.

One important aspect of the invention is the treatment of a subject with an abnormally high risk of ischemic stroke. As used herein, a subject with an abnormally high risk of ischemic stroke is a category determined according to routine medical practice. Typically, the risk factors associated with heart disease are the same as those associated with seizures. The main risk factors include hypertension, hypercholesterolemia and smoking. In addition, arterial fibrillation or recent myocardial infarction is an important risk factor.

[0117] The treatment of a stroke may be for a patient who has already experienced a stroke, or may be a prophylactic treatment. In the prophylactic case, treatment is for a subject with an abnormally increased risk of ischemic stroke as described above. If the subject has already experienced a stroke, treatment may include acute treatment. Acute treatment is when the onset of a condition begins,
Alternatively, it means administering the agent with the onset of a substantial change in the already existing condition.

Other subjects at high risk of experiencing a cardiovascular-related disorder are hypercholesterolemic and hypertriglyceridemic subjects. Hypercholesterolemic and hypertriglyceridemic subjects are associated with an increased incidence of premature coronary heart disease. Hypercholesterolemia subjects
Have LDL levels> 160 mg / dL or> 130 mg / dL and be male, premature coronary heart disease, smoking (more than 10 per day), hypertension, low HDL (<35 mg / dL)
At least two risk factors selected from the group consisting of diabetes mellitus, hyperinsulinemia, abdominal obesity, a family history of high lipoprotein (a), and a personal history of ceCNREBrovascular disease or obstructive peripheral vascular disease. Hypertriglyceridemic subjects have triglyceride (TG) levels> 250 mg / dL. Therefore,
A hyperlipidemic subject is defined as a subject whose cholesterol and triglyceride levels are equal to or exceed the limits set forth above for both hypercholesterolemic and hypertriglyceridemic subjects.

The present invention is not a CNREB activator or inhibitor, but acts synergistically or synergistically with such activators or inhibitors of the present invention to contain CNRE motif-containing Also encompasses co-administration of substances that can act to regulate gene expression (eg, renin, c-myc, collagen type II and T cell receptors). In one embodiment, the CNREB activator or inhibitor is administered substantially simultaneously with a non-CNREB activator or inhibitor that modulates expression of a CNRE motif-containing gene. “Simultaneously”
By administering a CNREB activator or inhibitor to a subject in need of such treatment is meant to be administered substantially in time with the administration of the non-CNREB activator or inhibitor, whereby: The non-CNREB activator or inhibitor may exert an enhancing effect on the expression regulating activity (CNRE motif-containing gene) of the CNREB activator or inhibitor. Thus, substantially simultaneously means that the CNREB activator or inhibitor is non-C
It is meant to be administered before, simultaneously with and / or after administration of the NREB activator or inhibitor. The CNREB activator or inhibitor can be administered as a polypeptide and / or as a nucleic acid expressing a CNRE binding polypeptide. In a preferred embodiment of the invention, the treatment of a disorder mediated by the renin-angiotensin system comprises a CNREB activator or inhibitor (preferably a CNREB inhibitor, in particular a CNREB inhibitor,
Together with a non-CNREB activator or inhibitor, including a renin-angiotensin inhibitor.

The outline of the angiotensin synthesis pathway in vivo is as follows. The process is initiated when the enzyme renin acts on angiotensinogen, a pseudoglobulin in plasma, resulting in the decapeptide angiotensin I. Angiotensin I
Is converted to angiotensin II (angiotensin [1-8] octapeptide) by angiotensin converting enzyme (ACE). The latter include various mammalian species,
For example, active pressors have been implicated as causative agents of some types of hypertension in humans.

A renin-angiotensin system inhibitor as defined herein is intended to prevent the production of angiotensin II from angiotensinogen or to inhibit angiotensin II
It is a compound that acts to prevent the activity of II. Such inhibitors are well known to those skilled in the art and include compounds that act to inhibit enzymes involved in angiotensin II production, including renin and ACE. They also include compounds that act on the substrates of these enzymes, as well as those that interfere with the activity of angiotensin II once produced. Examples of such compound classes include antibodies (eg, against renin or against ACE), amino acids and their analogs (including those bound to larger molecules), peptides (angiotensinogen and angiotensin).
I, including peptide analogs of I), prorenin-related analogs, phospholipids and much more. Among the most potent and useful renin-angiotensin system inhibitors are renin inhibitors, ACE inhibitors and angiotensin II antagonists.

Renin inhibitors are well known and include amino acids and their derivatives, peptides and their derivatives, and antibodies to renin. Examples of renin inhibitors that are the subject of US patents are as follows. Urea derivatives of peptides (US 5,116,835);
Amino acids linked by non-peptide bonds (US5114,937); di- and tripeptide derivatives (US5,106,835); amino acids and their derivatives (US5,104,8)
69 and 5,095,119); diolsulfonamides and sulfinyls (US 5,098)
Modified peptide (US5,095,006); peptidyl β-aminoacylaminodiol carbamate (US5,098,471); pyrrole imidazolone (US5,075,451).
); Fluorine and chlorine statins or statone containing peptides (US5
, 066,643); Peptidylaminodiol (US5,063,208 and US4,845,079)
N-morpholino derivatives (US5,055,466); pepstatin derivatives (US4,980,283)
); N-heterocyclic alcohols (US 4,885,292); monoclonal antibodies to renin (U
.S. 4,780,401); and various other peptides and their analogs (US 5,071,
837, US5,064,965, US5,063,207, US5,036,054, US5,036,053, US5,0
34,512 and US 4,894,437).

ACE inhibitors mediate in the angiotensin (renin) angiotensin I angiotensin II sequence by inhibiting angiotensin converting enzyme and reducing or eliminating the formation of the pressor substance angiotensin II. ACE inhibitors are useful as antihypertensives and for treating congestive heart failure.

ACE inhibitors are well known and include amino acids and their derivatives, peptides, including di- and tripeptides, and antibodies to ACE. A class of compounds known to be useful as ACE inhibitors is captopril (US Pat. No. 4,105,77).
Acylmercaptoproline and mercaptoalkanoylproline such as zofenopril (US Pat. No. 4,316,906) and enalapril (US Pat. No. 4,374,829), lisinopril (ibid.), Quinapril (quinapril)
) (U.S. Pat. No. 4,344,949), carboxyalkyl dipeptides such as ramipril (U.S. Pat. No. 4,587,258) and perindopril (U.S. Pat. No. 4,508,729); cilazapril (U.S. Pat. No. 4,512,924) and benazepril ( carboxyalkyl dipeptide mimetics such as benazepril (U.S. Pat. No. 4,410,520); fosinopril (U.S. Pat.
No. 337,201) and phosphinyl alkanoyl prolines such as trandolopril.

Angiotensin II inhibitors are compounds that interfere with the activity of angiotensin II. Angiotensin II inhibitors include angiotensin II antagonists and antibodies to angiotensin II. Preferred are AT ₁ specific antagonist. Angiotensin II antagonists are well known and include peptide and non-peptide compounds. Most angiotensin II antagonists are slightly modified homologues whose agonist activity is diminished by substitution of phenylalanine at position 8 with some other amino acid, and its stability is determined by in vivo degradation. Can be increased by other substitutions that delay Examples of angiotensin II antagonists include peptide compounds (eg, salaracin,
[San ¹ , Val ⁵ , Ala ⁸ ] angiotensin- (1-8) octapeptide and related analogs); N-substituted imidazol-2-ones (US 5,087,634); 2-n-butyl-4-chloro-1 -(
Imidazole acetate derivatives including 2-chlorobenzyl) imidazole-5-acetic acid (see, for example, Wong et al., J. Pharmacol. Exp. Ther. 247 (1), 1-7 (1988)); 4
, 5,6,7-Tetrahydro-1H-imidazo [4,5-c] pyridine-6-carboxylic acid and similar derivatives (US 4,816,463); N-2-tetrazole β-glucuronide analogues (US5,
085,992); substituted pyrroles, pyrazoles and triazoles (US 5,081,1)
27); Derivatives of phenyl and heterocycle such as 1,3-imidazole (US Pat. No. 5,073,56)
6); imidazo-fused 7-membered heterocycle (US 5,064,825); peptide (for example, US 4,7
72,684); antibodies to angiotensin II (eg, US Pat. No. 4,302,386); and aralkyl imidazole compounds such as biphenylmethyl-substituted imidazoles (eg, EP 253,310, Jan. 20, 1988). Other angiotensin II inhibitors currently being tested include ES-8891 (N-morpholinoacetyl- (1-naphthyl)-
L-alanyl- (4-thiazolyl) -L-alanyl (35,45) -4-amino-3-hydroxy-5
-Cyclohexapentanoyl-n-hexylamide, Sankyo Company Ltd., Tokyo, Japan, SK & F 108566 (E-? A? -2- [2-butyl-1-[(carboxyphenyl) methyl] 1H-imidazole- 5-yl] methylan] -2-thiophenpropanoic acid, SmithKlin
e Beecham Pharmaceuticals, PA), Losartan (DUP753 / MK954, DuPont Merck P
harmaceutical Co.), Remikirin (RO42-5892 , F.Hoffmann LaRoche AG), adenosine A ₂ agonists (Marion Merrell Dow) and some non-peptide heterocycles (G.
D.Searle & Company).

Disorders such as instinct hypertension, coronary artery disease and diabetes are associated with increased vascular smooth muscle tone that limits the regulation of local blood flow in the kidney, heart, brain and other parts of the vascular bed. Clinical and experimental findings suggest that locally produced angiotensin
It is suggested that an imbalance between II and nitric oxide (NO) results in inappropriate tone of vascular smooth muscle, increasing blood pressure and altering local blood flow. Indeed, administration of a nitric oxide synthase (NOS) inhibitor or angiotensin II increases vascular smooth muscle tone and / or contractility, and systemic blood pressure, and increases local blood flow to organs such as the kidney and heart. Decrease. Conversely, NO, angiotensin II antagonists, renin inhibitors and angiotensin converting enzyme (ACE) inhibitors reduce smooth muscle tone, increase local blood flow to these organs, and reduce systemic blood pressure.

Other agents that reduce the risk of cardiovascular injury and are useful with the agents of the present invention include:
Includes those selected from the group consisting of antithrombotics, antiplatelet drugs, fibrinolytics, lipid-lowering drugs, direct thrombin inhibitors, and glycoprotein IIb / IIIa receptor inhibitors.

Antithrombotic and / or fibrinolytic drugs include the interaction of plasminogen (prokallikrein, kininogen, factor XII, factor XIIIa, plasminogen proactivator and tissue plasminogen activator [TPA] Plasmin) streptokinase; urokinase; anisoylated plasminogen-streptokinase activator complex (Anisoylated Plasminogen-Strep)
tkinase Activator Complex); prourokinase (Pro-UK); rTPA (alteplase or actibase; r indicates recombinant); rPro-UK; avokinase (Ab
bokinase); Eminase; Sreptas anagrelide hydrochloride (Sreptas
e Anagrelide Hydrochloride); Bivalirudin; Dalteparin Sodium; Danaparoid Sodium
um); Dazoxiben Hydrochloride; Efegatran Sulfate (E
fegatran Sulfate); Enoxaparin Sodium; Ifetroban; Ifetroban Sodium; Tinzaparin Sodium; Retaplase; Trifenagrel (Tr)
ifenagrel); Warfarin; dextran.

Antiplatelet agents include Clopridogrel; Sulfinpyrazone;
Aspirin; dipyridamole; clofibrate; pyridinol carbamate;
PGE; Glucagon; Antiserotonin drugs; Caffeine; Theophylline; Pentoxifylline; Tielopidine; Anagrelide
Is included.

[0130] Lipid-lowering drugs include gemfibrozil, choristyramine (cho)
lystyramine), colestipol, nicotinic acid, probucol, lovastatin,
Fluvastatin, simvastatin (simvastatin), atorvastatin (atorvastatin), pravastatin, cilivastatin (cirivastatin) are included.

Direct thrombin inhibitors include hirudin, hirugen, hirulog (h
irulog), agatroban, PPACK, thrombin aptamer.

Glycoprotein IIb / IIIa receptor inhibitors are both antibodies and non-antibodies, ReoPro
(Abcixamab), lamifiban, tirofiban, but are not limited thereto.

[0133] One preferred agent is aspirin. The agents of the present invention can also be administered together with a diuretic. Diuretics include thiazide, furosemide, spironolactone, triamterin, amiloride.

The agents of the present invention can also be administered with a calcium channel antagonist. Calcium channel antagonists include nifedipine, amlodipine, felodipine XL, isradipine, nicardipine (Nicard
ipine), diltiazem, and verapamil.

The agents of the present invention can also be administered with a vasodilator. Vasodilators include alprostadil; Azaclorzine Hydrochloride; Bamethan Sulfate; Bamethan; Bepridil Hydrochloride; Buterizine; Buterizine; Cetiedil Citrate; Cromonal Hydrochloride; Clonitrate. Diltiazem hydrochloride; dipyridamole; droprenilamine; erythrityl tetranitrate; felodipine; flunarizine hydrochloride; fostedil; fexobenzine; inositol nicotinate inocitol; );
Mefenidyl fumarate; Mibefradil Dihydrochloride;
Mixifidine Hydrochloride; Mixidine; Nafuronyl oxalate; Nicardipine hydrochloride; Nicergoline; Nicorandil; Nicotinyl alcohol; Nifedipine; Nimodipine; Nisoldipine; Oxfenicine; Pentaerythritol nitrate; Pentoxifylline; Pentrinitrol; Perhexiline maleate; Pindolol; Pirsidomine; Pleniramine; Includes Luniacinate.

In another preferred embodiment of the present invention, a CNREB modulator, in particular a CNREB-1 inhibitor, of the present invention can be co-administered with an adenylate cyclase inhibitor. Adenylate cyclase inhibitors include H-89 (PKA inhibitor), cilostazol, dibutyryl cAMP (dbcAMP), β-adrenergic receptor agonist, milnacipran, 2 ′, 3′-dideoxyadenosine (2 ', 3'
-DDA), 2'-deoxyadenosine-3'-monophosphate, RMI 12330A or MDL 1230A, prostaglandin E2, SQ22536, guanosine 5'-O- (2-thiodiphosphate), polyphloretin and bromide Acetyloxytocin is included.

In another preferred embodiment of the invention, the CNREB modulator of the invention, in particular the CNREB-1 activator, can be co-administered with an adenylate cyclase activator. Adenylate cyclase activators include forskolin, NKH477,
Iloprost, 2-[(4-azido-2,6-diethylphenyl) imino]
Includes imidazolidine, GRF and betasol.

[0138] The invention also encompasses a method of increasing CNREB-1 activity in a subject. The method is
Administering a CNREB-1 activator that increases CNREB-1 activity to a subject in need of such treatment in an amount effective to increase CNREB-1 activity in the subject. In some embodiments, the amount is greater than the standard baseline level.
1 Enough to increase activity. “Standard baseline level” of CNREB-1 activity
Can be determined using one of the methods described elsewhere herein, for example, using an indirect assay such as detecting renin levels in cells. It is known in the art that a large number of age, gender (etc.) matched standard subjects can be sampled to obtain such control level values of CNREB activity. Once determined, such control levels can be used to compare the CNREB activity of a subject receiving the above therapy with a CNREB modulator (ie, a CNREB-1 activator) to increase the CNREB activity of the subject. You can check if it is equal to or exceeds the standard baseline level. In some embodiments, the subject has a condition that includes hypertension, impotence, spinal cord injury, toxic shock syndrome, or blood transfusion. In yet another embodiment, the CNREB-1 activator is a nucleic acid. In a preferred embodiment, the nucleic acid is the nucleic acid of SEQ ID NO: 1, SEQ ID NO: 1.
D NO: 3, SEQ ID NO: 5 and SEQ ID NO: 6. In yet another embodiment, the CNREB-1 activator is a polypeptide. In a preferred embodiment, the CNREB-1 activator comprises SEQ ID NO: 1
The nucleic acid of SEQ ID NO: 3, the nucleic acid of SEQ ID NO: 5 and the polypeptide of the expression product of the nucleic acid selected from the group consisting of the nucleic acid of SEQ ID NO: 6, or a functional fragment or variant thereof. is there. If a human subject is in need of such a treatment, a human homolog of the aforementioned mouse sequence is administered. In some embodiments, CNREB-
1 Administer the activator urgently. In some embodiments, the CNREB-1 activator is administered prophylactically. In yet another embodiment, the method further comprises co-administering an agent other than the CNREB-1 activator. Preferred non-CNREB-1 activators include fluorocortisone, potassium tablets, vasopressin analogues, somatostatin analogues, beta blockers, sympathomimetic agents, dopamine antagonists, venous constrictors and adenylate cyclase activators It is.

According to the foregoing method, the present invention provides erectile dysfunction (dysfunction) by affecting renin levels using a CNREB-related substance of the present invention (eg, a CNREB-1 activator).
Including treatment. Erectile dysfunction or impotence is a common disorder found in more than 10 million men in the United States. Mental etiology was thought to be the main cause of erectile dysfunction, but it is now thought that the majority of erectile dysfunction is caused by a potential organic disorder. I have. This conceptual change is supported by a particularly high rate of impotence in men with instinct hypertension, coronary artery disease and diabetes. In addition, the primary mechanism responsible for impotence is that smooth muscle tone and / or smooth muscle in the corpus cavernosum and penile artery, which prevents regulation of penile blood flow by physiological regulators.
Or an increase in contractility. A similar mechanism, namely increased tone and / or contractility of vascular smooth muscle, prevents regulation of blood flow in the annular, renal and other arteries of patients with hypertension, diabetes, etc.

Other potential organic causes of erectile dysfunction include endocrine disorders such as testicular dysfunction and hyperprolactinemia; eg, androgens, antihypertensives, anticholinergics, antidepressants, anti-depressants, Side effects of drugs such as psychotic drugs, central nervous system depressants and addictive or addictive drugs; Peyronie's disease, pre-painful priapism (previous p)
riapism), and penile disorders such as penile injury; for example, anterior temporal lobe injury, spinal cord disease, loss of sensory input, erectile nerve disease, and diabetic autonomic neuropathy (au
neurological diseases such as tonomic neuropathy); vascular diseases such as essential hypertension, aortic occlusion, atherosclerotic occlusion or stenosis of the vulvar artery, venous leakage, and diseases of the sinusoidal space.

As a modified vascular tissue, the corpus cavernosum (ccp) produces and secretes the same range of autocrine and paracrine regulators as normal vascular tissue.
However, the smooth muscle tone of ccp does not appear to be controlled in a similar manner as the vessel wall. At present, the tension or contractility of ccp is
It has been postulated to be adrenergic modulated and produce NO and endothelin locally. In ccp, most studies have focused on observing the flaccid effects of NO, vasoactive intestinal peptide (VIP), calcitonin gene-related peptide (CGRP) and parasympathetic innervation. Acts similarly on normal and ccp vascular smooth muscle.

Recently, it has been discovered that inhibitors of the renin-angiotensin system, as well as vascular tissue, produce and secrete angiotensin II in the corpus cavernosum of the penis, which plays an important role in modulating penile blood flow. (PCT International Patent Application W097 / 10821).
Angiotensin II antagonist or AC topically in the corpus cavernosum or systemically
Administration of E inhibitors has a strong effect on penile blood flow. Using this effect,
Erectile dysfunction can be ameliorated without the inconvenience and side effects of drugs used as intracavernous medicine. Therefore, such agents can be co-administered with the CNREB-1 activator of the present invention to ameliorate the above symptoms.

The present invention also includes a method for treating a subject having a cancer that expresses c-myc. If so, an effective amount of a CNREB-1 inhibitor that inhibits c-myc-mediated activity can be administered to the subject to slow or inhibit the growth of cancer cells that express c-myc. . CNREB-1 inhibitors include antisense CNREB-1 nucleic acids, dominant negative CNREB-1 nucleic acids, dominant negative CNREB-1 polypeptides, anti-CNREB-
Antibodies, anti-CNREB-1 antibody fragments, and functional fragments or variants thereof that inhibit CNREB-1 activity are included. In certain embodiments, the CNREB-1 inhibitor is a nucleic acid. In a preferred embodiment, the nucleic acid is the nucleic acid of SEQ ID NO: 1, the nucleic acid of SEQ ID NO: 3,
An antisense nucleic acid or a dominant negative nucleic acid of CNREB-1 nucleic acid selected from the group consisting of nucleic acid of ID NO: 5 and nucleic acid of SEQ ID NO: 6. The mode of administration and dosage of the agent will depend on the health insurance physician's knowledge of the particular stage of the condition being treated, the age and health of the subject being treated, combination therapy (if any), and the particular route of administration. And factors within expertise. In a preferred embodiment of the invention, c-myc-expressing cancers include: cholangiocarcinoma; brain cancer including glioblastoma and medulloblastoma; breast cancer; cervical cancer; choriocarcinoma; colon cancer; Membrane cancer; esophageal cancer; gastric cancer; hematological neoplasms including acute lymphocytic and myeloid leukemia; multiple myeloma;
AIDS-related leukemia and adult T-cell leukemia lymphoma; intraepithelial neoplasia, including Bowen's disease and breast Paget's disease; liver cancer; lung cancer; lymphoma, including Hodgkin's disease and lymphocytic lymphoma; neuroblastoma; squamous cell carcinoma Oral cancer including those arising from epithelial cells, stromal cells, germ cells and mesenchymal cells; pancreatic cancer; prostate cancer; rectal cancer; leiomyosarcoma, rhabdomyosarcoma, liposarcoma, fibrosarcoma and Sarcomas including osteosarcoma; skin cancers including melanoma, Kaposi's sarcoma, basal cell carcinoma and squamous cell carcinoma; Testicular cancer, including thyroid adenoma and medullary carcinoma; and renal carcinoma, including adenocarcinoma and Wilms tumor. In other embodiments, the cancer that expresses c-myc is an ectopically expressing CNREB factor. Ectopic expression is expressed in CNREB (CNREB
Overexpression of B-1 and / or CNREB-2).

In certain embodiments, the agent is another anti-cancer agent, such as: Acivicin
Aclarubicin; Acodazole hydrochloride (Acodazole); Acronin; Adozelesin; Aldesleukin; Altretamine (Altre)
tamine); Ambomycin; Amethantrone acetate
Aminoglutethimide; Amsacrine; Anastrozole
Anthramycin; Asparaginase; Asperlin; Azacytidine; Azetepa; Azolomycin (Azolo)
mycin); Batimastat; Benzodepa; Bicalutamide; Bisantrene hydrochloride; Bisnafide (Bi)
Bimelesin; Bizelesin; Bleomycin sulfate; Brequinar sodium; Bropirimine; Busulfan; Cactinomycin; Carsterone; Caracemide; Carbetimer;
Carubicin hydrochloride (Carubicin); Carzelesin; Cedafingol; Chlorambucil; Sirolemycin; Cisplatin; Cladribine; Crisnatol mesylate; Cyclophosphamide; Cytarabine; Dactinomycin; Daunorubicin hydrochloride; Decitabine; Dexomaplatin (Dexo
Dezaguanine; Dezaguanine; Dezaguanine mesylate; Diaziquone; Docetaxel (Docetaxel); Doxorubicin; Doxorubicin hydrochloride; Droloxifene; ); Deatrexate; Eflornithine hydrochloride; Elsamitrucin; Enloplatin; Enpromate
omate); Epipropidine; Epirubicin hydrochloride
Erbulozole; Esorubicin hydrochloride (Esorubicin); Estramustine; Estramustine sodium phosphate; Etanidazole
Etoposide; Etoposide phosphate; Etoprine; Fadrozole hydrochloride; Fazarabine; Fenretini
floxiuridine; fludarabine phosphate; fluorouracil; flurocitabine; fosquidone; fostriecin sodium; gemcitabine; gemcitabine hydrochloride; hydroxyurea; Ilmofosin; interferon α2a; interferon α-2b; interferon α-n1; interferon α-n3; interferon β-Ia; interferon γ-Ib; iproplatin (Iproplatin); irinotecan hydrochloride (Irinotecan); lanreotide acetate; (Letrozole
Leuprolide acetate; Liarozole hydrochloride; Lometrexol sodium; Lomustine; Lomustine; Losoxantrone hydrochloride; Masoprocol; Maytansine; Mechlorethamine hydrochloride; Melengestrol; Melphalan; Menogalil; Mercaptopurine; Methotrexate; Methotrexate sodium; Metoprine;
Mitomycin; Mitosper; Mitotane; Mitoxantrone hydrochloride; Mycophenolic acid; Nocodazole;
Nogalamycin; Ormaplatin; Oxisuran; Paclitaxel; Pegaspargase
gase); Peliomycin; Pentamustine; Pepromycin sulfate; Perfosfamide; Pipobroman; Piposulfan; Piroxantrone hydrochloride; Plicamycin; Plomestane; Porfimer sodium Porphyromycin; prednimustine; procarbazine hydrochloride; puromycin; puromycin hydrochloride; pyrazofurin; ribopurine (R)
Saffingol; Safingol hydrochloride; Semustine; Simtrazene; Sparfosate sodium; Sparsomycin; Spirogermanium hydrochloride (Spopol);
ogermanium); Spiromustine; Spiroplati
n); Streptonigrin; Streptozocin; Sulofenur; Talisomycin; Taxanes (Taxanes); Tecogalan sodium; Tegafar Tegafur; Teloxantrone hydrochloride; Teniposide
Teroxirone; test lactone; thiamiprine
); Thioguanine; Thiotepa; Tiazofurin; Tirapazamine; Topotecan hydrochloride (Topotecan); Toremifene citrate (Toremifiene); Trestrelone acetate; Triciribine phosphate; Trimetrexate Trimetrexate glucuronate; Triptorelin; Tubulozol hydrochloride
e); uracil mustard; uredepa; vapreotide
Verteporfin; Vinblastine sulfate; Vincristine sulfate; Vindesine; Vindesine sulfate; Vinepidine sulfate; Vinglycidine sulfate; Vinglycinate; Vinleurosine sulfate; Vinorelbine tartrate; Vinzolidine sulfate; borozole; Zeniplatin
Dinostatin; Zorubicin hydrochloride (Zorubicin);

Other anti-tumor compounds include: 20-epi-1,25 dihydroxyvitamin
D3; 5-ethynyluracil; abiraterone; abiruberin; acylfulvene; adecypenol; adzelesin (adozelesin); aldesleukin;
Altretamine; ambamustine; amidox; amifostine; aminolevulinic acid; amrubicin (amrubi)
cin); amsacrine; anagrelide; anagrezole; anastrozole; andrographolide; angiogenesis inhibitor; antagonist D; antagonist G; antarelix.
Anti-dorsal morphogenetic protein-1; anti-androgen, prostate cancer; anti-estrogens; anti-neoplaston; antisense oligonucleotides; aphidicolin glycinate; apoptosis gene modulators; ara-CDP-DL-PTBA; arginine deaminase; aslacrin
atamestane; atrimustine; axinastatin 1; axinastatin 2; axinastatin 3; azasterone; azatoxin; azatyrosine; baccatin III derivative; Batimastat; BCR / ABL antagonist;
Benzocholine, benzoyl staurosporine, β-lactam derivative, β-alethine, beta-clamycin B, betulinic acid, bFGF inhibitor, bicalutamide, bisantrene; Bisaziridinylspermine
Bisnafide; bistratene A; biseresin (
bizelesin); breflate; bropirimine; budotitane; buthionine (sulfoximine); calcipotriol; calphostin (calphostin) C; ; Carboxamide-
Amino-triazole; carboxamidotriazole; CaRest M3; CARN 700;
Cartilage-derived inhibitor; carzelesin; casein kinase inhibitor (ICOS
); Castanospermine; cecropin B; cetrorelix; chlorin; chloroquinoxaline sulfonamide;
Cicaprost; cis-porphyrin; cladribine
Clomifene analogs; clotrimazole; chorismycin (co);
llismycin) A; chorismycin B; combbretastatin A4;
Combretastatin analogues; conagenin; clambesidin (cramb
escidin) 816; crisnatol; cryptophycin
n) 8; a cryptophysin A derivative; curacin A; cyclopentane thraquiriones; cycloplatam;
cypemycin); cytarabine ocfosfate; cytolytic factor; cytostatin; dacliximab; decitabine (de)
citabine); dehydronindemin (didemnin) B; deslorelin
Dexifosfamide; dexrazoxane
Dexverapamil; diaziquone; didemnin B; didox; diethylnorspermine; dihydro-5-azacitidine; dihydrotaxol 9-; dioxamycin; diphenylspiromustine; docosanol (Docosanol); dolasetron (dol
asetron); doxifluridine; droxifen
e); dronabinol; duocarmycin SA;
Ebselen; ecomustine; edelfosine (edelf)
edrecolomab; eflornithine;
Elemene; emitefur; epirubicin
Epristeride; estrarnustine analogs; estrogen agonists; estrogen antagonists; etanidazole ());
etanidazole); etoposide phosphate; exemestane
); Fadrozole; fazarabine; fenretinide; filgrastim; finasteride (fin)
aslerilde); flavopiridol; fiezelastine (fiezela)
stine); fluasterone (fluasterone); fludarabine (fludarabine); fluorodaunorunicin hydrochloride (fluorodaunorunicin); forfenimex (fo
rfenimex); formestane; fostriecin
Fotemustine; gadolinium texaphyrin
Gallium nitrate; galocitabine; ganireli
gemcitabine; glutathione inhibitor; hepsulfam; heregulin; hexamethylenebisacetamide; hypericin; ibandronic acid; idarubicin; idoxifene; Hydramanton (idram
antone); ilmofosine; ilomastat; imidazoacridones; imiquimod; immunostimulatory peptide; insulin-like growth factor-I receptor inhibitor; interferon agonist; interferon; Iobenguane; iododoxorubicin; iporneanol, 4-
Irinotecan; iroplact; irsogladine;
irsogladine; isobengazole; isohomohalicondrin B; itasetron; jasplakinolide
jasplakinolide); kahalalide F; lamellarin-N
Triacetate; lanreotide; leinamycin
Lenograstim; lentinan sulfate; leptolstatin; letrozole letrozole; leukemia inhibitory factor;
Interferon; leuprolide + estrogen + progesterone; leuprorelin; levamisole; liarozole; a linear polyamine analog; a lipophilic disaccharide peptide; a lipophilic platinum compound;
lissoclinarnide) 7; lobaplatin; lombricine
); Lometrexol; lonidamine; losoxantrone; lovastatin; loxoribine
ribine); lutetotecn; lutetium texaphyrin
lysofylline; soluble peptide; maitansine; mannostatin A; marimast
at); masoprocol; maspin; matrilysin (matr)
ilysin) inhibitors; matrix metalloproteinase inhibitors; menogal (menoga)
ril); merbarone; meterelin; methioninase; metoclopramide; MIF inhibitor; mifepristone (mifep)
ristone); miltefosine; mirimostim; mismatched double-stranded RNA; mitoguazone; mitoctolol.
lactol); mitomycin analogs; mitonafide; mitoxin fibroblast growth factor-saporin; mitoxanthane (mitoxant)
Mofarotene; molgramostim; monoclonal antibody; human chorionic gonadotropin; lipid A monophosphate + myobacterium cell wall sk; mopidamol (mopidamol); a multidrug resistant gene inhibitor; Factor-I based therapy; mustard anticancer drug; micaperoxide (mycapero
xide) B; mycobacterium cell wall extract; myriaporone; N-
Acetyldinaline (acety1dinalline); N-substituted benzamide; nafarelin (n
afarelin); nagrestip; naloxone + pentazocine; napavin; naphterpin; nartograsm
tim); nedaplatin; nemorubicin; neridronic acid; neutral endopeptidase; nilutamid
e); nisamycin; nitric oxide modulator; nitroxide antioxidant; nitrrlyn; O6-benzylguanine; octeotide (octe)
otide); oxicenone; oligonucleotide; onapristone (on
apristone); ondansetron; oracin; oral cytokine inducer; ormaplatin; or osaterone
); Oxaliplatin; oxaunomycin;
Paclitaxel analogs; paclitaxel derivatives; parauamine (
palauamine); palmitoylrhizoxin; pamidronic acid; panaxytriol; panomifene; parabactin; pazelliptine
Pegaspargase; Perdesine; Pentosan sodium polysulfate; Pentostatin; Pentrozole
ozole); perflubron; perfosfamide (perfosfainlde)
); Perillyl alcohol; phenazinomycin
Phenylacetate; phosphatase inhibitor; picibanil; pilocarpine hydrochloride; pirarubicin; piritrexim; A platinum compound; a platinum-triamine complex; porfimer sodium;
Prostaglandin J2; Proteasome inhibitor; Protein A-based immune modulator; Protein kinase C inhibitor; microalgal; Protein tyrosine phosphatase inhibitor; Purine nucleoside phosphorylase inhibitor ; Purpurin
s); pyrazoloacridlne; pyridoxylated hemoglobin polyoxyethylene conjugate; raf antagonist; raltitrex
ram) tron; ras farricsyl protein transferase inhibitor; ras inhibitor; ras-GAP inhibitor; demethylated retelliptine; rhenium Re 186 etidronate; rhizoxin
); Ribozyme; RII retinamide; rogletimide
Rohitukine; romurtide; rokinimex (r
rubiginone B1; ruboxyl; safingol; saintopin; SarCNU; sarcophytol A; sargramostini A; Sdi I mimetic; (Senescence) -derived inhibitor 1; sense oligonucleotide; signal transduction inhibitor; signal transduction modulator; single-chain antigen binding protein; sizofiran; sobuzoxane; sodium borocaptate; Sorberol; somatomedin-binding protein; sonermin
Sparfosic acid; spicamycin D; spiromustine; splenopentin; spongistatin 1; squalamine; stem cell inhibitor; stem cell division inhibitor; stipamide ( stipiamide); stromelysin
Inhibitors; sufinosine; hyperactive vasoactive intestinal peptide antagonists; suradista; suramin; swainsonine; synthetic glycosaminoglycans; tallimustin
e); tamoxifen methiodide; tauromustin
e); taxanes; tazarotene; tecogala
n) Sodium; tegafur; tellurapyrylium
Telomerase inhibitor; temoporfin; temozolomide (temoporfin)
zolomide); teniposide; tetrachlorodecaoxide; tetrazomine; thaliblastine; thalidomide
thycoraline; thiocoraline; thrombopoietin; mimetic thrombopoietin; thymalfasin; thymopoietin receptor agonist; thymotrinan; thyroid-stimulating hormone;
tanocene) dichloride; topotecan; topsentin
Toremifene; totipotent stem cell factor; translation inhibitor; tretinoin; triacetyluridine; triciribine; trimetrexate; triptorelin; tropisetron; Tyrosine kinase inhibitors; tyrphostins; UBC inhibitors; ubenimex; urogenital sinus-derived growth inhibitory factor bypass kinase receptor antagonists; vapleotide (v
apreotide); variolin B; vector system, erythrocyte gene therapy;
Velaresol; velamine; verdins; verdins; verteporfin; vinorelbine; vinxaltine; vitaxin; ; Zilascorb
); Zinostatin stimalamer;

Anti-cancer co-enhancing agents: tricyclic antidepressants (eg, imipramine, desipramine, amitriptyline, clomipramine, trimipramine, doxepin, nortriptyline, protriptyline, amoxapine and maprotiline); non-tricyclic antidepressants ( Ca ⁺⁺ antagonists (eg, verapamil, nifedipine, nitrendipine and caroverine); calmodulin inhibitors (eg, prenylamine, trifluoroperazine and clomipramine); amphotericin B; tripanolanol analogs (eg, sertraline, trazodone and citalopram); Tamoxifen); antiarrhythmic agents (eg, reserpine); thiol blood loss agents (
For example, buthionine and sulfoximine) and multidrug-resistant reducing agents such as Cremaphor EL. The compounds of the present invention can also be administered with a cytokine, such as a granulocyte colony stimulating factor.

[0147] The invention also encompasses a method of modifying the expression of a CNRE-motif-containing gene. Examples of such genes include c-myc, renin, type II collagen, and T cell receptors. The method comprises contacting a cell expressing any of the genes with a CNREB-1 activity modulator in a cell in an amount effective to modify expression of any of the genes in the cell. . In some embodiments, contacting a mammalian cell expressing any of the above genes with a CNREB-1 activity modulator may occur in vitro or in vivo. In certain embodiments, the CNREB-1 activity modulator is a nucleic acid. In a further embodiment, the CNREB-1 activity modulator is a polypeptide. Although CNREB-1 has been described as an exemplary modulator, CNREB-2 can also be used. "modulation"
Means that the activity modulator can cause an increase or decrease in expression of the CNRE-motif-containing gene. One skilled in the art should be able to determine the effect of such a modulator. Typically, a CNREB-1 (or -2) activator is a CNREB-1 (or -2) activator
It should cause increased expression of the motif-containing gene. Conversely, CNREB-1 (or -2) inhibitors should cause a decrease in expression of CNRE-motif-containing genes in certain cell types. Another mode of action for such activators or inhibitors, however, may occur and will usually depend on the cell type in which the CNRE-motif-containing gene is expressed. The person skilled in the art will be familiar with such possibilities and can adapt according to the specific cell type and the requirements for the activity required by administering the CNREB modulator.

The pharmaceutical compositions described above are administered in effective amounts. The effective amount will depend on the mode of administration, the particular condition to be treated and the desired result. It is, as mentioned above,
It will also depend on the stage of the condition, the age and physical condition of the subject, the nature of the concomitant treatment, if any, and such factors as are well known to practitioners. For therapeutic use, it is in an amount sufficient to achieve the medically desired result. In some cases, this is a drop in blood pressure. In other cases, it is a reduction in the level of expression of renin. In yet another case, it is a decrease in c-myc expression or cell proliferation.

In general, the dosage of the active compounds according to the invention is from about 0.01 mg / kg per day to 1000 mg / kg per day. It is expected that dosages in the range of 50-500 mg / kg will be appropriate. Various routes of administration are available. The methods of the present invention may generally be practiced with any mode of medically acceptable administration, meaning any mode that produces an effective level of an active compound without causing clinically unacceptable adverse effects. Such modes of administration include oral, rectal, topical, intranasal, transdermal, or parenteral routes. The term "parenteral" includes subcutaneous, intravenous, intramuscular, or infusion. Intravenous or intramuscular routes are not particularly suitable for long-term treatment and prevention. They are however preferred in emergency situations. Oral administration is preferred for prophylactic treatment because of convenience to the patient and dosing schedule. When the peptide is used for therapy, in some embodiments, the desired route of administration is by pulmonary aerosol.
Techniques for the production of aerosol delivery systems containing peptides are well known to those skilled in the art. In general, such systems should utilize components that do not significantly impair the biological properties of the antibody, such as the ability to bind paratopes (eg, Sciarra and Cuti).
e, “Aerosols,” in Remington's Pharmaceutical Sciences , 18th edition, 1990, p.
pp. 1694-1712; incorporated by reference). One skilled in the art can readily determine various parameters and conditions for producing an antibody or peptide aerosol without resort to undue experimentation.

Compositions suitable for oral administration include separate units, for example, capsules, tablets,
It may be produced as a cough drop, each containing a predetermined amount of active agent. Other compositions include suspensions in aqueous or non-aqueous liquids such as syrups, elixirs or emulsions.

Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters.
For example, ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions or suspensions, including salts and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (eg, based on Ringer's dextrose), and the like. Preservatives and other additives may include, for example, antimicrobial agents, antioxidants, chelating agents, inert gases, and the like. Low dosages will result from other forms of administration, for example, intravenous administration. If the subject's response is inadequate at the time of initial medication application, a higher dose (or an effective higher dose by another more localized route of delivery) may be employed, as long as patient tolerance allows. . Multiple doses per day are intended to achieve appropriate systematic levels of the compound.

[0152] The CNREB polypeptide or fragment thereof may be optionally combined with a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" means one or more compatible solid or liquid fillers, diluents or encapsulating materials, suitable for human administration. The term "carrier" refers to a natural or synthetic organic or inorganic ingredient into which the active ingredients are combined to facilitate application. The components of the pharmaceutical composition can also be co-entrained with one another with the molecules of the invention in such a way that there are no interactions that substantially impair the desired drug efficacy.

When administered, the pharmaceutical preparations of the invention are applied in pharmaceutically-acceptable amounts and in pharmaceutically-acceptable compositions. Such preparations are routinely prepared with salt,
Buffers, preservatives, compatible carriers, and optionally other therapeutic agents may be included. When used in medicine, the salts should be pharmaceutically acceptable, but the pharmaceutically acceptable salts may be prepared by the convenient use of non-pharmaceutically acceptable salts. It is not excluded from the scope of the invention. Such pharmacologically or pharmaceutically acceptable salts include, but are not limited to, the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, maleic acid, acetic acid, salicylic acid, citric acid, formic acid , Malonic acid,
Including salts produced from succinic acid and the like. Also, pharmaceutically acceptable salts can be prepared as alkaline metal or alkaline earth metals, such as sodium, potassium or calcium salts.

While it is preferred that the CNREB polypeptide is produced recombinantly, such a polypeptide may be isolated from a biological extract. Recombinantly produced CNREB
A polypeptide can be a CNREB protein and another polypeptide, such as a protein-
A polypeptide capable of providing or enhancing protein binding, sequence-specific nucleic acid binding (eg, GAL4), a polypeptide capable of enhancing the stability of a CNREB polypeptide under assay conditions, or a polypeptide providing a detectable moiety, eg, green Includes chimeric proteins including fusions with fluorescent proteins. Polypeptides fused to CNREB polypeptides or fragments also provide a means for easily detecting the fusion protein, for example, by immunorecognition or by fluorescent labeling.

A variety of techniques may be used to introduce a nucleic acid of the present invention into cells, depending on whether the nucleic acid is introduced into a host in vitro or in vivo. Such techniques include transfection of nucleic acid-CaPO ₄ precipitates, transfection of nucleic acids linked to DEAE, transfection with a retrovirus including the nucleic acid of interest, liposome-mediated transfection and the like. For certain uses, it is preferred to target the nucleic acid to certain cells. In such instances, vehicles used to deliver the nucleic acids of the invention to cells (eg, retroviruses,
Or other viruses; liposomes) can have a targeting molecule linked thereto. For example, molecules such as antibodies specific for surface membrane proteins on target cells or ligands for receptors on target cells can be bound to or incorporated into a nucleic acid delivery vehicle. For example, when liposomes are employed to deliver the nucleic acids of the invention, proteins that bind to surface membrane proteins associated with endocytosis may be incorporated into liposome formulations for targeting and / or enhancing uptake. Such proteins include capsid proteins or fragments thereof that are attracted to a particular cell type, antibodies to proteins that undergo internalization in the circulation, proteins that target intracellular localization and enhance intracellular longevity, etc. . Polymer delivery systems have also been successfully used to deliver nucleic acids into cells and are known to those skilled in the art. Such systems even allow oral delivery of nucleic acids.

Other delivery systems can include time release systems, delayed release systems or sustained release systems. Such a system can avoid repeated administration of the CNREB modulator of the present invention, increasing convenience for subjects and physicians. Many types of release delivery systems are available and known to those skilled in the art. They include polymer-based systems such as poly (lactide-glycolide), copolyoxalates, polycaprolactones,
Including polyesteramides, polyorthoesters, polyhydroxybutyric acid, and polyanhydrides. Microcapsules of the polymer containing the drug are described, for example, in US Pat. No. 5,075,109. Delivery systems are non-polymeric: sterols,
For example, cholesterol, cholesterol esters and fatty acids or neutral fats,
Fats including, for example, mono-, di- and tri-glycerides; hydrogel release systems;
Silastic systems; peptide-based systems; wax coatings; compressed tablets using conventional binders and excipients; partially fused implants and the like. Particular examples include, but are not limited to, (a) an aggressive agent in which the reagents of the invention, such as those described in US Pat. Nos. 4,452,775, 4,675,189 and 5,736,152, are included, for example, in a form within a matrix. (Erosional) systems, and (b) US Patent 3,854,4
No. 80, 5,133,974 and 5,407,686, including diffusion systems in which the active ingredient penetrates from the polymer at a controlled rate. In addition, hardware delivery systems based on pumps can be used, some of which are adapted for implantation.

The use of a long-term sustained release implant is particularly suitable for the treatment of chronic conditions. Prolonged release as used herein means constructing and preparing an implant to deliver a therapeutic level of the active ingredient for at least 30 days, and preferably 60 days. Long-term sustained release implants are well known to those skilled in the art and include some of the above release systems.

[0158]

【Example】

Experimental Method Identification of CNREB-1 A mouse kidney yeast library was screened for renin CNREB using a yeast 1 hybrid cloning system and double positive c up to 1.5 kb.
A DNA clone was obtained and designated as KND9. KND9 cDNA does not contain the complete open reading frame (ORF) and lacks the initial ATG. The nucleotide sequence of KND9 (SEQ ID NO: 1) was obtained from GenBank (http://www.ncbi.nlm.nih.gov/cgi-bin/BLAST/nph/
newblast) in the mouse EST cDNA database, and the KND9 5'-terminal 282
The bp was found to be identical to the 3'-end of the mouse EST clone (AA415858-SEQ ID NO: 4). EST AA415858 is a mouse 2-cell embryo cell library (mouse 2-cell em
bryonic library).

Two primers (P1: nucleotides 1-22 of SEQ ID NO: 4, and P2: KND9-SEQ
The sequence complementary to nucleotides 300-322 of ID NO: 1) was used for RT-PCR. DBA / 2J
5 mg of total RNA from mouse kidney was used. Standard methods were used for reverse transcription of the cDNA. A single band of 528 bp was observed by RT-PCR. Cloning and sequencing of this band resulted in the clone EST-AA415858 (SEQ ID NO: 4) from KND9 (S
EQ ID NO: 1).

Sequence analysis of the CNREB-1 cDNA reveals that it encodes a 445 amino acid protein with 97% homology to rat RLD-1 (SEQ ID NO: 5) (SEQ ID NO: 2). (Apfel R. et al., Mol. Cell. Biol., 1994, 14: 7025-7035). CNREB-1
The cDNA also contains an 84 bp 5 'untranslated region (UTR), a 1335 bp open reading frame (ORF), and a 287 bp 3-UTR (standard polyadenylation signal and 41 b
p (including the poly-A tail region). From these results, the applicant has
It was concluded that NA encodes the full-length CNREB-1 (LXRα) protein. The ORF encodes a 445 amino acid protein (M _r 50,000), which has been confirmed using in vitro transcription / translation. Western blot analysis revealed that CNREB-1
It was demonstrated that the monoclonal antibody specifically recognized the translated CNREB-1 in vitro.

Identification of CNREB-2 Using the yeast one-hybrid cloning system, Applicants have also cloned strong double positive cDNA clones up to 0.7 kb in size. To obtain the complete mRNA of the KND42 clone, a mouse kidney cDNA library was 700 Pbp labeled with ³² P-labeled
KND42 cDNA. After three screening to obtain four positive clones from 10 ⁶ plaques. The longest one is 2.4 kb in size, which is called CNREB-
2 and analyzed by sequencing. CNREB-2 was thought to be a novel gene. Using this 2.4 kb cDNA as a probe for Northern blot hybridization, Applicants revealed that CNREB-2 was up to 3.5 kb.
Although mRNA is widely expressed in all tissues tested, bands up to 2.0 kb were found to be expressed only in intestine, kidney and liver. Applicant has two different CNREB
-2 It was assumed that the mRNA was due to alternative splicing of the mRNA or to be encoded by a different gene. Sequencing revealed that the 5'-end of CNREB-2 remained missing. To get the rest of the molecule,
Using a 2.4 kb fragment, the mouse genomic library was transferred to CNREB-
Screened in 2. Three positive clones were identified. Further sequencing analysis revealed two different splicing isoforms. The shorter isoform is CNREB-2S (SEQ ID NO: 17) and the longer isoform is CNREM-2L
(SEQ ID NO: 19), which encodes a polypeptide having the sequence of SEQ ID NO: 18 and SEQ ID NO: 20, respectively. No homology was found in the non-redundant GenBank gene database.

Expression of CNREB-1 in Prokaryotic Vectors Applicants have translated CNREB-1 cDNA into two different expression vectors: pcDNA3.1 (Invitrogen, C
arlsbad, CA) and pGEM3Zf (Promega, Madison, WI). in
In vitro transcription and translation assays revealed that the major 50 kDa protein and two small proteins (45 kDa and 40 kDa) were produced by both CNREB-1 expression plasmids (see FIG. 1). ). Applicants have assumed that 45 kDa and 40 kDa are generated by using an internal in-frame ATG to initiate translation. To confirm specific binding between CNRE and CNREB-1, Applicants performed a gel shift assay using ³² P-labeled Ren1-CNRE and CNREB-1 from transcription and translation in vitro. Applicants have demonstrated complex formation between CNRE and CNREB-1. Interestingly, the CNREB-1 complex was also formed with human c-myc, which shares the CNRE consensus element with Ren-1 CNRE and mouse renin CRE. MHC-1 CN
No complex formation was seen between RE and CNREB-1. All of these results are consistent with applicant's previous observations (Barreu G. et al., Proc. Natl. Acad. Sci. USA, 1
992, 89: 885-889; Horiuchi M. et al., J. Clin. Invest., 1993, 92: 1805-1811).

Transient Transfection and Reporter Gene Assay The AS4.1 cell line was derived from a renin-expressing renal tumor induced by SV40 T antigen-mediated tumorigenesis in transgenic mice. The system is ATCC (American Tis
sue Culture Collection, Manassas, VA). AS4.1 cells express high levels of renin mRNA. Therefore, AS4.1 cells were used as a model for studying renin gene expression.

The HeLa cell line (epithelial cervical cancer) was also purchased from ATCC. HeLa cells have high levels of c-
Express myc mRNA. Therefore, HeLa cells were used as a model for studying c-myc gene expression.

All cDNAs (CNREB-1 and controls) were cloned into eukaryotic expression vector pcDNA3.1 (Invitrogen, CA) for transfection. Transfection was performed using Fugene-6 (transfection agent) (Boehringer Mannheim, G.
ermany) and according to the manufacturer's protocol.

When the cells reached 80% confluence in the 6-well cell culture plate, 1 μg of each plasmid and 0.2 μg of β-galactosidase plasmid were added to 4 μl of Fugene-6
Was used for each well. β-galactosidase plasmid was used as a control for transfection efficiency. Total RNA was converted to Trizol reagent (GIB
CO BRL, Gaithersburg, MD) for 18 hours after transfection. Reporter gene assays were performed 36 hours after transfection according to the manufacturer's instructions. Both luciferase and chloramphenicol acetyltransferase (CAT) activities were performed with Promega, Madison, WI assay kits. The activity of each plasmid was normalized by the transfection efficiency of β-galactosidase.

Northern Blot Hybridization Analysis 10 μg of total RNA from the differently transfected samples described above was separated on a 1.0% agarose gel containing 2.6 M formaldehyde and blotted on a Zetaprobe membrane. 10% formamide, 3x SSPE (1X = 0.18 M NaCl, 0.1%
The membrane was prehybridized at 55 ° C. for 4 hours in a solution containing 01 M Na ₂ HPO ₄ , 1 mM EDTA), 0.1% SDS, 0.5% Blono, and 0.5 mg / ml denatured herring sperm DNA. Hybridization was performed using 10% dextran sulfate and [α- ³² P] -dATP-labeled mouse Nen cDNA or c-myc cDNA (NEN Dupont, MA; specific activity 3,000).
(Ci / mmol) fragment (Hay N., et al., Genes & Dev. 1989, 3: 293-303) in a similar solution at 55 ° C for 12 hours. Hybridization is performed in a hybridization incubator (Model 400, Robbins Scientific, Sunnyvale
, CA). The final stringent wash is 0.1X SSC at 65 ° C (1X = 0.15 MN
Performed in aCl, 0.015 M Na citrate), 1% SDS for 30 minutes. 0.24 to 9.49 Kb RNA ladder (BRL, Gaitherburg, MD) co-electrophoresed for the molecular size of the detected mRNA
Was measured from the mobility.

Results Total RNA isolated from CNREB-1 (LXRα) expressing heart, kidney, liver, intestine, lung, and spleen was separated by electrophoresis on a 1.0% formaldehyde (formadehyde) / agarose gel. Ze with RNA
It was transferred to a taprobe membrane and hybridized with ³² P-labeled CNREB-1 (hereinafter, used in place of LXRα) (upper) and GAPDH (lower) cDNA. 1.7 kb LXRα c
Using DNA as a probe, Northern blot hybridization was performed to
The tissue distribution of XRα was confirmed. LXRα mRNA is up to 1.8 kb in size, expressed in heart, kidney, liver, intestine, lung, and spleen (FIG. 1A).

Based on its isolation via a yeast one-hybrid system that specifically binds to the putative mouse renin CNRE , Applicants have hypothesized that the putative mouse renin CNRE It was identified as a binding protein. To test this in more detail, a gel mobility shift assay was performed using the LXRα protein produced by an in vitro transcription / translation system, probed with ³² P-labeled mouse renin CNRE, The formation of a specific complex was revealed (FIG. 1B). The gel mobility assay uses 2.0 μl of LXRα produced by the in vitro transcription / translation system.
Performed using aliquots and ³² P-labeled mouse renin CNRE (2.5 × 10 ⁴ cpm). ^{The 32} P-labeled probe is shown at the top of the figure. The names and amounts of proteins added to each reaction are also shown at the top of the figure. The CNRE: LXRα complex was dramatically reduced in response to dose by the addition of the LXRα monoclonal antibody (a gift from Dr. Lehmann), but remained stable after the addition of 50 μg of bovine serum albumin. (FIG. 1B).

Previous studies have shown that human LXRα forms a heterodimer with RXRα (a universal partner of nuclear receptor heterodimers) and the sequence 5′-AGGTCANNNNAGGTCA-3 ′ (DR- 4) (Apfel R. et al., Mol. Cel
l Biol., 1994, 14: 7025-7035). ^{Using the 32} P-labeled DR4 element, Applicants have confirmed specific complex formation between DR4 and LXRα / RXRα heterodimers. However, neither LXRα nor RXRα alone bound specifically to DR4, whereas RXR
α is not considered necessary for LXRα to bind to the CNRE sequence.

In summary, mouse renin CNRE is a specific cis-DNA binding element of LXRα (CNREB-1). LXRα, a novel cAMP-responsive transcription factor, has been well-proven for cAMP to stimulate renin mRNA expression and renin secretion in the kidney. No LXRα expression was detected in As4.1 cells by either Northern or Western blot analysis. This observation indicates that 8-bromo-cA
Consistent with previous reports that MP or forskolin did not affect renin mRNA levels in these cells. Therefore, it was hypothesized that LXRα was not expressed in As4.1 cells because of lack of cAMP reactivity for renin gene expression in As4.1 cells. To test this hypothesis, Applicants constructed a constitutive transfection product of As4.1 cells expressing LXRα. That is, LXRα was inserted upstream of the internal ribosome insertion sequence (IRES) of encephalomyocarditis virus (EMCV) that activates the GFP gene of the retrovirus vector pMX (FIG. 2A). The transfected cells should encode a 4 kb transcript upon translation, which contains a chimeric LXRα / GFP mRNA (FIG. 2B) encoding two different proteins (LXRα and GFP). At the same time, GFP mock constitutively expressed As4.1 cells were generated and used as controls in this study. Transfected products were isolated by FACS.

Using Northern blot analysis, applicants determined that renin mRNA levels were LXRα As4.1.
In cells, it rises about 2-fold under basic conditions, and in response to 8-bromo-cAMP stimulation, an additional 2.
It was revealed that it was induced 5-fold (Fig. 3). 10 μg RNA (control GFP
-As4.1 constitutively expressed cells and LXRα-As4.1 constitutively expressed cells were isolated from those treated with cAMP for 0, 1, and 6 hours) and separated by electrophoresis on a 1.0% formaldehyde (formadehyde) / agarose gel. The RNA was transferred to a Zetaprobe membrane and hybridized with a mouse renin cDNA probe (top). RNA agarose gel stained with ethidium bromide is shown at the bottom. The time and concentration of cAMP stimulation are shown at the top of the figure. On the other hand, renin mR
NA levels were not changed by cAMP in control GFP-As4.1 cells. This is consistent with other reports that renin gene expression does not respond to 8-bromo-cAMP stimulation in As4.1 cells.

Taken together, the results suggest that mouse renin CNRE exerts an upregulating effect on mouse renin gene expression and functions as a novel cAMP response element.

LXRα is a Novel cAMP-Reactive Transcription Factor for Human Renin Gene Expression Applicants have determined by further studies whether human CNRE can also bind to LXRα. In this study, the applicant ³² P- labeled human renin CNRE as a probe, followed by gel mobility shift assay using the LXRα protein produced by transcription / translation in vitro. The results revealed the formation of a specific complex. The CNRE: LXRα complex was destroyed by anti-LXRα antibodies, but not by control mouse IgG (FIG. 4).

The applicant then asked whether the CNRE: LXRα interaction mediated the cAMP transfer of the human renin gene. Applicants filed a human renin gene promoter (nt-580 to nt + 16 bp) fused to a luciferase reporter gene (phRen580; Dr. Pin.
et a) were transfected into As4.1 cells with either a control vector (pcDNA3.1) or an LXRα expression vector. As4.1 cells were co-transfected with the luciferase-driven human renin gene promoter (phRen580) and either pcDNA3.1 or LXRα plasmid. 48 hours after transfection, cells were stimulated with 10 ⁻³ M 8-bromo-cAMP for 6 hours.
Luciferase activity is expressed in relative units with pcDNA3.1 without cAMP as 100%,
Transfection efficiencies were expressed as mean (± SD, n = 6) of assays normalized with β-gal activity as an internal standard. Applicants have demonstrated that LXRα stimulates the expression of the human renin gene promoter / luciferase chimeric expression vector with and without cAMP stimulation 1.6-fold and 3.0-fold, respectively (FIG. 5). This result indicates that LXRα transactivates the human renin gene upon cAMP stimulation.

Equivalents Those skilled in the art will recognize many equivalents to the specific embodiments of the invention described herein or will be able to ascertain using only routine experimentation. Such equivalents are intended to be included within the scope of the following claims. All references disclosed herein are incorporated by reference in their entirety.

[Sequence list]

[Brief description of the drawings]

FIG. 1A shows LXRα in various tissues from adult C57BL / 6J mice.
(CNREB-1) Northern blot hybridization analysis of mRNA transcript levels; FIG. 1B shows specific binding of LXRα (CNREB-1) to mouse renin CNRE.

FIG. 2 shows the construction of an LXRα stable transformant of As4.1 cells. FIG. 2A is a scheme diagram of the LXRα (CNREB-1) stable expression vector; FIG. 2B is a Northern blot analysis of LXRα in As4.1 stable cells.

FIG. 3 is a Northern blot showing that cAMP induces renin expression via CNREB-1.

FIG. 4 is a gel mobility shift assay showing specific binding of LXRα to human renin CNRE.

FIG. 5 is a bar graph showing that LXRα is involved in cAMP-stimulated transactivation of the cAMP-induced human renin gene.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61K 45/06 A61P 9/04 4C086 48/00 9/12 4H045 A61P 9/00 43/00 111/09/04 C07K 14/47 9/12 C12N 1/15 43/00 111 1/19 C07K 14/47 1/21 C12N 1/15 C12Q 1/02 1/19 1/68 A 1/21 G01N 33/15 Z 5 / 10 33/50 Z C12Q 1/02 C12N 15/00 ZNAA 1/68 A61K 37/02 G01N 33/15 37/48 33/50 C12N 5/00 A (72) Inventor Dazo, Victor Jay 02459 Massachusetts, USA , Newton, Dudley Road 110 (72) Inventor Koichi Tamura 20 Apartment, Brookline, Chapel Street, 02146, Massachusetts, USA Tobe-208 F-term (reference) 2G045 AA34 AA35 CB01 CB25 DA13 DA36 FB02 FB03 FB08 4B024 AA01 AA11 BA80 CA04 DA01 DA02 DA05 DA11 EA04 GA11 4B063 QA01 QA05 QA13 QA18 QQ20 QR55 QR80 QS34 AXA ABA AXA ABA XA ABA XA ABA XA ABA XA 4C084 AA02 AA13 AA17 AA20 BA44 CA25 DC01 MA01 MA02 NA05 NA14 ZA392 ZA421 ZA832 ZB261 ZC022 ZC202 ZC502 4C086 AA01 AA02 EA16 MA01 MA02 MA04 MA07 NA05 NA14 ZA42 ZB26 ZC02 ZC20 4H040 AA10A30A10A

Claims (98)

[Claims] 1. A nucleic acid of SEQ ID NO: 7; a nucleic acid of SEQ ID NO: 17; and SE
A nucleic acid molecule that hybridizes under stringent conditions to a molecule selected from the group consisting of nucleic acids of Q ID NO: 19, and that encodes a CNREB-2 polypeptide; (C) a nucleic acid molecule which differs from the nucleic acid molecule (a) or (b) in the codon sequence due to the degeneracy of the genetic code, d) An isolated nucleic acid molecule comprising: (a), (b), or the complement of (c). 2. The isolated nucleic acid molecule of claim 1, comprising SEQ ID NO: 17. 3. The nucleic acid molecule of SEQ ID NO: 17, or a fragment thereof.
4. The isolated nucleic acid molecule of claim 1. 4. A nucleic acid of SEQ ID NO: 7; a nucleic acid of SEQ ID NO: 17;
An isolated nucleic acid molecule selected from the group consisting of: a unique fragment of a nucleic acid molecule selected from the group consisting of nucleic acids of ID NO: 19; (b) the complement of (a); However, the unique fragment has the following sequence: (1) SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID
SEQ ID NO: 14, SEQ ID NO: 15 or SEQ ID NO: 16, (2) the complement of (1), (3) a fragment of (1) and (2) Such an isolated nucleic acid molecule comprising a sequence of adjacent nucleotides that are not identical to any selected sequence. 5. The sequence of adjacent nucleotides comprises: (1) at least two adjacent nucleotides that are non-identical to the sequence group; (2) at least three adjacent nucleotides that are non-identical to the sequence group; At least four contiguous nucleotides that are non-identical to the sequence group; (4) at least five contiguous nucleotides that are non-identical to the sequence group; (5) at least six contiguous nucleotides that are non-identical to the sequence group 5. The isolated nucleic acid molecule of claim 4, wherein the isolated nucleic acid molecule is selected from the group consisting of: nucleotides, (6) at least seven contiguous nucleotides that are non-identical to the sequence. 6. The fragment is at least 8 nucleotides, 10 nucleotides, 12 nucleotides, 14 nucleotides, 16 nucleotides, 18 nucleotides, 20 nucleotides, 22 nucleotides, 24 nucleotides, 26 nucleotides, 28 nucleotides, 30 nucleotides, 50 nucleotides, 75 nucleotides. 5. The isolated nucleic acid molecule of claim 4, having a size selected from the group consisting of: 100 nucleotides, and 200 nucleotides. 7. The isolated nucleic acid molecule of claim 4, wherein the molecule encodes an immunogenic polypeptide. 8. An expression vector comprising the isolated nucleic acid molecule of claim 1 operably linked to a promoter. 9. An expression vector comprising the isolated nucleic acid molecule of claim 4 operably linked to a promoter. 10. A host cell transformed or transfected with the expression vector of claim 8. 11. A host cell transformed or transfected with the expression vector of claim 9. 12. An isolated polypeptide encoded by an isolated nucleic acid molecule according to any one of claims 1 to 3, wherein the polypeptide, or a fragment of the polypeptide, has CNREB-2 activity. . 13. The isolated polypeptide of claim 12, wherein the isolated polypeptide is encoded by the isolated nucleic acid molecule of claim 2. 14. The isolated polypeptide comprises amino acids 1-4 of SEQ ID NO: 18.
14. The isolated polypeptide of claim 13, comprising a polypeptide having a sequence of 34. 15. An isolated polypeptide encoded by an isolated nucleic acid molecule according to any one of claims 1 to 3, wherein the polypeptide, or a fragment of the polypeptide, is immunogenic. 16. The fragment or fragment portion binds to a human antibody.
2. The polypeptide according to item 1. 17. An isolated binding polypeptide that selectively binds to a polypeptide encoded by the isolated nucleic acid molecule of any one of claims 1-3. 18. The isolated binding polypeptide of claim 17, which binds to a polypeptide having the amino acid sequence of SEQ ID NO: 18. 19. An antibody or an antibody fragment selected from the group consisting of an Fab fragment, an F (ab) ₂ fragment or a fragment containing a CDR region, which is selective for a polypeptide having the amino acid sequence of SEQ ID NO: 18. 19. The isolated binding polypeptide of claim 18, which is: 20. An isolated fragment comprising a fragment of the polypeptide of claim 12 that is long enough to represent a unique sequence in the human genome and identifying a polypeptide having CNREB-2 activity. Polypeptide, wherein said fragment is SE
Q ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ
Such an isolated polypeptide, except for the sequence of adjacent amino acids encoded by the isolated nucleic acid of ID NO: 15, or SEQ ID NO: 16. 21. (a) providing a nucleic acid molecule comprising SEQ ID NO: 17 or a fragment thereof of at least eight contiguous nucleotides; and (b) stringent screening using the nucleic acid molecule of (a) as a probe. Obtaining, under conditions, a nucleic acid molecule encoding a polypeptide that is a candidate having CNREB-2 activity, and (c) expressing the nucleic acid molecule isolated in (b) in a host cell to produce the polypeptide. And measuring the CNREB-2 activity of the expressed polypeptide,
A method of isolating a nucleic acid molecule encoding a polypeptide having CNREB-2 activity, wherein the presence of the activity is indicative of an isolated nucleic acid molecule encoding the polypeptide having CNREB-2 activity. 22. Contacting a mammalian cell in a cell with an amount of a CNREB-1 inhibitor effective to reduce renin expression in the mammalian cell, comprising: expressing renin in the mammalian cell expressing renin. Ways to reduce. 23. The method of claim 22, wherein said contacting a mammalian cell with a CNREB-1 inhibitor is performed in vitro. 24. The method of claim 22, wherein said contacting a mammalian cell with a CNREB-1 inhibitor is performed in vivo. 25. The method of claim 22, wherein the CNREB-1 inhibitor is a nucleic acid. 26. The nucleic acid of SEQ ID NO: 1, the nucleic acid of SEQ ID NO: 3, the nucleic acid of SEQ ID NO: 1
26. The method of claim 25, wherein the method is an antisense nucleic acid or a dominant negative nucleic acid of a CNREB-1 nucleic acid selected from the group consisting of a nucleic acid of NO: 5 and a nucleic acid of SEQ ID NO: 6. 27. The method of any one of claims 22 to 26, further comprising co-administering an effective amount of adenylate cyclase to reduce renin expression in mammalian cells. 28. A method of reducing CNREB-1 activity in a subject, comprising the steps of: providing a subject in need of such treatment with an effective amount of CNREB-1 in the subject to reduce CNREB-1 activity; The method comprising administering a -1 inhibitor. 29. The method of claim 28, wherein the amount is sufficient to reduce CNREB-1 activity to below normal basal levels. 30. The method of claim 28, wherein the subject has a reverse cardiovascular condition. 31. The method of claim 28, wherein the subject has hypertension. 32. The method of claim 28, wherein the subject has congestive heart failure. 33. The method of claim 28, wherein the CNREB-1 inhibitor is a nucleic acid. 34. The nucleic acid of SEQ ID NO: 1, the nucleic acid of SEQ ID NO: 3, the nucleic acid of SEQ ID NO: 3.
34. The method of claim 33, wherein the method is an antisense nucleic acid or a dominant negative nucleic acid of a CNREB-1 nucleic acid selected from the group consisting of a nucleic acid of NO: 5 and a nucleic acid of SEQ ID NO: 6. 35. The method of any one of claims 28 to 34, wherein the CNREB-1 inhibitor is administered rapidly. 36. The method according to any one of claims 28 to 34, wherein the CNREB-1 inhibitor is administered prophylactically. 37. An agent other than a CNREB-1 inhibitor selected from the group consisting of a diuretic, an anti-adrenergic agent, a vasodilator, a calcium influx blocker, and an angiotensin converting enzyme inhibitor, comprising the steps of: 29. The method of claim 28, further comprising co-administering an amount effective to reduce one activity. 38. The method of claim 28, further comprising co-administering an effective amount of adenylate cyclase to reduce CNREB-1 activity in the subject. 39. A method of increasing CNREB-1 activity in a mammalian cell, comprising contacting the mammalian cell with an amount of a CNREB-1 activator effective to increase CNREB-1 activity in the mammalian cell. The above method, comprising: 40. The method of claim 39, wherein the CNREB-1 activator is a nucleic acid. 41. The nucleic acid of SEQ ID NO: 1, the nucleic acid of SEQ ID NO: 3, the nucleic acid of SEQ ID NO: 1
42. The nucleic acid of NO: 5 and the nucleic acid of SEQ ID NO: 6 are selected from the group consisting of:
The method described in. 42. The method of claim 39, wherein the agent is a polypeptide. 43. The agent is a nucleic acid of SEQ ID NO: 1, a nucleic acid of SEQ ID NO: 3, a nucleic acid of SEQ ID NO: 3.
40. The method of claim 39, which is a polypeptide or a functional fragment or variant thereof, which is an expression product of a nucleic acid selected from the group consisting of a nucleic acid of O: 5 and a nucleic acid of SEQ ID NO: 6. 44. The method of claim 39, wherein contacting the mammalian cell with the CNREB-1 activator is performed in vitro. 45. The method of claim 39, wherein contacting the mammalian cell with the CNREB-1 activator is performed in vivo. 46. A method of increasing CNREB-1 activity in a subject, comprising the steps of: providing a subject in need of such treatment with an effective amount of CNREB-1 in the subject to increase CNREB-1 activity; The method comprising administering -1 activator. 47. The method of claim 46, wherein the CNREB-1 activator is a nucleic acid. 48. The nucleic acid of SEQ ID NO: 1, the nucleic acid of SEQ ID NO: 3, the nucleic acid of SEQ ID NO: 1
48. The nucleic acid of NO: 5 and the nucleic acid of SEQ ID NO: 6 are selected from the group consisting of:
The method described in. 49. The method of claim 46, wherein the CNREB-1 activator is a protein. 50. The CNREB-1 activator is selected from the group consisting of a nucleic acid of SEQ ID NO: 1, a nucleic acid of SEQ ID NO: 3, a nucleic acid of SEQ ID NO: 5, and a nucleic acid of SEQ ID NO: 6. 47. The method according to claim 46, which is a polypeptide or a functional fragment or variant thereof, which is an expression product of the nucleic acid to be produced. 51. The method of claim 46, wherein the amount is sufficient to increase CNREB-1 activity to above normal basal levels. 52. The method of claim 46, wherein the CNREB-1 activator is administered rapidly. 53. The method of claim 46, wherein the CNREB-1 activator is administered prophylactically. 54. A drug selected from the group consisting of flurocortisone, potassium tablets, vasopressin analogs, somatostatin analogs, β-blockers, sympathomimetics, dopamine antagonists, and venous constrictors, and adenylate cyclase activators. 47. The method of claim 46, further comprising co-administering an agent other than the CNREB-1 inhibitor. 55. A method comprising: (a) collecting a test sample from an individual; (b) measuring the expression level of CNREB-1 in the test sample; and (c) measuring the expression level of CNREB-1 and a control. In comparison, CNREB-
1. A method for determining a CNREB-1 expression level in a subject, comprising: determining the expression level. 56. The method according to claim 55, wherein the expression of CNREB-1 in (b) is CNREB-1 mRNA expression. 57. The method of claim 55, wherein the expression of CNREB-1 in (b) is CNREB-1 polypeptide expression. 58. The method of claim 55, wherein the increased level of CNREB-1 expression relative to a control is indicative of the subject's susceptibility to the occurrence of a renin-angiotensin system-mediated disorder. 59. The method of claim 58, wherein the renin-angiotensin system mediated disorder is hypertension. 60. The method of claim 58, wherein the renin-angiotensin system mediated disorder is a cardiovascular disorder. 61. A method for determining a subject's susceptibility to the occurrence of a renin-angiotensin system-mediated disorder, comprising: (a) characterizing a CNREB-1 nucleic acid sequence in a test sample taken from the subject; (B) comparing the CNREB-1 nucleic acid sequence of the test sample with the CNREB-1 nucleic acid sequence of the control sample, wherein the CNREB-1 nucleic acid sequence of the control sample is compared with the CNREB-1 nucleic acid sequence of the control sample. Such a method, wherein the observed mutation or match in the CNREB-1 nucleic acid sequence is indicative of the subject's susceptibility to the occurrence of a renin-angiotensin system-mediated disorder. 62. The CNREB-1 nucleic acid sequence in a test sample is compared to the CNR in a control sample.
62. The method of claim 61, wherein the observed mutation is apparent at first sight when compared to the EB-1 nucleic acid sequence. 63. The CNREB-1 nucleic acid sequence in a test sample is compared to the CNR in a control sample.
62. The method of claim 61, wherein the observed match is apparent at first glance when compared to the EB-1 nucleic acid sequence. 64. The method of claim 61, wherein the comparison is between CNREB-1 mRNA molecules. 65. The mutation in CNREB-1 mRNA is a nucleic acid of SEQ ID NO: 1, SEQ ID NO: 3.
The nucleic acid of SEQ ID NO: 5, and the nucleic acid of SEQ ID NO: 6 are detected by hybridization of mRNA from a test sample to a CNREB-1 nucleic acid selected from the group consisting of: the method of. 66. The method of claim 65, wherein the comparison is made between CNREB-1 cDNA sequences, and wherein said comparison is made by hybridization of a CNREB-1 cDNA probe to genomic DNA isolated from a test sample. 67. (a) subjecting the genomic DNA isolated from the test sample of the subject to Southern hybridization with a CNREB-1 cDNA probe; and (b) (i) applying CNREB to the test sample of the test sample. 67. The method of claim 66, further comprising: comparing hybridization of the -1 cDNA probe to (ii) hybridization of the CNREB-1 cDNA probe to a control sample. 68. The method according to claim 66, wherein the CNREB-1 cDNA probe detects a restriction fragment length polymorphism. 69. The method of claim 61, wherein the comparison is made between CNREB-1 nucleic acid sequences, and wherein the comparison is performed by determining the sequence of at least a portion of the CNREB-1 cDNA in the test sample using the polymerase chain reaction. Wherein the CNREB- determined from the wild-type CNREB-1 nucleic acid sequence shown in SEQ ID NO: 1.
1 The method, wherein the deviation in the cDNA is indicative of the susceptibility of the subject to the occurrence of a renin-angiotensin system-mediated disorder. 70. A CNREB-1 cDNA or CNR isolated from a molecule (a) test sample, wherein the mutations in the CNREB-1 nucleic acid sequence hybridize to each other to form a duplex.
62. The method of claim 61, wherein the mismatch is detected by identifying a mismatch between EB-1 mRNA and a molecule (b) a nucleic acid probe complementary to a human wild-type CNREB-1 nucleic acid sequence. 71. The method of claim 1, further comprising the steps of: (a) amplifying the CNREB-1 cDNA sequence in a test sample; (b) 63. The method of claim 61, wherein the amplified CNREB-1 cDNA sequence is detected by hybridizing with a nucleic acid probe comprising the CNREB-1 sequence. 72. A method for comparing CNREB-1 nucleic acid sequences, molecularly cloning the CNREB-1 gene in a test sample, and sequencing all or part of the cloned CNREB-1 gene. 62. The method of claim 61, wherein the mutation of the -1 nucleic acid sequence is detected. 73. The method of claim 61, wherein detecting a mutation in the CNREB-1 nucleic acid sequence comprises screening for a deletion mutation. 74. The method of claim 61, wherein detecting a mutation in the CNREB-1 nucleic acid sequence comprises screening for a point mutation. 75. The method of claim 61, wherein detecting a mutation in the CNREB-1 nucleic acid sequence comprises screening for insertional mutations. 76. The method of claim 61, wherein the test sample is a tissue. 77. The tissue may be brain, heart, breast, colon, bladder, uterus, prostate, stomach, testis, ovary, pancreas, pituitary, adrenal gland, thyroid, salivary gland, mammary gland, kidney, liver, small intestine, spleen, 77. A member selected from the group consisting of thymus, bone marrow, trachea, and lung.
The method described in. 78. The method of claim 61, wherein the test sample is a biological bodily fluid. 79. The biological fluid is amniotic fluid, aqueous humor, bile, blood, bronchoalveolar lavage, bronchial fluid, cerebrospinal fluid, follicular fluid, gingival crevicular fluid, middle ear fluid, peritoneal fluid, pleural fluid. 79. The method of claim 78, wherein the method is selected from the group consisting of, prostate fluid, saliva, semen, serum, sweat, synovial fluid, tears, culture supernatant, and urine. 80. A method of modulating c-myc expression in a cell, comprising: a cell that expresses c-myc, and an amount of c-myc effective to modulate c-myc expression in the cell. Contacting an agent that modulates CNREB-1 activity in the cell. 81. The method of claim 80, wherein said agent is a nucleic acid. 82. The method of claim 80, wherein said agent is a polypeptide. 83. contacting a cell expressing c-myc with an agent in vitro;
81. The method of claim 80. 84. The method of claim 80, wherein contacting the agent with cells expressing c-myc is performed in vivo. 85. A method of modulating the expression of type II collagen in a cell, comprising the steps of: expressing a cell expressing type II collagen; and an amount effective to modulate type II collagen expression in the cell. Contacting an agent that modulates CNREB-1 activity in the cell. 86. The method of claim 85, wherein said agent is a nucleic acid. 87. The method of claim 85, wherein said agent is a polypeptide. 88. The method according to claim 85, wherein the agent is contacted with cells expressing type II collagen in vitro. 89. The method of claim 85, wherein the contact of the agent with cells expressing type II collagen is performed in vivo. 90. A method of modulating the expression of a T cell receptor in a cell, comprising the steps of: expressing a cell that expresses a T cell receptor in an amount effective to modulate T cell expression in the cell. Contacting an agent that modulates CNREB-1 activity in a cell. Embodiment 91. The method of embodiment 90 wherein the agent is a nucleic acid. 92. The method of claim 90, wherein said agent is a polypeptide. 93. The method of claim 90, wherein the agent is contacted with cells expressing the T cell receptor in vitro. 94. The method of claim 90, wherein the contacting of the agent with cells expressing T cells is performed in vivo. 95. A pharmaceutically effective amount of CNREB-1 for inhibiting CNREB-1 activity
A pharmaceutical composition comprising: and a pharmaceutically acceptable carrier. 96. The pharmaceutical composition according to claim 95, further comprising a pharmaceutically effective amount of adenylate cyclase to inhibit CNREB-1 activity. 97. The pharmaceutical composition according to claim 95 or 96, wherein the CNREB-1 inhibitor is a nucleic acid. 98. The nucleic acid of SEQ ID NO: 1, the nucleic acid of SEQ ID NO: 3, the nucleic acid of SEQ ID NO: 1
100. The pharmaceutical composition according to claim 97, which is an antisense nucleic acid or a dominant negative nucleic acid of CNREB-1 nucleic acid selected from the group consisting of nucleic acid of NO: 5 and nucleic acid of SEQ ID NO :.