Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/575—Hormones
  • C07K14/58—Atrial natriuretic factor complex; Atriopeptin; Atrial natriuretic peptide [ANP]; Cardionatrin; Cardiodilatin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
  • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/156—Polymorphic or mutational markers
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/158—Expression markers
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
  • G01N2333/575—Hormones
  • G01N2333/58—Atrial natriuretic factor complex; Atriopeptin; Atrial natriuretic peptide [ANP]; Brain natriuretic peptide [BNP, proBNP]; Cardionatrin; Cardiodilatin
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2800/00—Detection or diagnosis of diseases
  • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

• the present invention is related to novel nucleotide and protein sequences that are variants of BNP, and assays and methods of use thereof.
• ECM extracellular matrix
• Such factors may include early inflammatory actions, a local increase in fibroblast cell populations, modulation of the synthetic function of f ⁇ broblasts, and altered regulation of the biosynthesis and degradation of collagen.
• the pathophysiological response to the tissue trauma seen in fibrosis results in the formation of abnormal tissues which do not duplicate the functionality of the original organ tissue, so that the repair of tissue trauma does not lead to a complete restoration of organ capacity and function.
• a f ⁇ brotic process which results from pathophysiological responses to tissue trauma is cardiac fibrosis.
• Cardiac fibrosis has a number of causes, which lead to the deposition of fibrotic tissue.
• cardiac fibrosis may result from heart failure, hypertension and other cardiac pathological/disease states.
• fibrotc tissue in the heart is characterized by the deposition of abnormally large amounts of extracellular matrix components, including collagen, as well as other matrix proteins. Therefore, the cardiac fibrotic process needs to be inhibited in order to prevent damage to the cardiac tissue and hence to the ability of the heart to function.
• Cardiac fibroblasts are important to the cardiac fibrotic process because they produce interstitial proteins and other myocardial components which have been implicated in heart failure (Hess et al, Circ, 63:360-371 (1981); Villari et al, Am J.
• Cardiac surgery also may cause cardiac fibrosis; such fibrosis may also lead to the requirement for an additional operation, which is often associated with higher morbidity and mortality. Detection and/or quantitation of cardiac fibrosis is therefore very important for preventing or treating such fibrosis.
• BNP Brain Natriuretic Peptide
• BNP and its related natriuretic peptide ANP contain a 17- amino acid ring structure and are produced by the cardiac atria in response to volume overload and by ventricles in response to pressure overload, respectively (Broomsma et al, 2001.
• BNP was an attractive target for development as a therapeutic agent for heart failure and/or as a diagnostic marker. Both ANP and BNP are formed as pre-pro-polypeptides. Human BNP is derived from the 134- aa precursor preproBNP.
• a 26-aa signal peptide sequence is cleaved from the N-terminus of preproBNP.
• the remaining proBNPl- 108 prohormone is further cleaved by corin, a membrane -bound serine protease, into an N-terminal pro-BNPl-76 fragment and the active 32-peptide , C -terminal proBNP77-108 hormone termed BNP (Azzay et al, 2003. Heart Failure Review 8, 315-320.).
• BNP C -terminal proBNP77-108 hormone
• BNP has the important ability to decrease left ventricular filling pressures without a resultant reflex tachycardia, reflex vasoconstriction, and further activation of vasoconstricting neurohumoral systems. BNP also has lusiotropic effects and has been demonstrated to inhibit cardiac fibrosis.
• natriuretic peptides also appear to exhibit an antimitogenic effect in the heart and other organ systems, suggesting a potential role in the modulation of cell growth. Additional evidence suggests a direct vasodilatory effect on the coronary arteries with a reduction in myocardial oxygen consumption. In addition to these cardiac and vascular properties, BNP has a direct effect on renal hemodynamics and function. Increased glomerular filtration is the result of an unbalanced vasodilatation of the afferent arterioles and vasoconstriction of the efferent arterioles.
• the A-type natriuretic peptide receptor is expressed in a variety of tissues, including kidney, blood vessels, adrenal glands, heart, lungs, adipose tissue, eye, pregnant uterus and placenta. Clearance of ANP and BNP from the blood is effected in two ways: through a special clearance receptor, the C-type natriuretic receptor, and through enzymatic degradation by neutral endopeptidases (Broomsma et al, 2001. Cardiovascular Research 51, 442-449).
• the natriuretic peptides are characterized by a 17 aa central ring structure, which is formed by a disulfide bridge and is suggested to be necessary for the binding of these peptides to their respective receptors and for their biological activity (Azzay et al, 2003. Heart Failure Review 8, 315-320.).
• ANP and BNP are both expressed more in Atria than Ventrcles.
• BNP has a more favorable expression in ventricles (atria:ventricle expression ration 3:1 compared with 40:1 to ANP). In a situation of a failing heart both BNP and ANP expression is increased 100- fold above normal levels (Trends Endocrinol Metab. 2003 Nov;14(9):411-6).
• BNP rise is often larger and more rapid than ANP, and it emerged as a superior marker for heart failure and left- ventricular dysfunction (Azzay et al, 2003. Heart Failure Review 8, 315- 320.).
• BNP was shown to have a diagnostic benefit for few different clinical purposes: 1. Elevated plasma levels of BNP are found in conditions of increased cardiac wall stress. In congestive heart failure (CHF), circulating concentrations of BNP are clearly elevated and this elevation reflects the severity of the condition. Therefore it can be used to establish prognosis in patients with heart failure. In addition, it provides a too 1 to monitor changes in the severity of failure without using more sophisticated diagnostic modalities like Echo imaging. 2.
• CHF congestive heart failure
• natriuretic peptides are excellent prognostic indicators for survival in heart failure. 3. Detection of response to treatment : a continued increase in plasma concentration would be indicative of unsuccessful, and a decrease of successful, treatment. 4. In myocardial infarction ANP and BNP concentrations are also elevated, and are of prognostb value for indicating patients most at risk. 5. BNP measurements help to differentiate between cardiac versus non-cardiac causes of dyspnea (Lancet. 1994 Feb 19;343(8895):440-4). However there is limited evidence that it can be a useful marker for the very early detection of cardiac damage or heart failure when patients are still asymptomatic. The N-terminal proBNP (ntBNP) is more stable and its serum levels rise more then
• BNP therefore it is theoretically a better marker for BNP overproduction than BNP itself. Yet, its added value over BNP diagnostic wise is minor.
• LVEF left ventricular ejection fraction
• ntBNP showed no difference in the diagnostic properties.
• ntBNP showed slight improvement over BNP — receiver operating characteristics area under curve of 0.82 for ntBNP and 0.794 for BNP (Eur J Heart Fail. 2004 Mar 15;6(3):295-300).
• SUMMARY OF THE INVENTION The background art does not teach or suggest va riants of BNP. The background art also does not teach or suggest variants of BNP that are useful as diagnostic markers.
• the background art also does not teach or suggest variants of BNP that are useful as diagnostic markers for cardiac diseases and/or pathology.
• the present invention overcomes these deficiencies of the background art by providing BNP variants, which may optionally be used as diagnostic markers.
• BNP variants are useful as diagnostic markers for cardiac diseases and/or pathology, including but not limited to for heart failure and left verntricular disfunction.
• cardiac disease and/or pathology and/or condition and/or disorder may comprise one or more of Myocardial infarct, acute coronary syndrome, angina pectoris (stable and unstable), cardiomyopathy, myocarditis, congestive heart failure or any type of heart failure, the detection of reinfarction, the detection of success of thrombolytic therapy after Myocardial infarct, Myocardial infarct after surgery, assessing the size of infarct in Myocardial infarct, the differential diagnosis of heart related conditions from lung related conditions (as pulmonary embolism), the differential diagnosis of Dyspnea, and cardiac valve related conditions.
• cardiac disease includes any type of cardiac pathology and/or disorder and/or damage, including both chronic and acute damage, as well as progression from acute to chronic damage of the heart, and also propagation of one acute event to another acute event.
• An example of the latter may occur when an infarct is followed by another infarct in a relatively short period of time, such as within 24 hours for example.
• An infarct may also lead to acute heart failure immediately after the infarct, as another example.
• These non- limiting examples are intended to demonstrate that cardiac disease may also comprise a plurality of acute events.
• These variant markers may be described as "BNP variant disease markers”. According to one embodiment of the present invention markers are specifically released to the bloodstream under disease conditions according to one of the above differential variant marker conditions.
• the variant marker is detected in a biological sample which may optionally be taken from a subject (patient).
• suitable biological samples include but are not limited to blood, serum, plasma, blood cells, urine, sputum, saliva, stool, spinal fluid, lymph fluid, the external secretions of the skin, respiratory, intestinal, and genitourinary tracts, tears, milk, neuronal tissue, and any human organ or tissue.
• the biological sample comprises cardiac tissue and/or a semm sample and/or a urine sample and/or any other tissue or liquid sample.
• the sample can optionally be diluted with a suitable eluant before contacting the sample to the antibody, if an antibody is used.
• tmhmm from Center for Biological Sequence Analysis, Technical University of Denmark DTU, http://www.cbs.dtu.dk/services/TMHMM/TMHMM2.0b.guide.php
• tmpred from EMBnet, maintained by the ISREC Bionformatics group and the LICR Infonnation Technology Office, Ludwig Institute for Cancer Research, Swiss Institute of Bioinfon ⁇ atics, http://www.ch.embnet.org/software/TMPRED_form.html
• signalp_hmm and signalpjm both from Center for Biological Sequence Analysis, Technical University of Denmark DTU, http://www.cbs.dtu.dk/services/SignalP/background/prediction.php
• signalp_hmm and signalp_nn refer to two modes of operation for the program SignalP: hmm refers to Hidden Markov Model, while nn refers to neural networks. Localization was also determined through manual inspection of known protein localization and/or gene structure, and the use of heuristics by the individual inventor. In some cases for the manual inspection of cellular localization prediction inventors used the ProLoc computational platform [Einat Hazkani-Covo, Erez Levanon, Galit Rotman, Dan Graur and Amit Novik; (2004) Evolution of multicellularity in metazoa: comparative analysis of the subcellular localization of proteins in Saccharomyces, Drosophila and Caenorhabditis.
• protein domains e.g., prediction of trans- membranous regions and localization thereof within the protein
• pi protein length
• amino acid composition homology to pre-annotated proteins
• recognition of sequence patterns which direct the protein to a certain organelle such as, nuclear localization signal, NLS, mitochondria localization signal
• signal peptide and anchor modeling and using unique domains from Pfam that are specific to a single compartment.
• T - > C means that the SNP results in a change at the position given in the table from T to C.
• M - > Q means that the SNP has caused a change in the corresponding amino acid sequence, from methionine (M) to glutamine (Q). If, in place of a letter at the right hand side for the nucleotide sequence SNP, there is a space, it indicates that a frames ift has occurred. A frameshift may also be indicated with a hyphen (-). A stop codon is indicated with an asterisk at the right hand side (*).
• a comment may be found in parentheses after the above description of the SNP itself.
• This comment may include an FTId, which is an identifier to a SwissProt entry that was created with the indicated SNP.
• An FTId is a unique and stable feature identifier, which allows to construct links directly from position- specific annotation in the feature table to specialized protein- related databases.
• the header of the first column is "SNP position(s) on amino acid sequence", representing a position of a known mutation on amino acid sequence.
• SNPs may optionally be used as diagnostic markers according to the present invention, alone or in combination with one or more other SNPs and/or any other diagnostic marker.
• Prefened embodiments of the present invention comprise such SNPs, including but not limited to novel SNPs on the known (WT or wild type) protein sequences given below, as well as novel nucleic acid and/or amino acid sequences formed through such SNPs, and/or any SNP on a variant amino acid and/or nucleic acid sequence described herein.
• the below list relates to abbreviations on the histogram showing EST expression of this cluster (tissues not shown had neglible or no expression).
• E-PAN endocrinejpancreas
• E-PT endocrine jarathyroid thyroid
• GI gastrointestinal tract
• HN head and neck
• LN lymph node
• PNS peripheral nervous system
• TCELL immune T cells
• THYM thymus
• oligonucleotides which are embodiments of the present invention, for example as amplicons, hybridization units and/or from which primers and/or complementary oligonucleotides may optionally be derived, and/or for any other use.
• cardiac disease includes any type of cardiac pathology and/or disorder and/or damage, including both chronic and acute damage, as well as progression from acute to chronic damage of the heart, and also propagation of one acute event to another acute event. An example of the latter may occur when an infarct is followed by another infarct in a relatively short period of time, such as within 24 hours for example .
• an infarct may also lead to acute heart failure immediately after the infarct, as another example.
• cardiac disease may also comprise a plurality of acute events.
• the term "marker” in the context of the present invention refers to a nucleic acid fragment, a peptide, or a polypeptide, which is differentially present in a sample taken from patients having a cardiac disease, such as acute cardiac damage for example, as compared to a comparable sample taken from subjects who do not have cardiac disease.
• the phrase "differentially present” refers to differences in the quantity of a marker present in a sample taken from patients having cardiac disease as compared to a comparable sample taken from patients who do not have cardiac disease.
• a nucleic acid fragment may optionally be differentially present between the two samples if the amount of the nucleic acid fragment in one sample is significantly different from the amount of the nucleic acid fragment in the other sample, for example as measured by hybridization and/or NAT-based assays.
• a polypeptide is differentially present between the two samples if the amount of the polypeptide in one sample is significantly different from the amount of the polypeptide in the other sample. It should be noted that if the marker is detectable in one sample and not detectable in the other, then such a marker can be considered to be differentially present.
• a marker such as a protein or fragment thereof
• a marker could optionally be present in a blood sample from the patient, indicating the presence of damage; lack of presence of such a marker (and/or presence at a low level) would therefore optionally and preferably indicate a lack of such damage.
• chronically damaged heart might cause a low level of the marker to be present in the blood sample, while acute damage would cause a high level to be present.
• One of ordinary skill in the art could easily determine such relative levels of the markers; further guidance is provided in the description of each individual marker below.
• diagnostic means identifying the presence or nature of a pathologic condition. Diagnostic methods differ in their sensitivity and specificity.
• the "sensitivity” of a diagnostic assay is the percentage of diseased individuals who test positive (percent of "true positives”). Diseased individuals not detected by the assay are “false negatives.” Subjects who are not diseased and who test negative in the assay are termed “tme negatives.”
• the "specificity” of a diagnostic assay is 1 minus the false positive rate, where the "false positive” rate is defined as the proportion ofthose without the disease who test positive. While a particular diagnostic method may not provide a definitive diagnosis of a condition, it suffices if the method provides a positive indication that aids in diagnosis.
• Diagnosing refers to classifying a disease or a symptom, determining a severity of the disease, monitoring disease progression, forecasting an outcome of a disease and/or prospects of recovery.
• the term “detecting” may also optionally encompass any of the above. Diagnosis of a disease according to the present invention can be effected by determining a level of a polynucleotide or a polypeptide of the present invention in a biological sample obtained from the subject, wherein the level determined can be conelated with predisposition to, or presence or absence of the disease.
• a "biological sample obtained from the subject” may also optionally comprise a sample that has not been physically removed from the subject, as described in greater detai below.
• the term "level” refers to expression levels of RNA and/or protein or to DNA copy number of a marker of the present invention.
• the level of the marker in a biological sample obtained from the subject is different (i.e., increased or decreased) from the level of the same variant in a similar sample obtained from a healthy individual (examples of biological samples are described herein).
• Numerous well known tissue or fluid collection methods can be utilized to collect the biological sample from the subject in order to determine the level of DNA, RNA and/or polypeptide of the variant of interest in the subject. Examples include, but are not limited to, fine needle biopsy, needle biopsy, core needle biopsy and surgical biopsy (e.g., brain biopsy), and lavage.
• a "test amount" of a marker refers to an amount of a marker present in a sample being tested.
• a test amount can be either in absolute amount (e.g., microgram/ml) or a relative amount (e.g., relative intensity of signals).
• a "test amount” of a marker refers to an amount of a marker in a subject's sample that is consistent with a diagnosis of cardiac disease.
• a test amount can be either in absolute amount (e.g., microgram/ml) or a relative amount (e.g., relative intensity of signals).
• a "control amount" of a marker can be any amount or a range of amounts to be compared against a test amount of a marker.
• a control amount of a marker can be the amount of a marker in a patient with cardiac disease or a person without cardiac disease.
• a control amount can be either in absolute amount (e.g., microgram/ml) or a relative amount (e.g., relative intensity of signals).
• Detect refers to identifying the presence, absence or amount of the object to be detected.
• a “label” includes any moiety or item detectable by spectroscopic, photo chemical, biochemical, immunochemical, or chemicalmeans.
• useful labels include 32P, 35S, fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonly used in an ELISA), biotin-streptavadin, dioxigenin, haptens and proteins for which antisera or monoclonal antibodies are available, or nucleic acid molecules with a sequence complementary to a target.
• the label often generates a measurable signal, such as a radioactive, chromogenic, or fluorescent signal, that can be used to quantify the amount of bound label in a sample.
• the label can be incorporated in or attached to a primer or probe either covalently, or through ionic, van der Waals or hydrogen bonds, e.g., incorporation of radioactive nucleotides, or biotinylated nucleotides that are recognized by streptavadin.
• the label may be directly or indirectly detectable. Indirect detection can involve the binding of a second label to the first label, directly or indirectly.
• the label can be the ligand of a binding partner, such as biotin, which is a binding partner for streptavadin, or a nucleotide sequence, which is the binding partner for a complementary sequence, to which it can specifically hybridize.
• the binding partner may itself be directly detectable, for example, an antibody may be itself labeled with a fluorescent molecule.
• the binding partner also may be indirectly detectable, for example, a nucleic acid having a complementary nucleotide sequence can be a part of a branched DNA molecule that is in turn detectable through hybridization with other labeled nucleic acid molecules (see, e.g, P. D. Fahrlander and A. Klausner, Bio/Technology 6:1165 (1988)). Quantitation of the signal is achieved by, e.g, scintillation counting, densitometry, or flow cytometry.
• Exemplary detectable labels include but are not limited to magnetic beads, fluorescent dyes, radiolabels, enzymes (e.g, horse radish peroxide, alkaline phosphatase and others commonly used in an ELISA), and calorimetric labels such as colloidal gold or colored glass or plastic beads.
• the marker in the sample can be detected using an indirect assay, wherein, for example, a second, labeled antibody is used to detect bound marker-specific antibody, and/or in a competition or inhibition assay wherein, for example, a monoclonal antibody which binds to a distinct epitope of the marker are incubated simultaneously with the mixture.
• Immunoassay is an assay that uses an antibody to specifically bind an antigen.
• the immunoassay is characterized by the use of specific binding properties of a particular antibody to isolate, target, and/or quantify the antigen.
• the specified antibodies bind to a particular protein at least two times greater than the background (non-specific signal) and do not substantially bind in a significant amount to other proteins present in the sample.
• Specific binding to an antibody under such conditions may require an antibody that is selected for its specificity for a particular protein.
• polyclonal antibodies raised to seminal basic protein from specific species such as rat, mouse, or human can be selected to obtain only those polyclonal antibodies that are specifically immunoreactive with seminal basic protein and not with other proteins, except for polymorphic variants and alleles of seminal basic protein. This selection may be achieved by subtracting out antibodies that cross-react with seminal basic protein molecules from other species.
• a variety of iimnunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein.
• solid -phase ELISA immunoassays are routinely used to select antibodies specifically immunoreactive with a protein (see, e.g, Harlow & Lane, Antibodies, A Laboratory Manual (1988), for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity).
• a specific or selective reaction will be at least twice background signal or noise and more typically more than 10 to 100 times background.
• any of the above nucleic acid and/or amino acid sequences further comprises any sequence having at least about 70%, preferably at least about 80%, more preferably at least about 90%, most preferably at least about 95% homology thereto.
• All nucleic acid sequences and/or amino acid sequences shown herein as embodiments of the present invention relate to their isolated form, as isolated polynucleotides (including for all transcripts), oligonucleotides (including for all segments, amplicons and primers), peptides (including for all tails, bridges, insertions or heads, optionally including other antibody epitopes as described herein) and/or polypeptides
• an isolated polynucleotide comprising a nucleic acid sequence selected from the group consisting of: HUMNATPEP_PEA_1_T1, HUMNATPEP_PEA_1_T2, HUMNATPEP_PEA_1_T3 or HUMNATPEP_PEA_1_T4.
• an isolated polynucleotide segment comprising a nucleic acid sequence selected from the group consisting of: HUMNATPEP_PEA_l_node _0, HUMNATPEP_PEA_l_node_l, HUMNATPEP_PEA_l_node_2, HUMNATPEP_PEA_l_node_3,
• an isolated polypeptide comprising an amino acid sequence selected from the group consisting of: HUMNATPEP PEA 1 P2, HUMNATPEP PEA 1 P3 or HUMNATPEP PEA 1 P7.
• an isolated chimeric polypeptide encoding for HUMNATPEP_PE _1_P2 comprising a first amino acid sequence being at least 90 % homologous to
• an isolated polypeptide encoding for a tail of HUMNATPEP_PEA_1_P2 comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence GKHPLPPRPPSPIPVCDTVRVTLGFWSGN TL in HUMNATPEP_PEA_1_P2.
• an isolated chimeric polypeptide encoding for HUMNATPEP_PEA_1_P3 comprising a first amino acid sequence being at least 90 % homologous to
• HUMNATPEP_PEA_1_P3 • According to prefened embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for HUMNATPEP_PEA_1JP7, comprising a first amino acid sequence being at least 90 % homologous to
• MVLYTLRAPRSPKMVQGSGCFGRKMDRISSSSGLGCKVLRRH conesponding to amino acids 93 - 134 of ANFB_HUMAN, which also conesponds to amino acids 1 - 42 of
• HUMNATPEPJPEA _1_P7 there is provided an antibody capable of specifically binding to an epitope of an amino acid sequence as described herein.
• the antibody is capable of differentiating between a splice variant having said epitope and a conesponding known protein.
• a kit for detecting heart disorders comprising a kit detecting overexpression of a splice variant as described herein.
• the kit comprises a NAT-based technology.
• the kit further comprises at least one primer pair capable of selectively hybridizing to a nucleic acid sequence as described herein.
• the kit further comprises at least one oligonucleotide capable of selectively hybridizing to a nucleic acid sequence as described herein.
• the kit comprises an antibody as described herein.
• the kit further comprises at least one reagent for performing an ELISA or a Western blot. According to prefened embodiments of the present invention, there is provided a method for detecting heart disorders, comprising detecting overexpression of a splice variant according to any of the above claims.
• detecting overexpression is performed with a NAT-based technology.
• detecting overexpression is performed with an immunoassay.
• the immunoassay comprises an antibody as described herein.
• a biomarker capable of detecting a BNP variant-detectable disease comprising any of the above nucleic acid sequences or a fragment thereof, or any of the above amino acid sequences or a fragment thereof.
• a metiiod for screening for variant-detectable disease comprising detecting cells affected by a BNP variant- detectable disease with a biomarker or an antibody or a method or assay as described herein.
• a method for diagnosing a BNP variant detectable disease comprising detecting cells affected by a BNP variant- detectable disease with a biomarker or an antibody or a method or assay as described herein.
• a method for monitoring disease progression and/or treatment efficacy and/or detection of acute over chronic exacerbation of a BNP variant-detectable disease comprising detecting cells affected by a BNP variant-detectable disease with a biomarker or an antibody or a method or assay as described herein.
• a method of selecting a therapy for a BNP variant-detectable disease comprising detecting cells affected by a BNP variant-detectable disease with a biomarker or an antibody or a method or assay as described herein and selecting a therapy according to said detection.
• the BNP variant-detectable disease comprises heart failure and/or left ventricular disfunction.
• a nucleic acid constmct comprising the isolated polynucleotide as described herein.
• the nucleic acid constmct further comprises a promoter for regulating transcription of the isolated polynucleotide in sense or antisense orientation.
• the nucleic acid constmct further comprises a positive and a negative selection marker for selecting for homologous recombination events.
• a host cell comprising the nucleic acid construct as described herein.
• an isolated polypeptide comprising an amino acid sequence at least 70 % identical to a polypeptide as described herein, as determined using the LALIGN software of EMBnet Switzerland (http://www.ch.embnet.org/index.html) using default parameters or an active portion thereof.
• an oligonucleotide specifically hybridizable with a nucleic acid sequence encoding a polypeptide as described herein.
• a pharmaceutical composition comprising a therapeutically effective amount of a polypeptide as described herein and a pharmaceutically acceptable canier or diluent.
• a method of treating BNP- related disease in a subject comprising upregulating in the subject expression of a polypeptide as described herein, thereby treating the BNP- related disease in a subject.
• upregulating expression of said polypeptide is effected by: (i) administering said polypeptide to the subject; and/or (ii) administering an expressible polynucleotide encoding said polypeptide to the subject.
• an isolated oligonucleotide comprising an amplicon selected from the group consisting of SEQ ID NOs: 20, 23 or 26.
• a primer pair comprising a pair of isolated oligonucleotides capable of amplifying an amplicon or segment as described herein.
• the primer pair comprises a pair of isolated oligonucleotides selected from the group consisting of: SEQ NOs 18 and 19; 21 and 22; 24 and 25; or 27 and 28.
• SEQ NOs 18 and 19 are isolated oligonucleotides selected from the group consisting of: SEQ NOs 18 and 19; 21 and 22; 24 and 25; or 27 and 28.
• SEQ NOs 18 and 19 are isolated oligonucleotides selected from the group consisting of: SEQ NOs 18 and 19; 21 and 22; 24 and 25; or 27 and 28.
• Figure 1 shows a schematic summary of quantitative real-time PCR analysis.
• Figure 2 shows expression of ESTs in each category, as "parts per million”.
• Figure 3 A shows a comparison of the genomic stmcture for the variant transcript HUMNATPEP_PEA_1_T1 and the known or "WT" transcript.
• Figure 3B shows a comparison of the stmcture of the variant protein HUMNATPEP_PEA_1_P2 in comparison to the stmcture of the known or "WT” protein.
• Figure 4 shows the expression of ANFB_HUMAN Natriuretic peptide transcripts detectable by HUMNATPEP seg5 amplicon in heart tissue samples as opposed to other tissues.
• Figure 5 is a histogram showing specific expression of HUMNATPEP seg2 transcripts in heart tissue samples as opposed to other tissues.
• Figure 6 is a histogram showing relative expression of the above- indicated Homo sapiens natriuretic peptide precursor B (NPPB) known protein transcripts in heart tissue samples as opposed to other tissues.
• Figure 7 presents RT_PCR results of known BNP transcript and the
• HUMNATPEP_PEA_1_T1 splice variant as described above.
• the expression of known BNP transcript was found to occur in normal heart tissue, while no expression of HUMNATPEP_PEA_1_T1 variant was detected in this tissue panel.
• Specific expression of the HUMNATPEP_PEA_1_T1 variant in focal fibrosis heart tissue was demonstrated.
• N means normal heart tissue;
• F means fibrotic heart tissue;
• Neg means negative control, without reverse transcriptase.
• DESCRIPTION OF PREFERRED EMBODIMENTS The present invention overcomes these deficiencies of the background art by providing BNP variants, which may optionally be used as diagnostic markers.
• these BNP variants are useful as diagnostic markers for cardiac diseases and/or pathology, including but not limited to heart failure and left ventricular disfimction.
• the variants of the present invention may optionally be used, additionally or alternatively, for therapeutic uses, including but not limited to, diuretic, natriuretic, vascular smooth muscle relaxing and vasodilation actions, and lowering blood volume and blood pressure. These variants may optionally be used for therapeutic treatment of heart failure.
• the present invention is of novel markers for cardiac disease that are both sensitive and accurate.
• Biomo lecular sequences (amino acid and/or nucleic acid sequences) uncovered using the methodology of the present invention and described herein can be efficiently utilized as tissue or pathological markers and/or as drugs or drug targets for treating or preventing a disease. These markers are specifically released to the bloodstream under conditions of cardiac disease and/or cardiac pathology, including but not limited to cardiac damage, and/or are otherwise expressed at a much higher level and/or specifically expressed in heart.
• the method of the present invention identifies clusters (genes) which are characterized in that the transcripts are differentially expressed in heart muscle tissue compared with other normal tissues, preferably in comparison to skeletal muscle tissue.
• heart muscle tissue differentially expressed proteins are potential acute heart damage markers. Leakage of intracellular content can also occur in chronic damage to the heart muscle, therefore proteins selected according to this method are potential markers for chronic heart conditions.
• proteins selected according to this method are potential markers for chronic heart conditions.
• BNP brain natriuretic peptide
• ANF atrial natriuretic factor
• BNP and ANF are not only differentially expressed in heart tissue, they are also overexpressed dramatically (hundreds of times greater expression) when heart failure occurs.
• Other heart specific secreted proteins might present similar overexpression in chronic damage.
• the markers described herein are overexpressed in heart as opposed to muscle, as described in greater detail below.
• the measurement of these markers, alone or in combination, in patient samples provides information that the diagnostician can conelate with a probable diagnosis of cardiac disease and /or cardiac pathology, including but not limited to cardiac damage.
• the present invention therefore also relates to diagnostic assays for cardiac disease and/or cardiac pathology, including but not limited to cardiac damage, and methods of use of such markers for detection of cardiac disease and/or cardiac pathology, including but not limited to cardiac damage (alone or in combination), optionally and preferably in a sample taken from a subject (patient), which is more preferably some type of blood sample.
• the present invention therefore also relates to diagnostic assays for cardiac disease and/or cardiac pathology, including but not limited to cardiac damage, and methods of use of such markers for detection of cardiac disease and/or cardiac pathology, including but not limited to cardiac damage (alone or in combination), optionally and preferably in a sample taken from a subject (patient), which is more preferably some type of blood sample.
• the present invention relates to bridges, tails, heads and/or insertions, and/or analogs, homologs and derivatives of such peptides. Such bridges, tails, heads and/or insertions are described in greater detail below with regard to the Examples.
• a "tail” refers to a peptide sequence at the end of an amino acid sequence that is unique to a splice variant according to the present invention. Therefore, a splice variant having such a tail may optionally be considered as a chimera, in that at least a first portion of the splice variant is typically highly homologous (often 100% identical) to a portion of the conesponding known protein, while at least a second portion of the variant comprises the tail.
• a “head” refers to a peptide sequence at the beginning of an amino acid sequence that is unique to a splice variant according to the present invention.
• a splice variant having such a head may optionally be considered as a chimera, in that at least a first portion of the splice variant comprises the head, while at least a second portion is typically highly homologous (often 100% identical) to a portion of the conesponding known protein.
• an edge portion refers to a connection between two portions of a splice variant according to the present invention that were not joined in the wild type or known protein.
• An edge may optionally arise due to a join between the above "known protein" portion of a variant and the tail, for example, and/or may occur if an internal portion of the wild type sequence is no longer present, such that two portions of the sequence are now contiguous in the splice variant that were not contiguous in the known protein.
• a "bridge” may optionally be an edge portion as described above, but may also include a join between a head and a "known protein” portion of a variant, or a join between a tail and a "known protein” portion of a variant, or a join between an insertion and a "known protein” portion of a variant.
• a bridge between a tail or a head or a unique insertion, and a "known protein" portion of a variant comprises at least about 10 amino acids, more preferably at least about 20 amino acids, most preferably at least about 30 amino acids, and even more preferably at least about 40 amino acids, in which at least one amino acid is from the tail/head/insertion and at least one amino acid is from the "known protein" portion of a variant.
• the bridge may comprise any number of amino acids from about 10 to about 40 amino acids (for example, 10, 11, 12, 13...37, 38, 39, 40 amino acids in length, or any number in between).
• bridges cannot be extended beyond the length of the sequence in either direction, and it should be assumed that every bridge description is to be read in such manner that the bridge length does not extend beyond the sequence itself. Furthermore, bridges are described with regard to a sliding window in certain contexts below.
• a bridge between two edges may optionally be described as follows: a bridge portion of CONTIG- NAME_P1 (representing the name of the protein), comprising a polypeptide having a length "n", wherein n is at least about 10 amino acids in length, optionally at least about 20 amino acids in length, preferably at least about 30 amino acids in length, more preferably at least about 40 amino acids in length and most preferably at least about 50 amino acids in length, wherein at least two amino acids comprise XX (2 amino acids in the center of the bridge, one from each end of the edge), having a structure as follows (numbering according to the sequence of C0NTIG-NAME_P1) : a sequence starting from any of amino acid numbers 49- x to 49 (for example); and ending at any of amino acid numbers 50 + ((n-2) - x) (for example), in which x varies from 0 to n2.
• this invention provides antibodies specifically recognizing the splice variants and polypeptide fragments thereof of this invention. Preferably such antibodies differentially recognize splice variants of the present invention but do not recognize a conesponding known protein (such known proteins are discussed with regard to their splice variants in the Examples below).
• this invention provides an isolated nucleic acid molecule encoding for a splice variant according to the present invention, having a nucleotide sequence as set forth in any one of the sequences listed herein, or a sequence complementary thereto.
• this invention provides an isolated nucleic acid molecule, having a nucleotide sequence as set forth in any one of the sequences listed herein, or a sequence complementary thereto.
• this invention provides an oligonucleotide of at least about 12 nucleotides, specifically hybridizable with the nucleic acid molecules of this invention.
• this invention provides vectors, cells, liposomes and compositions comprising the isolated nucleic acids of this invention.
• this invention provides a method for detecting a splice variant according to the present invention in a biological sample, comprising: contacting a biological sample with an antibody specifically recognizing a splice variant according to the present invention under conditions whereby the antibody specifically interacts with the splice variant in the biological sample but do not recognize known conesponding proteins (wherein the known protein is discussed with regard to its splice variant(s) in the Examples below), and detecting said interaction; wherein the presence of an interaction conelates with the presence of a splice variant in the biological sample.
• this invention provides a method for detecting a splice variant nucleic acid sequences in a biological sample, comprising: hybridizing the isolated nucleic acid molecules or oligonucleotide fragments of at least about a minimum length to a nucleic acid material of a biological sample and detecting a hybridization complex; wherein the presence of a hybridization complex correlates with the presence of a splice variant nucleic acid sequence in the biological sample.
• the splice variants described herein are non- limiting examples of markers for diagnosing cardiac disease and/or cardiac pathology, including but not limited to cardiac damage.
• Each splice variant marker of the present invention can be used alone or in combination, for various uses, including but not limited to, prognosis, prediction, screening, early diagnosis, determination of progression, therapy selection and treatment monitoring of cardiac disease and/or cardiac pathology, including but not limited to cardiac damage.
• any marker according to the present invention may optionally be used alone or combination.
• Such a combination may optionally comprise a plurality of markers described herein, optionally including any subcombination of markers, and/or a combination featuring at least one other marker, for example a known marker.
• such a combination may optionally and preferably be used as described above with regard to determining a ratio between a quantitative or semi-quantitative measurement of any marker described herein to any other marker described herein, and/or any other known marker, and/or any other marker.
• the known marker comprises the "known protein" as described in greater detail below with regard to each cluster or gene.
• a splice variant protein or a fragment thereof, or a splice variant nucleic acid sequence or a fragment thereof may be featured as a biomarker for detecting cardiac disease and/or cardiac pathology, including but not limited to cardiac damage, such that a biomarker may optionally comprise any of the above.
• the present invention optionally and preferably encompasses any amino acid sequence or fragment thereof encoded by a nucleic acid sequence conesponding to a splice variant protein as described herein.
• Any oligopeptide or peptide relating to such an amino acid sequence or fragment thereof may optiona Uy also (additionally or alternatively) be used as a biomarker, including but not limited to the unique amino acid sequences of these proteins that are depicted as tails, heads, insertions, edges or bridges.
• the present invention also optionally encompasses antibodies capable of recognizing, and/or being elicited by, such oligopeptides or peptides.
• the present invention also optionally and preferably encompasses any nucleic acid sequence or fragment thereof, or amino acid sequence or fragment thereof, corresponding to a splice variant of the present invention as described above, optionally for any application. Non- limiting examples of methods or assays are described below.
• the present invention also relates to kits based upon such diagnostic methods or assays.
• Nucleic acid sequences and Oligonucleotides Various embodiments of the present invention encompass nucleic acid sequences described hereinabove; fragments thereof, sequences hybridizable therewith, sequences homologous thereto, sequences encoding similar polypeptides with different codon usage, altered sequences characterized by mutations, such as deletion, insertion or substitution of one or more nucleotides, either naturally occurring or artificially induced, either randomly or in a targeted fashion.
• the present invention encompasses nucleic acid sequences described herein; fragments thereof, sequences hybridizable therewitii, sequences homologous thereto [e.g, at least 50 %, at least 55 %, at least 60%, at least 65 %, at least 70 %, at least 75 %, at least 80 %, at least 85 %, at least 95 % or more say 100 % identical to the nucleic acid sequences set forth below], sequences encoding similar polypeptides with different codon usage, altered sequences characterized by mutations, such as deletion, insertion or substitution of one or more nucleotides, either naturally occuning or man induced, either randomly or in a targeted fashion.
• the present invention also encompasses homologous nucleic acid sequences (i.e., which form a part of a polynucleotide sequence of the present invention) which include sequence regions unique to the polynucleotides of the present invention.
• the present invention also encompasses novel polypeptides or portions thereof, which are encoded by the isolated polynucleotide and respective nucleic acid fragments thereof described hereinabove.
• a "nucleic acid fragment" or an "oligonucleotide” or a "polynucleotide” are used herein interchangeably to refer to a polymer of nucleic acids.
• a polynucleotide sequence of the present invention refers to a single or double stranded nucleic acid sequences which is isolated and provided in the form of an RNA sequence, a complementary polynucleotide sequence (cDNA), a genomic polynucleotide sequence and/or a composite polynucleotide sequences (e.g, a combination of the above).
• cDNA complementary polynucleotide sequence
• genomic polynucleotide sequence e.g, a combination of the above.
• composite polynucleotide sequences e.g, a combination of the above.
• the phrase "complementary polynucleotide sequence” refers to a sequence, which results from reverse transcription of messenger RNA using a reverse transcriptase or any other RNA dependent DNA polymerase. Such a sequence can be subsequently amplified in vivo or in vitro using a DNA dependent DNA polymerase.
• genomic polynucleotide sequence refers to a sequence derived (isolated) from a chromosome and thus it represents a contiguous portion of a chromosome.
• composite polynucleotide sequence refers to a sequence, which is composed of genomic and cDNA sequences.
• a composite sequence can include some exonal sequences required to encode the polypeptide of the present invention, as well as some intronic sequences interposing therebetween.
• the intronic sequences can be of any source, including of other genes, and typically will include conserved splicing signal sequences. Such intronic sequences may further include cis acting expression regulatory elements.
• Prefened embodiments of the present invention encompass oligonucleotide probes.
• An example of an oligonucleotide probe which can be utilized by the present invention is a single stranded polynucleotide which includes a sequence complementary to the unique sequence region of any variant according to the present invention, including but not limited to a nucleotide sequence coding for an amino sequence of a bridge, tail, head and/or insertion according to the present invention, and/or the equivalent portions of any nucleotide sequence given herein (including but not limited to a nucleotide sequence of a node, segment or amplicon described herein).
• an oligonucleotide probe of the present invention can be designed to hybridize with a nucleic acid sequence encompassed by any of the above nucleic acid sequences, particularly the portions specified above, including but not limited to a nucleotide sequence coding for an amino sequence of a bridge, tail, head and/or insertion according to the present invention, and/or the equivalent portions of any nucleotide sequence given herein (including but not limited to a nucleotide sequence of a node, segment or amplicon described herein).
• Oligonucleotides designed according to the teachings of the present invention can be generated according to any oligonucleotide synthesis method known in the art such as enzymatic synthesis or solid phase synthesis.
• Oligonucleotides used according to this aspect of the present invention are those having a length selected from a range of about 10 to about 200 bases preferably about 15 to about 150 bases, more preferably about 20 to about 100 bases, most preferably about 20 to about 50 bases.
• the oligonucleotide of the present invention features at least 17, at least 18, at least 19, at least 20, at least 22, at least 25, at least 30 or at least 40, bases specifically hybridizable with the biomarkers of the present invention.
• the oligonucleotides of the present invention may comprise heterocylic nucleosides consisting of purines and the pyrimidines bases, bonded in a 3' to 5' phosphodiester linkage.
• oligonucleotides are those modified at one or more of the backbone, internucleoside linkages or bases, as is broadly described hereinunder.
• Specific examples of prefened oligonucleotides useful according to this aspect of the present invention include oligonucleotides containing modified backbones or non-natural internucleoside linkages.
• Oligonucleotides having modified backbones include those that retain a phosphorus atom in the backbone, as disclosed in U.S. Pat.
• Prefened modified oligonucleotide backbones include, for example, phosphorothioates, chiral phosphorofhioates, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having nonnal 3'-5' linkages, 2'-5' linked analogs of these, and those having inverted polarity wherein the adjacent pairs o f nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'.
• modified oligonucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages.
• morpholino linkags formed in part from the sugar portion of a nucleoside
• siloxane backbones sulfide, sulfoxide and sulfone backbones
• formacetyl and thioformacetyl backbones methylene formacetyl and thioformacetyl backbones
• alkene containing backbones sulfamate backbones
• sulfonate and sulfonamide backbones amide backbones
• others having mixed N, O, S and CHj component parts, as disclosed in U.S. Pat. Nos. 5,034,506; 5,166,315;
• oligonucleotides which can be used according to the present invention, are those modified in both sugar and the internucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups. The base units are maintained for complementation with the appropriate polynucleotide target.
• An example for such an oligonucleotide mimetic includes peptide nucleic acid (PNA).
• PNA peptide nucleic acid
• United States patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is herein incorporated by reference.
• Other backbone modifications, which can be used in the present invention are disclosed in U.S. Pat.
• Oligonucleotides of the present invention may also include base modifications or substitutions.
• "unmodified” or “natural” bases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U).
• Modified bases include but are not limited to other synthetic and natural bases such as 5-methylcytosine (5-m ⁇ -C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2- aminoadenine, 6- methyl and other alkyl derivatives of adenine and guanine, 2 -propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2- thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8- halo, 8-amino, 8-tl ⁇ iol, 8-thioalkyl, 8- hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5- trifluoromethyl and other
• Further bases particularly useful for increasing the binding affinity of the oligomeric compounds of the invention include 5- substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2- aminopropyladenine, 5-pro ⁇ ynyluracil and 5- ⁇ ropynylcytosine.
• 5 -methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6- 1.2 °C and are presently prefened base substitutions, even more particularly when combined with 2'-0- methoxyethyl sugar modifications.
• oligonucleotides of the invention involves chemically linking to the oligonucleotide one or more moieties or conjugates, which enhance the activity, cellular distribution or cellular uptake of the oligonucleotide.
• moieties include but are not limited to lipid moieties such as a cholesterol moiety, cholic acid, a fhioefher, e.g, hexyl-S-tritylthiol, a thiocholesterol, an aliphatic chain, e.g, dodecandiol oêtcyl residues, a phospholipid, e.g, di-hexadecykrac-glycerol or triethylammonium 1,2-di-O- hexadecyl-rac-glycero-3-H-phosphonate, a polyamine or a polyethylene glycol chain, or adamantane acetic acid, a palmityl
• oligonucleotides of the present invention may include further modifications for more efficient use as diagnostic agents and/or to increase bioavailability, therapeutic efficacy and reduce cytotoxicity.
• a nucleic acid construct according to the present invention may be used, which includes at least a coding region of one of the above nucleic acid sequences, and further includes at least one cis acting regulatory element.
• cis acting regulatory element refers to a polynucleotide sequence, preferably a promoter, which binds a trans acting regulator and regulates the transcription of a coding sequence located downstream thereto.
• Any suitable promoter sequence can be used by the nucleic acid constmct of the present invention.
• the promoter utilized by the nucleic acid constmct of the present invention is active in the specific cell population transformed.
• cell type-specific and/or tissue -specific promoters include promoters such as albumin that is liver specific, lymphoid specific promoters [Calame et al, (1988) Adv. Immunol. 43:235-275]; in particular promoters of T-cell receptors [Winoto et al, (1989) EMBO J. 8:729-733] and immunoglobulins; [Banerji et al. (1983) Cell 33729-740], neuron- specific promoters such as the neurofilament promoter [Byrne et al. (1989) Proc. Natl. Acad. Sci.
• promoters such as albumin that is liver specific, lymphoid specific promoters [Calame et al, (1988) Adv. Immunol. 43:235-275]; in particular promoters of T-cell receptors [Winoto et al, (1989) EMBO J. 8:729-733] and immunoglobulin
• the nucleic acid construct of the present invention can further include an enhancer, which can be adjacent or distant to the promoter sequence and can function in up regulating the transcription therefrom.
• the nucleic acid constmct of the present invention preferably further includes an appropriate selectable marker and/or an origin of replication.
• the nucleic acid constmct utilized is a shuttle vector, which can propagate both in E.
• the construct according to the present invention can be, for example, a plasmid, a bacmid, a phagemid, a cosmid, a phage, a vims or an artificial chromosome.
• suitable constructs include, but are not limited to, pcDNA3, pcDNA3.1 (+/-), pGL3, PzeoSV2 (+/-), pDisplay, pEF/myc/cyto, pCMV/myc/cyto each of which is commercially available from Invitrogen Co. (www.invitrogen.com).
• retroviral vector and packaging systems are those sold by Clontech, San Diego, Calif, includingRetro-X vectors pLNCX and pLXSN, which permit cloning into multiple cloning sites and the trasgene is transcribed from CMV promoter.
• Vectors derived from Mo-MuLV are also included such as pBabe, where the transgene will be transcribed from the 5 'LTR promoter.
• Cunently prefened in vivo nucleic acid transfer techniques include transfection with viral or non- viral constmcts, such as adenovims, lentivirus, Herpes simplex I vims, or adeno-associated vims (AAV) and lipid-based systems.
• lipids for lipid-mediated transfer of the gene are, for example, DOTMA, DOPE, and DC -Choi [Tonkinson et al. Cancer Investigation, 14(1): 54-65 (1996)].
• the most prefened constructs for use in gene therapy are vimses, most preferably adenovimses, AAV, lentiviruses, or retrovimses.
• a viral constmct such as a retroviral constmct includes at least one transcriptional promoter/enhancer or locus -defining element(s), or other elements that control gene expression by other means such as alternate splicing, nuclear RNA export, or post- translational modification of messenger.
• Such vector constmcts also include a packaging signal, long terminal repeats (LTRs) or portions thereof, and positive and negative strand primer binding sites appropriate to the vims used, unless it is already present in the viral construct.
• a constmct typically includes a signal sequence for secretion of the peptide from a host cell in which it is placed.
• the signal sequence for this purpose is a mammalian signal sequence or the signal sequence of the polypeptide variants of the present invention.
• the construct may also include a signal that directs polyadenylation, as well as one or more restriction sites and a translation termination sequence.
• such constructs will typically include a 5' LTR, a tRNA binding site, a packaging signal, an origin of second- strand DNA synthesis, and a 3' LTR or a portion thereof.
• Other vectors can be used that are non-viral, such as cationic lipids, polylysine, and dendrimers.
• Hybridization assays Detection of a nucleic acid of interest in a biological sample may optionally be effected by hybridization-based assays using an oligonucleotide probe (non- limiting examples of probes according to the present invention were previously described).
• Traditional hybridization assays include PCR, RT-PCR, Rea ime PCR, RNase protection, in-situ hybridization, primer extension, Southern blots (DNA detection), dot or slot blots (DNA, RNA), and Northern blots (RNA detection) (NAT type assays are described in greater detail below). More recently, PNAs have been described (Nielsen et al. 1999, Cunent Opin. Biotechnol. 10:71-75).
• kits containing probes on a dipstick setup and the like Other detection methods include kits containing probes on a dipstick setup and the like.
• Hybridization based assays which allow the detection of a variant of interest (i.e., DNA or RNA) in a biological sample rely on the use of oligonucleotides which can be 10, 15, 20, or 30 to 100 nucleotides long preferably from 10 to 50, more preferably from 40 to 50 nucleotides long.
• the isolated polynucleotides (oligonucleotides) of the present invention are preferably hybridizable with any of the herein described nucleic acid sequences under moderate to stringent hybridization conditions.
• Moderate to stringent hybridization conditions are characterized by a hybridization solution such as containing 10 % dextrane sulfate, 1 M NaCI, 1 % SDS and 5 x l ⁇ 6 cpm 32 P labeled probe, at 65 °C, with a final wash solution of 0.2 x SSC and 0.1 % SDS and final wash at 65 °C and whereas moderate hybridization is effected using a hybridization solution containing 10 % dextrane sulfate, 1 M NaCI, 1 % SDS and 5 x 10 6 cpm 32 P labeled probe, at 65 °C, with a final wash solution of 1 x SSC and 0.1 % SDS and final wash at 50 °C.
• a hybridization solution such as containing 10 % dextrane sulfate, 1 M NaCI, 1 % SDS and 5 x l ⁇ 6 cpm 32 P labeled probe, at 65 °C
• moderate hybridization is effected using
• hybridization of short nucleic acids can be effected using the following exemplary hybridization protocols which can be modified according to the desired stringency;
• hybridization temperature 0.1 % nonfat dried milk, hybridization temperature.
• hybridization duplexes are separated from unhybridized nucleic acids and the labels bound to the duplexes are then detected.
• labels refer to radioactive, fluorescent, biological or enzymatic tags or labels of standard use in the art.
• a label can be conjugated to either the oligonucleotide probes or the nucleic acids derived from the biological sample.
• Probes can be labeled according to numerous well known methods.
• Non- limiting examples of radioactive labels include 3H, 14C, 32P, and 35S.
• detectable markers include ligands, fluorophores, chemiluminescent agents, enzymes, and antibodies.
• oligonucleotides of the present invention can be labeled subsequent to synthesis, by incorporating biotinylated dNTPs or rNTP, or some similar means (e.g, photo- cross- linking a psoralen derivative of biotin to RNAs), followed by addition of labeled streptavidin (e.g, phycoerythrin- conjugated streptavidin) or the equivalent.
• oligonucleotide probes when fluorescently- labeled oligonucleotide probes are used, fluorescein, lissamine, phycoerythrin, rhodamine (Perkin Elmer Cetus), Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy7, FluorX (Amersham) and others [e.g, Kricka et al. (1992), Academic Press San Diego, Calif] can be attached to the oligonucleotides. Those skilled in the art will appreciate that wash steps maybe employed to wash away excess target DNA or probe as well as unbound conjugate. Further, standard heterogeneous assay formats are suitable for detecting the hybrids using the labels present on the oligonucleotide primers and probes.
• probes can be labeled according to numerous well known methods.
• radioactive nucleotides can be incorporated into probes of the invention by several methods.
• Non- limiting examples of radioactive labels include 3 H, I C, 32 P, and M S.
• Probes of the invention can be utilized with naturally occuning sugar-phosphate backbones as well as modified backbones including phosphorothioates, dithionates, alkyl phosphonates and a-nucleotides and the like. Probes of the invention can be constructed of either ribonucleic acid (RNA) or deoxyribonucleic acid (DNA), and preferably of DNA.
• RNA ribonucleic acid
• DNA deoxyribonucleic acid
• NAT-based assays Detection of a nucleic acid of interest in a biological sample may also optionally be effected by NAT-based assays, which involve nucleic acid amplification technology, such as PCR for example (or variations thereof such as realtime PCR for example).
• a "primer" defines an oligonucleotide which is capable of annealing to (hybridizing with) a target sequence, thereby creating a double stranded region which can serve as an initiation point for DNA synthesis under suitable conditions.
• Amplification of a selected, or target, nucleic acid sequence may be carried out by a number of suitable methods. See generally Kwoh et al, 1990, Am. Biotechnol. Lab.
• amplification techniques include polymerase chain reaction (PCR), ligase chain reaction (LCR), strand displacement amplification (SDA), transcription-based amplification, the q3 replic ase system and NASB A (Kwoh et al, 1989, Proc. Natl. Acad. Sci. USA 86, 1173 - 1177; Lizardi et al, 1988, BioTechnology 6:1197-1202; Malek et al, 1994, Methods Mol. Biol, 28:253-260; and Sambrook et al, 1989, supra).
• PCR polymerase chain reaction
• LCR ligase chain reaction
• SDA strand displacement amplification
• amplification pair refers herein to a pair of oligonucleotides (oligos) of the present invention, which are selected to be used together in amplifying a selected nucleic acid sequence by one of a number of types of amplification processes, preferably a polymerase chain reaction.
• amplification processes include ligase chain reaction, strand displacement amplification, or nucleic acid sequence- based amplification, as explained in greater detail below.
• the oligos are designed to bind to a complementary sequence under selected conditions.
• amplification of a nucleic acid sample from a patient is amplified under conditions which favor the amplification of the most abundant differentially expressed nucleic acid.
• RT-PCR is carried out on an mRNA sample from a patient under conditions which favor the amplification of the most abundant mRNA.
• the amplification of the differentially expressed nucleic acids is carried out simultaneously. It will be realized by a person skilled in the art that such methods could be adapted for the detection of differentially expressed proteins instead of differentially expressed nucleic acid sequences.
• the nucleic acid i.e. DNA or RNA
• for practicing the present invention may be obtained according to well known methods.
• Oligonucleotide primers of the present invention may be of any suitable length, depending on the particular assay format and the particular needs and targeted genomes employed.
• the oligonucleotide primers are at least 12 nucleotides in length, preferably between 15 and 24 molecules, and they may be adapted to be especially suited to a chosen nucleic acid amplification system
• the oligonucleotide primers can be designed by taking into consideration the melting point of hybridization thereof with its targeted sequence (Sambrook et al, 1989, Molecular Cloning - A Laboratory Manual, 2nd Edition, CSH Laboratories; Ausubel et al, 1989, in Cunent Protocols in Molecular Biology, John Wiley & Sons Inc., N.Y.).
• antisense oligonucleotides may be employed to quantify expression of a splice isoform of interest. Such detection is effected at the pre- mRNA level. Essentially the ability to quantitate transcription from a splice site of interest can be effected based on splice site accessibility. Oligonucleotides may compete with splicing factors for the splice site sequences. Thus, low activity of the antisense oligonucleotide is indicative of splicing activity.
• the polymerase chain reaction and other nucleic acid amplification reactions are well known in the art (various non- limiting examples of these reactions are described in greater detail below).
• the pair of oligonucleotides according to this aspect of the present invention are preferably selected to have compatible melting temperatures (Tm), e.g, melting temperatures which differ by less than that 7 °C, preferably less than 5 °C, more preferably less than 4 °C, most preferably less than 3 °C, ideally between 3 °C and 0 °C.
• Tm melting temperatures
• PCR can be used to directly increase the concentration of the target to an easily detectable level.
• This process for amplifying the target sequence involves the introduction of a molar excess of two oligonucleotide primers which are complementary to their respective strands of the double -stranded target sequence to the DNA mixture containing the desired target sequence. The mixture is denatured and then allowed to hybridize. Following hybridization, the primers are extended with polymerase so as to form complementary strands. The steps of denaturation, hybridization (annealing), and polymerase extension (elongation) can be repeated as often as needed, in order to obtain relatively high concentrations of a segment of the desired target sequence.
• LCR Ligase Chain Reaction
• LAR The ligase chain reaction [LCR; sometimes refened to as "Ligase Amplification Reaction” (LAR)] has developed into a well-recognized alternative method of amplifying nucleic acids.
• LCR has also been used in combination with PCR to achieve enhanced detection of single -base changes: see for example Segev, PCT Publication No. W09001069 Al (1990).
• the four oligonucleotides used in this assay can pair to form two short ligatable fragments, there is the potential for the generation of target-independent background signal.
• the use of LCR for mutant screening is limited to the examination of specific nucleic acid positions.
• an oligonucleotide primer is used to add a phage RNA polymerase promoter to the 5' end of the sequence of interest.
• a cocktail of enzymes and substrates that includes a second primer, reverse transcriptase, RNase H, RNA polymerase and ribo-and deoxyribonucleoside triphosphates, the target sequence undergoes repeated rounds of transcription, cDNA synthesis and second- strand synthesis to amplify the area of interest.
• the use of 3SR to detect mutations is kinetically limited to screening small segments of DNA (e.g, 200-300 base pairs).
• Q-Beta (Q ⁇ ) Replicase In this method, a probe which recognizes the sequence of interest is attached to the replicatable RNA template for Q ⁇ replicase.
• a previously identified major problem with false positives resulting from the replication of unhybridized probes has been addressed through use of a sequence-specific ligation step.
• available thermostable DNA ligases are not effective on this RNA substrate, so the ligation must be perfonned by T4 DNA ligase at low temperatures (37 degrees C). This prevents the use of high temperature as a means of achieving specificity as in the LCR, the ligation event can be used to detect a mutation at the junction site, but not elsewhere.
• a successful diagnostic method must be very specific.
• a straight- forward method of controlling the specificity of nucleic acid hybridization is by controlling the temperature of the reaction. While the 3SR/NASBA, and Q ⁇ systems are all able to generate a large quantity of signal, one or more of the enzymes involved in each cannot be used at high temperature (i.e., > 55 degrees C). Therefore the reaction temperatures cannot be raised to prevent non-specific hybridization of the probes. If probes are shortened in order to make them melt more easily at low temperatures, the likelihood of having more than one perfect match in a complex genome increases. For these reasons, PCR and LCR cunently dominate the research field in detection technologies.
• n is the number of cycles
• y is the overall efficiency, or yield of the reaction. If every copy of a target DNA is utilized as a template in every cycle of a polymerase chain reaction, then the mean efficiency is 100 %. If 20 cycles of PCR are performed, then the yield will be
• reaction conditions reduce the mean efficiency to 85 %, then the yield in those 20 cycles will be only 1.8520, or 220,513 copies of the starting material.
• a PCR running at 85 % efficiency will yield only 21 % as much final product, compared to a reaction ranning at 100 % efficiency.
• a reaction that is reduced to 50 % mean efficiency will yield less than 1 % of the possible product.
• routine polymerase chain reactions rarely achieve the theoretical maximum yield, and PCRs are usually run for more than 20 cycles to compensate for the lower yield.
• One method of the detection of allele- specific variants by PCR is based upon the fact that it is difficult for Taq polymerase to synthesize a DNA strand when there is a mismatch between the template strand and the 3' end of the primer.
• An allele -specific variant may be detected by the use of a primer that is perfectly matched with only one of the possible alleles; the mismatch to the other allele acts to prevent the extension of the primer, thereby preventing the amplification of that sequence.
• This method has a substantial limitation in that the base composition of the mismatch influences the ability to prevent extension across the mismatch, and certain mismatches do not prevent extension or have only a minimal effect.
• a similar 3'- mismatch strategy is used with greater effect to prevent ligation in the
• the direct detection method may be, for example a cycling probe reaction (CPR) or a branched DNA analysis.
• Cycling probe reaction uses a long chimeric oligonucleotide in whic h a central portion is made of RNA while the two termini are made of DNA. Hybridization of the probe to a target DNA and exposure to a thermostable RNase H causes the RNA portion to be digested. This destabilizes the remaining DNA portions of the duplex, releasing the remainder of the probe from the target DNA and allowing another probe molecule to repeat the process. The signal, in the form of cleaved probe molecules, accumulates at a linear rate. While the repeating process increases the signal, the RNA portion of the oligonucleotide is vulnerable to RNases that may carried through sample preparation.
• Branched DNA involves oligonucleotides with branched structures that allow each individual oligonucleotide to carry 35 to 40 labels (e.g, alkaline phosphatase enzymes). While this enhances the signal from a hybridization event, signal from non-specific binding is similarly increased.
• labels e.g, alkaline phosphatase enzymes
• the detection of at least one sequence change may be accomplished by, for example restriction fragment length polymo ⁇ hism (RFLP analysis), allele specific oligonucleotide (ASO) analysis, Denaturing/Temperature Gradient Gel Electrophoresis (DGGE/TGGE), Single- Strand Conformation Polymo ⁇ hism (SSCP) analysis or Dideoxy fmge ⁇ rinting (ddF).
• RFLP analysis restriction fragment length polymo ⁇ hism
• ASO allele specific oligonucleotide
• DGGE/TGGE Denaturing/Temperature Gradient Gel Electrophoresis
• SSCP Single- Strand Conformation Polymo ⁇ hism
• ddF Dideoxy fmge ⁇ rinting
• nucleic acid segments for mutations.
• One option is to determine the entire gene sequence of each test sample (e.g, a bacterial isolate). For sequences under approximately 600 nucleotides, this may be accomplished using amplified material (e.g, PCR reaction products). This avoids the time and expense associated with cloning the segment of interest. However, specialized equipment and highly trained personnel are required, and the method is too labor- intense and expensive to be practical and effective in the clinical setting.
• a given segment of nucleic acid may be characterized on several other levels. At the lowest resolution, the size of the molecule can be determined by electrophoresis by comparison to a known standard ran on the same gel.
• a more detailed picture of the molecule may be achieved by cleavage with combinations of restriction enzymes prior to electrophoresis, to allow construction of an ordered map.
• the presence of specific sequences within the fragment can be detected by hybridization of a labeled probe, or the precise nucleotide sequence can be determined by partial chemical degradation or by primer extension in the presence of chain-terminating nucleotide analogs.
• Restriction fragment length polymorphism RFLP: For detection of single -base differences between like sequences, the requirements of the analysis are often at the highest level of resolution. For cases in which the position of the nucleotide in question is known in advance, several methods have been developed for examining single base changes without direct sequencing.
• a change in the pattern of digestion can be used as a diagnostic tool (e.g., restriction fragment length polymo ⁇ hism [RFLP] analysis).
• RFLP restriction fragment length polymo ⁇ hism
• Single point mutations have been also detected by the creation or destruction of RFLPs. Mutations are detected and localized by the presence and size of the RNA fragments generated by cleavage at the mismatches.
• Single nucleotide mismatches in DNA heteroduplexes are also recognized and cleaved by some chemicals, providing an alternative strategy to detect single base substitutions, generically named the "Mismatch Chemical Cleavage" (MCC).
• MCC Mismatch Chemical Cleavage
• RFLP analysis When RFLP analysis is used for the detection of point mutations, it is, by its nature, limited to the detection of only those single base changes which fall within a restriction sequence of a known restriction endonuclease. Moreover, the majority of the available enzymes have 4 to 6 base-pair recognition sequences, and cleave too frequently for many large-scale DNA manipulations. Thus, it is applicable only in a small fraction of cases, as most mutations do not fall within such sites. A handful of rare- cutting restriction enzymes with 8 base-pair specificities have been isolated and these are widely used in genetic mapping, but these enzymes are few in number, are limited to the recognition of G+C-rich sequences, and cleave at sites that tend to be highly clustered.
• Allele specific oligonucleotide ASO: If the change is not in a recognition sequence, then allele -specific oligonucleotides (ASOs), can be designed to hybridize in proximity to the mutated nucleotide, such that a primer extension or ligation event can bused as the indicator of a match or a mis- match. Hybridization with radioactively labeled allelic specific oligonucleotides (ASO) also has been applied to the detection of specific point mutations.
• the method is based on the differences in the melting temperature of short DNA fragments differing by a single nucleotide. Stringent hybridization and washing conditions can differentiate between mutant and wild -type alleles.
• the ASO approach applied to PCR products also has been extensively utilized by various researchers to detect and characterize point mutations in ras genes and gsp/gip oncogenes. Because of the presence of various nucleotide changes in multiple positions, the ASO method requires the use of many oligonucleotides to cover all possible oncogenic mutations. With either of the techniques described above (i.e., RFLP and ASO), the precise location of the suspected mutation must be known in advance of the test.
• DGGE/TGGE Denaturing/Temperature Gradient Gel Electrophoresis
• variants can be distinguished, as differences in melting properties of homoduplexes versus heteroduplexes differing in a single nucleotide can detect the presence of mutations in the target sequences because of the conesponding changes in their electrophoretic mobilities.
• the fragments to be analyzed usually PCR products, are "clamped” at one end by a long stretch of G-C base pairs (30-80) to allow complete denaturation of the sequence of interest without complete dissociation of the strands.
• the attachment of a GC "clamp" to the DNA fragments increases the fraction of mutations that can be recognized by DGGE. Attaching a GC clamp to one primer is critical to ensure that the amplified sequence has a low dissociation temperature.
• TGGE uses a thennal gradient rather than a chemical denaturant gradient.
• TGGE requires the use of specialized equipment which can generate a temperature gradient pe ⁇ endicularly oriented relative to the electrical field.
• TGGE can detect mutations in relatively small fragments of DNA therefore scanning of large gene segments requires the use of multiple PCR products prior to running the gel.
• Single-Strand Conformation Polymorphism SSCP: Another common method, called “Single-Strand Conformation Polymo ⁇ hism" (SSCP) was developed by Hayashi, Sekya and colleagues and is based on the observation that single strands of nucleic acid can take on characteristic confonnations in non-denaturing conditions, and these conformations influence electrophoretic mobility.
• the complementary strands assume sufficiently different structures that one strand may be resolved from the other. Changes in sequences within the fragment will also change the conformation, consequently altering the mobility and allowing this to be used as an assay for sequence variations.
• the SSCP process involves denaturing a DNA segment (e.g, a PCR product) that is labeled on both strands, followed by slow electrophoretic separation on a non- denaturing polyacrylamide gel, so that intra-molecular interactions can fonn and not be disturbed during the mn. This technique is extremely sensitive to variations in gel composition and temperature. A serious limitation of this method is the relative difficulty encountered in comparing data generated in different laboratories, under apparently similar conditions.
• Dideoxy fingerprinting (ddF): The dideoxy finge ⁇ rinting (ddF) is another technique developed to scan genes for the presence of mutations.
• the ddF technique combines components of Sanger dideoxy sequencing with SSCP.
• a dideoxy sequencing reaction is perfomied using one dideoxy terminator and then the reaction products are electrophoresed on nondenaturing polyacrylamide gels to detect alterations in mobility of the termination segments as in SSCP analysis.
• ddF is an improvement over SSCP in terms of increased sensitivity
• ddF requires the use of expensive dideoxynucleotides and this technique is still limited to the analysis of fragments of die size suitable for SSCP (i.e., fragments of 200-300 bases for optimal detection of mutations).
• all of these methods are limited as to the size of the nucleic acid fragment that can be analyzed.
• sequences of greater than 600 base pairs require cloning, with the consequent delays and expense of either deletion sub- cloning or primer walking, in order to cover the entire fragment.
• SSCP and DGGE have even more severe size limitations. Because of reduced sensitivity to sequence changes, these methods are not considered suitable for larger fragments.
• SSCP is reportedly able to detect 90 % of single-base substitutions within a 200 base-pair fragment, the detection drops to less than 50 % for 400 base pair fragments. Similarly, the sensitivity of DGGE decreases as the length of the fragment reaches 500 base-pairs.
• the ddF technique as a combination of direct sequencing and SSCP, is also limited by the relatively small size of the DNA that can be screened.
• the step of searching for any of the nucleic acid sequences described here, in tumor cells or in cells derived from a cancer patient is effected by any suitable technique, including, but not limited to, nucleic acid sequencing, polymerase chain reaction, ligase chain reaction, self- sustained synthetic reaction, Q ⁇ -Replicase, cycling probe reaction, branched DNA, restriction fragment length polymo ⁇ hism analysis, mismatch chemical cleavage, heteroduplex analysis, allele-specific oligonucleotides, denaturing gradient gel electrophoresis, constant denaturant gel electrophoresis, temperature gradient gel electrophoresis and dideoxy fmge ⁇ rinting.
• any suitable technique including, but not limited to, nucleic acid sequencing, polymerase chain reaction, ligase chain reaction, self- sustained synthetic reaction, Q ⁇ -Replicase, cycling probe reaction, branched DNA, restriction fragment length polymo ⁇ hism analysis, mismatch chemical cleavage, heteroduplex analysis, allele-specific oligonu
• Detection may also optionally be performed with a chip or other such device.
• the nucleic acid sample which includes the candidate region to be analyzed is preferably isolated, amplified and labeled with a reporter group.
• This reporter group can be a fluorescent group such as phycoerythrin.
• the labeled nucleic acid is then incubated with the probes immobilized on the chip using a fluidics station, describe the fabrication of fluidics devices and particularly microcapillary devices, in silicon and glass substrates. Once the reaction is completed, the chip is inserted into a scanner and patterns of hybridization are detected. The hybridization data is collected, as a signal emitted from the reporter groups already inco ⁇ orated into the nucleic acid, which is now bound to the probes attached to the chip.
• the identity of the nucleic acid hybridized to a given probe can be determined. It will be appreciated that when utilized along with automated equipment, the iiove described detection methods can be used to screen multiple samples for a disease and/or pathological condition both rapidly and easily.
• polypeptide amino acid sequences and peptides
• polypeptide amino acid sequences and peptides
• polypeptide polypeptide
• peptide amino acid residues
• protein polymer of amino acid residues.
• the terms apply to amino acid polymers in which one or more amino acid residue is an analog or mimetic of a conesponding naturally occuning amino acid, as well as to naturally occurring amino acid polymers.
• Polypeptides can be modified, e.g, by the addition of carbohydrate residues to form glycoproteins.
• polypeptide polypeptide
• peptide and protein
• Polypeptide products can be biochemically synthesized such as by employing standard solid phase techniques.
• Such methods include but are not limited to exclusive solid phase synthesis, partial solid phase synthesis methods, fragment condensation, classical solution synthesis. These methods are preferably used when the peptide is relatively short (i.e., 10 kDa) and/or when it cannot be produced by recombinant techniques (i.e., not encoded by a nucleic acid sequence) and therefore involves different chemistry.
• Solid phase polypeptide synthesis procedures are well known in the art and further described by John Monow Stewart and Janis Dillaha Young, Solid Phase Peptide Syntheses (2nd Ed, Pierce Chemical Company, 1984). Synthetic polypeptides can optionally be purified by preparative high performance liquid clromatography [Creighton T. (1983) Proteins, structures and molecular principles.
• the present invention also encompasses polypeptides encoded by the polynucleotide sequences of the present invention, as well as polypeptides according to the amino acid sequences described herein.
• the present invention also encompasses homologues of these polypeptid es, such homologues can be at least 50 %, at least 55 %, at least 60%, at least 65 %, at least 70 %, at least 75 %, at least 80 %, at least 85 %, at least 95 % or more say 100 % homologous to the amino acid sequences set forth below, as can be determined using BlasfP software of the National Center of Biotechnology Information (NCBI) using default parameters, optionally and preferably including the following: filtering on (this option filters repetitive or low- complexity sequences from the query using the Seg (protein) program), scoring matrix is BLOSUM62 for proteins, word size is 3, E value is 10, gap costs are 11, 1 (initialization and extension), and number of alignments shown is 50.
• NCBI National Center of Biotechnology Information
• nucleic acid sequence homology is determined using BlastN software of the National Center of Biotechnology Information (NCBI) using default parameters, which preferably include using the DUST filter program, and also preferably include having an E value of 10, filtering low complexity sequences and a word size of 11.
• NBI National Center of Biotechnology Information
• the present invention also encompasses fragments of the above described polypeptides and polypeptides having mutations, such as deletions, insertions or substitutions of one or more amino acids, either naturally occuning or artificially induced, either randomly or in a targeted fashion.
• peptides identified according the present invention may be degradation products, synthetic peptides or recombinant peptides as well as peptidomimetics, typically, synthetic peptides and peptoids and semipeptoids which are peptide analogs, which may have, for example, modifications rendering the peptides more stable while in a body or more capable of penetrating into cells.
• Natural aromatic amino acids, T ⁇ , Tyr and Phe may be substituted for synthetic non- natural acid such as Phenylglycine, TIC, naphthylelanine (Nol), ring-methylated derivatives of Phe, halogenated derivatives of Phe or o-mefhyl-Tyr.
• the peptides of the present invention may also include one or more modified amino acids or one or more non-amino acid monomers (e.g. fatty acids, complex carbohydrates etc).
• amino acid or “amino acids” is understood to include the 20 naturally occuning amino acids; those amino acids often modified post-translationally in vivo, including, for example, hydroxyproline, phosphoserine and phosphothreonine; and other unusual amino acids including, but not limited to, 2-aminoadipic acid, hydroxylysine, isodesmosine, nor-valine, nor-leucine and omithine.
• amino acid includes both D- and L-amino acids.
• the peptides of the present invention are preferably utilized in diagnostics which require the peptides to be in soluble form, the peptides of the present invention preferably include one or more non- natural or natural polar amino acids, including but not limited to serine and threonine which are capable of increasing peptide solubility due to their hydroxyl- containing side chain.
• the peptides of the present invention are preferably utilized in a linear fonn, although it will be appreciated that in cases where cyclicization does not severely interfere with peptide characteristics, cyclic forms of the peptide can also be utilized.
• the peptides of present invention can be biochemically synthesized such as by using standard solid phase techniques.
• Antibodies refers to a polypeptide ligand that is preferably substantially encoded by an immunoglobulin gene or immunoglobulin genes, or fragments thereof, which specifically binds and recognizes an epitope (e.g, an antigen).
• the recognized immunoglobulin genes include the kappa and lambda light chain constant region genes, the alpha, gamma, delta, epsilon and mu heavy chain constant region genes, and the myriad- immunoglobulin variable region genes.
• Antibodies exist, e.g, as intact immunoglobulins or as a number of well characterized fragments produced by digestion with various peptidases. This includes, e.g, Fab' and F(ab)' 2 fragments.
• antibody also includes antibody fragments either produced by the modification of whole antibodie s or those synthesized de novo using recombinant DNA methodologies. It also includes polyclonal antibodies, monoclonal antibodies, chimeric antibodies, humanized antibodies, or single chain antibodies. "Fc" portion of an antibody refers to that portion of an immunoglobulin heavy chain that comprises one or more heavy chain constant region domains, CHI, CH2 and CH3, but does not include the heavy chain variable region.
• Fab the fragment which contains a monovalent antigen-binding fragment of an antibody molecule
• Fab' the fragment of an antibody molecule that can be obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain
• two Fab' fragments are obtained per antibody molecule
• (Fab')2 the fragment of the antibody that can be obtained by treating whole antibody with the enzyme pepsin without subsequent reduction
• F(ab')2 is a dimer of two Fab' fragments held together by two disulfide bonds
• Fv defined as a genetically engineered fragment containing the variable region of the light chain and the variable region of the heavy chain expressed as two chains
• SCA Single chain antibody
• Antibody fragments according to the present invention can be prepared by proteolytic hydrolysis of the antibody or by expression in E. coli or mammalian cells (e.g. Chinese hamster ovary cell culture or other protein expression systems) of DNA encoding the fragment.
• Antibody fragments can be obtained by pepsin or papain digestion of whole antibodies by conventional methods.
• antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment denoted F(ab')2.
• This fragment can be further cleaved using a thiol reducing agent, and optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages, to produce 3.5S Fab' monovalent fragments.
• a thiol reducing agent optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages
• an enzymatic cleavage using pepsin produces two monovalent Fab' fragments and an Fc fragment directly.
• Fv fragments comprise an association of VH and VL chains. This association may be noncovalent, as described in Inbar et al. [Proc. Nat'l Acad. Sci. USA 69:2659-62 (19720].
• the variable chains can be linked by an intermolecular disulfide bond or cross- linked by chemicals such as glutaraldehyde.
• the Fv fragments comprise VH and VL chains connected by a peptide linker.
• sFv single- chain antigen binding proteins
• stmctural gene comprising DNA sequences encoding the VH and VL domains connected by an oligonucleotide.
• the structural gene is inserted into an expression vector, which is subsequently introduced into a host cell such as E. coli.
• the recombinari host cells synthesize a single polypeptide chain with a linker peptide bridging the two V domains.
• CDR peptides (“minimal recognition units") can be obtained by constructing genes encoding the CDR of an antibody of interest. Such genes are prepared, for example, by using the polymerase chain reaction to synthesize the variable region from RNA of antibody-producing cells. See, for example, Larrick and Fry [Methods, 2: 106-10 (1991)].
• Humanized forms of non-human (e.g, murine) antibodies are chimeric molecules of immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab') or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin.
• Humanized antibodies include human immunoglobulins (recipient antibody) in which residues from a complementary detemiining region (CDR) of the recipiert are replaced by residues from a CDR of a non- human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity.
• CDR complementary detemiining region
• Fv framework residues of the human immunoglobulin are replaced by conesponding non-human residues.
• Humanized antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences.
• the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions conespond to those of a nonhuman immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence.
• the humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin [Jones et al.
• a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues are often refened to as import residues, which are typically taken from an import variable domain. Humanization can be essentially performed following the method of Winter and co-workers [Jones et al. Nature, 321:522-525 (1986); Riechmann et al.
• humanized antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the conesponding sequence from a non- human species.
• humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.
• Human antibodies can also be produced using various techniques known in the art, including phage display libraries [Hoogenboom and Winter, J. Mol.
• human antibodies can be made by introduction of human immunoglobulin loci into transgenic animals, e.g, mice in which the endogenous immunoglobulin genes have been partially or completely inactivated.
• the antibody of this aspect of the present invention specifically binds at least one epitope of the polypeptide variants of the present invention.
• epitope refers to any antigenic determinant on an antigen to which the paratope of an antibody binds.
• Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or carbohydrate side chains and usually have specific three dimensional stmctural characteristics, as well as specific charge characteristics.
• a unique epitope may be created in a variant due to a change in one or more post-translational modifications, including but not limited to glycosylation and/or phosphorylation, as described below. Such a change may also cause a new epitope to be created, for example through removal of glycosylation at a particular site.
• An epitope according to the present invention may also optionally comprise part or all of a unique sequence portion of a variant according to the present invention in combination with at least one other portion of the variant which is not contiguous to the unique sequence portion in the linear polypeptide itself, yet which are able to form an epitope in combination.
• One or more unique sequence portions may optionally combine with one or more other non- contiguous portions of the variant (including a portion whbh may have high homology to a portion of the known protein) to form an epitope.
• Immunoassays In another embodiment of the present invention, an immunoassay can be used to qualitatively or quantitatively detect and analyze markers in a sample.
• This method comprises: providing an antibody that specifically binds to a marker; contacting a sample with the antibody; and detecting the presence of a complex of the antibody bound to the marker in the sample.
• an antibody that specifically binds to a marker purified protein markers can be used.
• Antibodies that specifically bind to a protein marker can be prepared using any suitable methods known in the art. After the antibody is provided, a marker can be detected and/or quantified using any of a number of well recognized immunological binding assays.
• Useful assays include, for example, an enzyme immune assay (EIA) such as enzyme-linked immunosorbent assay (ELISA), a radioimmune assay (RJA), a Western blot assay, or a slot blot assay see, e.g, U.S. Pat Nos. 4,366,241; 4,376,110; 4,517,288; and 4,837,168).
• EIA enzyme immune assay
• ELISA enzyme-linked immunosorbent assay
• RJA radioimmune assay
• a sample obtained from a subject can be contacted with the antibody that specifically binds the marker.
• the antibody can be fixed to a solid support to facilitate washing and subsequent isolation of the complex, prior to contacting the antibody with a sample.
• solid supports include but are not limited to glass or plastic in the form of, e.g, a microtiter plate, a stick, a bead, or a microbead.
• Antibodies can also be attached to a solid support. After incubating the sample with antibodies, the mixture is washed and the antibody- marker complex formed can be detected. This can be accomplished by incubating the washed mixture with a detection reagent.
• the marker in the sample can be detected using an indirect assay, wherein, for example, a second, labeled antibody is used to detect bound marker-specific antibody, and/or in a competition or inhibition assay wherein, for example, a monoclonal antibody which binds to a distinct epitope of the marker are incubated simultaneously with the mixture.
• incubation and/or washing steps may be required after each combination of reagents. Incubation steps can vary from about 5 seconds to several hours, preferably from about 5 minutes to about 24 hours. However, the incubation time will depend upon the assay format, marker, volume of solution, concentrations and the like.
• the immunoassay can be used to determine a test amount of a marker in a sample from a subject.
• a test amount of a marker in a sample can be detected using the immunoassay methods described above. If a marker is present in the sample, it will fonn an antibody-marker complex with an antibody that specifically binds the marker under suitable incubation conditions described above.
• the amount of an antibody- marker complex can optionally be determined by comparing to a standard.
• the test amount of marker need not be measured in absolute units, as long as the unit of measurement can be compared to a control amount and/or signal.
• Such antibodies are directed, for example, to the unique sequence portions of the polypeptide variants of the present invention, including but not limited to bridges, heads, tails and insertions described in greater detail below. Prefened embodiments of antibodies according to the present invention are described in greater detail with regard to the section entitled
• Radio -immunoassay In one version, this method involves precipitation of the desired substrate and in the methods detailed hereinbelow, with a specific antibody and 125 radiolabelled antibody binding protein (e.g, protein A labeled with I ) immobilized on a precipitable carrier such as agarose beads. The number of counts in the precipitated pellet is proportional to the amount of substrate.
• a labeled substrate and an unlabelled antibody binding protein are employed. A sample containing an unknown amount of substrate is added in varying amounts. The decrease in precipitated counts from the labeled substrate is proportional to the amount of substrate in the added sample.
• Enzyme linked immunosorbent assay Tins method involves fixation of a sample (e.g, fixed cells or a proteinaceous solution) containing a protein subsfrate to a surface such as a well of a microtiter plate. A substrate specific antibody coupled to an enzyme is applied and allowed to bind to the substrate. Presence of the antibody is then detected and quantitated by a colorimetric reaction employing the enzyme coupled to the antibody. Enzymes commonly employed in this method include horseradish peroxidase and alkaline phosphatase. If well calibrated and within the linear range of response, the amount of substrate present in the sample is proportional to the amount of color produced. A substrate standard is generally employed to improve quantitative accuracy.
• Western blot This method involves separation of a substrate from other protein by means of an acrylamide gel followed by transfer of the substrate to a membrane (e.g, nylon or PVDF). Presence of the substrate is then detected by antibodies specific to the substrate, which are in turn detected by antibody binding reagents.
• Antibody binding reagents may be, for example, protein A, or other antibodies. Antibody binding reagents may be radiolabelled or enzyme linked as described hereinabove. Detection may be by autoradiograpby, colorimetric reaction or chemiluminescence. This method allows both quantitation of an amount of substrate and determination of its identity by a relative position on the membrane which is indicative of a migration distance in the acrylamide gel during electrophoresis.
• Immunohistochemical analysis This method involves detection of a substrate in situ in fixed cells by substrate specific antibodies.
• the substrate specific antibodies may be enzyme linked or linked to fluorophores. Detection is by microscopy and subjective evaluation. If enzyme linked antibodies are employed, a colorimetric reaction may be required.
• Fluorescence activated cell sorting FACS: This method involves detection of a substrate in situ in cells by substrate specific antibodies. The substrate specific antibodies are linked to fluorophores. Detection is by means of a cell sorting machine which reads the wavelength of light emitted from each cell as it passes through a light beam. This method may employ two or more antibodies simultaneously.
• Radio -imaging Methods include but are not limited to, positron emission tomography (PET) single photon emission computed tomography (SPECT). Both of these techniques are non- invasive, and can be used to detect and/or measure a wide variety of tissue events and/or functions, such as detecting cancerous cells for example. Unlike PET, SPECT can optionally be used with two labels simultaneously. SPECT has some other advantages as well, for example with regard to cost and the types of labels that can be used. For example, US Patent No. 6,696,686 describes the use of SPECT for detection of breast cancer, and is hereby inco ⁇ orated by reference as if fully set forth herein.
• Display Libraries According to still another aspect of the present invention there is provided a display library comprising a plurality of display vehicles (such as phages, viruses or bacteria) each displaying at least 6, at least 7, at least 8, at least 9, at least 10, 10-15, 12- 17, 15-20, 15-30 or 20-50 consecutive amino acids derived from the polypeptide sequences of the present invention. Methods of constmcting such display libraries are well known in the art.
• display vehicles such as phages, viruses or bacteria
• the BNP variants of the present invention and compositions derived therefrom can be used to treat a subject having, being diagnosed with or predisposed to a BNP- related disease, such as cardiac disease.
• the subject according to the present invention is a mammal, preferably a human which is diagnosed with one of the diseases described hereinabove, or alternatively is predisposed to having one of the diseases described hereinabove.
• the term "treating” refers to preventing, curing, reversing, attenuating, alleviating, minimizing, suppressing or halting the deleterious effects of the BNP-related disease.
• Treating according to the present invention is effected by specifically upregulating the expression in the subject of at least one of the polypeptides of the present invention.
• active portion refers to an amino acid sequence portion which is capable of displaying one or more functions of the BNP polypeptides of the present invention.
• Upregulating methods and agents Upregulating expression of the BNP variants of the present invention may be effected via the administration of at least one of the exogenous polynucleotide sequences of the present invention (e.g, SEQ ID NOs: 1-3, 9-10 and/or 12-14) ligated into a nucleic acid expression constmct designed for expression of coding sequences in eukaryotic cells (e.g, mammalian cells).
• the exogenous polynucleotide sequence may be a DNA or RNA sequence encoding the variants of the present invention or active portions thereof.
• the nucleic acid constmct can be administered to the individual employing any suitable mode of administration, described hereinbelow (i.e., in- vivo gene therapy).
• the nucleic acid construct is introduced into a suitable cell via an appropriate gene delivery vehicle/method (transfection, transduction, homologous recombination, etc.) and an expression system as needed and then the modified cells are expanded in culture and returned to the individual (i.e., ex-vivo gene therapy).
• the nucleic acid construct of the present invention further includes at least one cis acting regulatory element.
• cis acting regulatory element refers to a polynucleotide sequence, preferably a promoter, which binds a trans acting regulator and regulates the transcription of a coding sequence located downstream thereto. Any suitable promoter sequence can be used by the nucleic acid construct of the present invention.
• the promoter utilized by the nucleic acid construct of the present invention is active in the specific cell population transfonned.
• cell type-specific and/or tissue -specific promoters include promoters such as albumin that is liver specific [Pinkert et al, (1987) Genes Dev. 1:268-277], lymphoid specific promoters [Calame et al, (1988) Adv. Immunol. 43:235-275]; in particular promoters of T-cell receptors
• the nucleic acid constmct of the present invention can further include an enhancer, which can be adjacent or distant to the promoter sequence and can function in up regulating the transcription therefrom.
• the nucleic acid constmct of the present invention preferably further includes an appropriate selectable marker and/or an origin of replication.
• the nucleic acid constmct utilized is a shuttle vector, which can propagate both in E. coli (wherein the construct comprises an appropriate selectable marker and origin of replication) and be compatible for propagation in cells, or integration in a gene and a tissue of choice.
• constmct can be, for example, a plasmid, a bacmid, a phagemid, a cosmid, a phage, a vims or an artificial chromosome.
• suitable constmcts include, but are not limited to, pcDNA3, pcDNA3.1 (+/-), pGL3, PzeoSV2 (+/-), pDisplay, pEF/myc/cyto, pCMV/myc/cyto each of which is commercially available from Invitrogen Co. (www.invitrogen.com).
• retroviral vector and packaging systems are those sold by Clontech, San Diego, Calif, including Retro -X vectors pLNCX and pLXSN, which permit cloning into multiple cloning sites and the transgene is transcribed from CMV promoter.
• Vectors derived from Mo-MuLV are also included such as pBabe, where the transgene will be transcribed from the 5 'LTR promoter.
• Cunently prefened in vivo nucleic acid transfer techniques include transfection with viral or non- viral constmcts, such as adenovims, lentivirus, He ⁇ es simplex I vims, or adeno-associated vims (AAV) and lipid-based systems.
• lipids for lipid -mediated transfer of the gene are, for example, DOTMA, DOPE, and DC -Choi [Tonkinson et al. Cancer Investigation, 14(1): 54-65 (1996)].
• the most prefened constructs for use in gene therapy are vimses, most preferably adenovimses, AAV, lentivimses, or retrovimses.
• a viral constmct such as a retroviral construct includes at least one transcriptional promoter/enhancer or locus -defining element(s), or other elements that control gene expression by other means such as alternate splicing, nuclear RNA export, or post- translational modification of messenger.
• Such vector constructs also include a packaging signal, long terminal repeats (LTRs) or portions thereof, and positive and negative strand primer binding sites appropriate to the vims used, unless it is already present in the viral construct.
• a constmct typically includes a signal sequence for secretion of the peptide from a host cell in which it is placed.
• the signal sequence for this pti ⁇ ose is a mammalian signal sequence or the signal sequence of the polypeptide variants of the present invention.
• the construct may also include a signal that directs polyadenylation, as well as one or more restriction sites and a translation termination sequence.
• constmcts will typically include a 5' LTR, a tRNA binding site, a packaging signal, an origin of second- strand DNA synthesis, and a 3' LTR or a portion thereof.
• Other vectors can be used that are non- viral, such as cationic lipids, polylysine, and dendrimers.
• the present methodology may also be effected by specifically upregulating the expression of the variants of the present invention endogenously in the subject.
• Agents for upregulating endogenous expression of specific splice variants of a given gene include antisense oligonucleotides, which are drected at splice sites of interest, thereby altering the splicing pattern of the gene.
• Two alternatively spliced isoforms are generated from the IL-5R gene, which include (i.e., long form) or exclude (i.e., short form) exon 9.
• the long form encodes for the intact membrane-bound receptor, while the shorter form encodes for a secreted soluble nonfunctional receptor.
• 2-O-MOE-oligonucleotides specific to regions of exon 9 Kanas and co-workers (supra) were able to significantly decrease the expression of the wild type receptor and increase the expression of the shorter isoforms.
• Design and synthesis of oligonucleotides which can be used according to the present invention are described hereinbelow and by Sazani and Kole (2003) Progress in Molecular and Subcellular Biology 31:217-239.
• upregulation may be effected by administering to the subject at least one polypeptide agent of the polypeptides of the present invention or an active portion thereof, as described hereinabove.
• administration of polypeptides is preferably confined to small peptide fragments (e.g, about 100 amino acids).
• An agent capable of up regulating a BNP polypeptide may also be any compound which is capable of increasing the transcription and/or translation of an endogenous DNA or mRNA encoding the BNP polypeptide and thus increasing endogenous BNP activity.
• An agent capable of upregulating a BNP may also be an exogenous polypeptide including at least a functional portion (as described hereinabove) of the BNP.
• Upregulation of BNP can be also achieved by introducing at least one BNP substrate.
• Non- limiting examples of such agents include HOXC10 (Gabellini D, et al, 2003; EMBO J. 22: 3715-24), human securin and cyclin Bl (Tang Z, et al, 2001; Mol. Biol. Cell. 12: 3839- 51), cyclins A, geminin H, and Cut2p (Bastians H, et al, 1999; Mol. Biol. Cell. 10: 3927- 3941).
• BNP-expressing cells can be any suitable cells, such as lung, ovary, bone manow which are derived from the individual and are transfected ex vivo with an expression vector containing the polynucleotide designed to express BNP as described hereinabove.
• Administration of the BNP- expressing cells of the present invention can be effected using any suitable route such as intravenous, intra peritoneal, and intra ovary.
• the BNP-expressing cells of the present invention are introduced to the individual using intravenous and/or intra organ administrations.
• BNP-expressing cells of the present invention can be derived from either autologous sources such as self bone manow cells or from allogeneic sources such as bone manow or other cells derived from non-autologous sources. Since non-autologous cells are likely to induce an immune reaction when administered to the body several approaches have been developed to reduce the likelihood of rejection of non-autologous cells. These include either suppressing the recipient immune system or encapsulating the non-autologous cells or tissues in immunoisolating, semipermeable membranes before transplantation. Encapsulation techniques are generally classified as microencapsulation, involving small spherical vehicles and macroencapsulation, involving larger flat- sheet and hollow- fiber membranes (Uludag, H. et al.
• microcapsules are known in the arts and include for example those disclosed by Lu MZ, et al. Cell encapsulation with alginate and alpha - phenoxycinnamylidene-acetylate poly(allylamine). Biotechnol Bioeng. 2000, 70: 479-83, Chang TM and Prakash S. Procedures for microencapsulation of enzymes, cells and genetically engineered microorganisms. Mol Biotechnol.
• microcapsules are prepared by complexing modified collagen with a ter-polymer shell of 2-hydroxyethyl methylacrylate (HEMA), methacrylic acid (MAA) and methyl methacrylate (MMA), resulting in a capsule thickness of 2-5 ⁇ m.
• HEMA 2-hydroxyethyl methylacrylate
• MAA methacrylic acid
• MMA methyl methacrylate
• Such microcapsules can be further encapsulated with additional 2-5 ⁇ m ter-polymer shells in order to impart a negatively charged smooth surface and to minimize plasma protein abso ⁇ tion (Chia, S.M.
• microcapsules are based on alginate, a marine polysaccharide (Sambanis, A. Encapsulated islets in diabetes treatment. Diabetes Thechnol. Ther. 2003, 5: 665-8) or its derivatives.
• microcapsules can be prepared by the polyelectrolyte complexation between the polyanions sodium alginate and sodium cellulose sulphate with the polycation poly(methylene-co-guanidine) hydrochloride in the presence of calcium chloride. It will be appreciated that cell encapsulation is improved when smaller capsules are used.
• Downregulating methods and agents Downregulation of BNP can be effected on the genomic and/or the transcript level using a variety of molecules which interfere with transcription and/or translation (e.g, antisense, siRNA, Ribozyme, DNAzyme), or on the protein level using e.g, antagonists, enzymes that cleave the polypeptide and the like.
• agents capable of downregulating expression level and/or activity of BNP are an antibody or antibody fragment capable of specifically binding BNP.
• the antibody specifically binds at least one epitope of a BNP as described hereinabove.
• RNA interference is a two step process.
• the first step which is termed as the initiation step, input dsRNA is digested into 21 -23 nucleotide (nt) small interfering RNAs (siRNA), probably by the action of Dicer, a member of the RNase III family of dsRN A- specific ribonucleases, which processes (cleaves) dsRNA (introduced directly or via a transgene or a vims) in an ATP -dependent manner.
• nt nucleotide
• siRNA small interfering RNAs
• siRNA duplexes bind to a nuclease complex to from the
• RNA- induced silencing complex An ATP -dependent unwinding of the siRNA duplex is required for activation of the RISC.
• the active RISC then targets the homologous transcript by base pairing interactions and cleaves the mRNA into 12 nucleotide fragments from the 3' terminus of the siRNA [Hutvagner and Zamore Cun. Opin. Genetics and Development 12:225-232 (2002); Hammond et al. (2001) Nat. Rev. Gen. 2:110-119 (2001); and Sha ⁇ Genes. Dev. 15:485-90 (2001)].
• each RISC contains a single siRNA and an RNase [Hutvagner and Zamore Cun. Opin. Genetics and Development 12:225-232 (2002)]. Because of the remarkable potency of RNAi, an amplification step within the RNAi pathway has been suggested. Amplification could occur by copying of the input dsRNAs which would generate more siRNAs, or by replication of the siRNAs formed. Alternatively or additionally, amplification could be effected by multiple turnover events of the RISC [Hammond et al. Nat. Rev. Gen. 2:110-119 (2001), Sha ⁇ Genes. Dev. 15:485-90 (2001); Hutvagner and Zamore Cun.
• RNAi molecules suitable for use with the present invention can be effected as follows. First, the BNP transcript mRNA sequence is scanned downstream of the AUG start codon for AA dinucleotide sequences. Occunence of each AA and the 3' adjacent 19 nucleotides is recorded as potential siRNA target sites.
• siRNA target sites are selected from the open reading frame, as untranslated regions (UTRs) are richer in regulatory protein binding sites.
• UTR-binding proteins and/or translation initiation complexes may interfere with binding of the siRNA endonuclease complex [Tuschl, T. 2001, ChemBiochem. 2:239-245].
• siRNAs directed at untranslated regions may also be effective, as demonstrated for GAPDH wherein siRNA directed at the 5' UTR mediated about 90 % decrease in cellular GAPDH mRNA and completely abolished protein level (www.ambion.com/techlib/tn/91/912.html).
• potential target sites are compared to an appropriate genomic database (e.g, human, mouse, rat etc.) using any sequence alignment software, such as the BLAST software available from the NCBI server (www.ncbi.nlm.nih.gov/BLAST/). Putative target sites which exhibit significant homology to other coding sequences are filtered out. Qualifying target sequences are selected as template for siRNA synthesis. Prefened sequences are those including low G/C content as these have proven to be more effective in mediating gene silencing as compared to those with G/C content higher than 55 %. Several target sites are preferably selected along the length of the target gene for evaluation. For better evaluation of the selected siRNAs, a negative control is preferably used in conjunction.
• Negative control siRNA preferably include the same nucleotide composition as the siRNAs but lack significant homologyto the genome. Thus, a scrambled nucleotide sequence of the siRNA is preferably used, provided it does not display any significant homology to any other gene.
• Another agent capable of downregulating a BNP transcript is a DNAzyme molecule capable of specifically cleaving an mRNA transcript or DNA sequence of the BNP. DNAzymes are single- stranded polynucleotides which are capable of cleaving both single and double stranded target sequences (Breaker, R.R. and Joyce, G. Chemistry and Biology 1995;2:655; Santoro, S.W. & Joyce, G.F. Proc. Natl, Acad. Sci. USA 1997;943:4262). A general model (the "10-23" model) for the DNAzyme has been proposed. "10-23"
• DNAzymes have a catalytic domain of 15 deoxyribonucleotides, flanked by two substrate- recognition domains of seven to nine deoxyribonucleotides each. This type of DNAzyme can effectively cleave its substrate RNA at ⁇ urine:pyrimidine junctions (Santoro, S.W. & Joyce, G.F. Proc. Natl, Acad. Sci. USA 199; for rev of DNAzymes see Kliachigian, LM [Cun Opin Mol Ther 4: 1 19-21 (2002)]. Examples of constmction and amplification of synthetic, engineered DNAzymes recognizing single and double -stranded target cleavage sites have been disclosed in U.S. Pat. No.
• DNAzymes of similar design directed against the human Urokinase receptor were recently observed to inhibit Urokinase receptor expression, and successfully inhibit colon cancer cell metastasis in vivo (Itoh et al, 20002, Abstract 409, Ann Meeting Am Soc Gen Ther. www.asgt.org).
• DNAzymes complementary to bcr-abl oncogenes were successful in inhibiting the oncogenes expression in leukemia cells, and lessening relapse rates in autologous bone manow transplant in cases of CML and ALL.
• Downregulation of a BNP transcript can also be effected by using an antisense polynucleotide capable of specifically hybridizing with an mRNA transcript encoding the BNP.
• Design of antisense molecules which can be used to efficiently downregulate a BNP must be effected while considering two aspects important to the antisense approach. The first aspect is delivery of the oligonucleotide into the cytoplasm of the appropriate cells, while the second aspect is design of an oligonucleotide which specifically binds the designated mRNA within cells in a way which inhibits translation thereof.
• antisense oligonucleotides suitable for the treatment of cancer have been successfully used [Hohnund et al, Cun Opin Mol Ther 1 :372-85 (1999)], while treatment of hematological malignancies via antisense oligonucleotides targeting c- myb gene, p53 and BcL2 had entered clinical trials and had been shown to be tolerated by patients [Gerwitz Cun Opin Mol Ther 1:297-306 (1999)]. More recently, antsense- mediated suppression of human heparanase gene expression has been reported to inhibit pleural dissemination of human cancer cells in a mouse model [Uno et al, Cancer Res 61:7855-60 (2001)].
• Another agent capable of downregulating a BNP transcript is a ribozyme molecule capable of specifically cleaving an mRNA transcript encoding a BNP. Ribozymes are being increasingly used for the sequence- specific inhibition of gene expression by the cleavage of mRNAs encoding proteins of interest [Welch et al, Cun Opin Biotechnol.
• ribozymes have been exploited to target viral RNAs in infectious diseases, dominart oncogenes in cancers and specific somatic mutations in genetic disorders [Welch et al, Clin Diagn Virol. 10:163-71 (1998)]. Most notably, several ribozyme gene therapy protocols for HIV patients are already in Phase 1 trials. More recently, ribozymes have been used for transgenic animal research, gene target validation and pathway elucidation. Several ribozymes are in various stages of clinical trials.
• ANGIOZYME was the first chemically synthesized ribozyme to be studied in human clinical trials. ANGIOZYME specifically inhibits formation of the VEGF-r (Vascular Endothelial Growth Factor receptor), a key component in the angiogenesis pathway. Ribozyme Pharmaceuticals, Inc., as well as other firms have demonstrated the importance of anti-angiogenesis therapeutics in animal models.
• HEPTAZYME a ribozyme designed to selectively destroy Hepatitis C Vims (HCV) RNA, was found effective in decreasing Hepatitis C viral RNA in cell culture assays (Ribozyme Pharmaceuticals, Inco ⁇ orated - WEB home page).
• Another agent capable of downregulating BNP would be any molecule which binds to and/or cleaves BNP. Such molecules can be BNP antagonists, or BNP inhibitory peptide. It will be appreciated that a non- functional analogue of at least a catalytic or binding portion of BNP can be also used as an agent which downregulates BNP. Another agent which can be used along with the present invention to downregulate BNP is a molecule which prevents BNP activation or substrate binding.
• Each of the upregulating or downregulating agents described hereinabove or the expression vector encoding BNP can be administered to the individual per se or as part of a pharmaceutical composition which also includes a physiologically acceptable carrier.
• the pu ⁇ ose of a pharmaceutical composition is to facilitate administration of the active ingredient to an organism.
• a "pharmaceutical composition” refers to a preparation of one or more of the active ingredients described herein with other chemical components such as physiologically suitable carriers and excipients.
• the pu ⁇ ose of a pharmaceutical composition is to facilitate administration of a compound to an organism.
• active ingredient refers to the preparation accountable for the biological effect.
• physiologically acceptable canier and “pharmaceutically acceptable carrier” wliich may be interchangeably used refer to a carrier or a diluent that does not cause significant initation to an organism and does not abrogate the biobgical activity and properties of the administered compound.
• an adjuvant is included under these phrases.
• One of the ingredients included in the pharmaceutically acceptable carrier can be for example polyethylene glycol (PEG), a biocompatible polymer with a wide range of solubility in both organic and aqueous media (Mutter et al. (1979).
• excipient refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient. Examples, without limitation, of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols. Techniques for formulation and administration of drags may be found in
• Suitable routes of administration may, for example, include oral, rectal, transmucosal, especially transnasal, intestinal or parenteral delivery, including intramuscular, subcutaneous and intramedullary injections as well as intrathecal, direct intraventricular, intravenous, inrtaperitoneal, intranasal, or intraocular injections.
• one may administer a preparatio n in a local rather than systemic manner, for example, via injection of the preparation directly into a specific region of a patient's body.
• Phamiaceutical compositions of the present invention may be manufactured by processes well known in the art, e.g, by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.
• Phamiaceutical compositions for use in accordance with the present invention may be formulated in conve ntional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into preparations which, can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.
• the active ingredients of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological salt buffer.
• physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological salt buffer.
• penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
• the compounds can be formulated readily by combining the active compounds with pharmaceutically acceptable caniers well known in the art. Such caniers enable the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for oral ingestion by a patient.
• Pharmacological preparations for oral use can be made using a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries if desired, to obtain tablets or dragee cores.
• Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacantii, methyl cellulose, hydroxypropylmethy cellulose, sodium carbomethylcellulose; and/or physiologically acceptable polymers such as polyvinylpymolidone (PVP).
• PVP polyvinylpymolidone
• disintegrating agents may be added, such as cross- linked polyvinyl pynolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
• Dragee cores are provided with suitable coatings.
• suitable coatings For this pu ⁇ ose, concentrated sugar solutions may be used which may optionally contain gum arabic, talc, polyvinyl pynolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures.
• Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.
• compositions which can be used orally, include push fit capsules made of gelatin as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol.
• the push- fit capsules may contain the active ingredients in admixture with filler such as lactose, binders such as starches, lubricants such as talc or magnesium stearate and, optionally, stabilizers.
• the active ingredients may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols.
• stabilizers may be added. All formulations for oral administration should be in dosages suitable for the chosen route of administration.
• compositions may take the form of tablets or lozenges formulated in conventional manner.
• active ingredients for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from a pressurized pack or a nebulizer with the use of a suitable propellant, e.g, dichlorodifluoromethane, trichlorofluoromefhane, dichloro-tetrafluoroefhane or carbon dioxide.
• a suitable propellant e.g, dichlorodifluoromethane, trichlorofluoromefhane, dichloro-tetrafluoroefhane or carbon dioxide.
• the dosage unit may be determined by providing a valve to deliver a metered amount.
• Capsules and cartridges of, e.g, gelatin for use in a dispenser may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
• the preparations described herein may be formulated for parenteral administration, e.g, by bolus injection or continuous infusion.
• Formulations for injection may be presented in unit dosage form, e.g, in ampoules or in multidose containers with optionally, an added preservative.
• the compositions may be suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
• Pharmaceutical compositions for parenteral administration include aqueous solutions of the active preparation in water-soluble fonn.
• suspensions of the active ingredients may be prepared as appropriate oily or water based injection suspensions.
• Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acids esters such as ethyl oleate, triglycerides or liposomes.
• Aqueous injection suspensions may contain substances, which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran.
• the suspension may also contain suitable stabilizers or agents which increase the solubility of the active ingredients to allow for the preparation of highly concentrated solutions.
• the active ingredient may be in powder form for constitution with a suitable vehicle, e.g, sterile, pyrogenfree water based solution, before use.
• compositions suitable for use in context of the present invention include compositions wherein the active ingredients are contained in an amount effective to achieve the intended pturpose. More specifically, a therapeutically effective amount means an amount of active ingredients effective to prevent, alleviate or ameliorate symptoms of disease or prolong the survival of the subject being treated. Determination of a therapeutically effective amount is well within the capability of those skilled in the art. For any preparation used in the methods of the invention, the therapeutically effective amount or dose can be estimated initially from in vitro assays.
• a dose can be formulated in animal models and such information can be used to more accurately determine useful doses in humans.
• Toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro, in cell cultures or experimental animals. The data obtained from these in vitro and cell culture assays and animal studies can be used in formulating a range of dosage for use in human.
• the dosage may vary depending upon the dosage form employed and the route of administration utilized. The exact fonuulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See e.g, Fingl, et al, 1975, in "The Pha ⁇ nacological Basis of Therapeutics", Ch. 1 p.l).
• dosing can be of a single or a plurality of administrations, with course of treatment lasting from several days to several weeks or until cure is effected or diminution of the disease state is achieved.
• the amount of a composition to be administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc.
• Compositions including the preparation of the present invention formulated in a compatible pharmaceutical earner may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition.
• compositions of the present invention may, if desired, be presented in a pack or dispenser device, such as an FDA approved kit, which may contain one or more unit dosage forms containing the active ingredient.
• the pack may, for example, comprise metal or plastic foil, such as a blister pack.
• the pack or dispenser device may be accompanied by instructions for administration.
• the pack or dispenser may also be accommodated by a notice associated with the container in a fonn prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the compositions or human or veterinary administration.
• Such notice for example, may be of labeling approved by the U.S. Food and Drag Administration for prescription drags or of an approved product insert.
• GenBank sequences the human EST sequences from the EST (GBEST) section and the human mRNA sequences from the primate (GBPRI) section were used; also the human nucleotide RefSeq mRNA sequences were used (see for example www.ncbi.nlm.nih.gov/Genbank/GenbankOverview.html and for a reference to the EST section, see www.ncbi.nlm.nih.gov/dbEST/; a general reference to dbEST, the EST database in GenBank, may be found in Boguski et al, Nat Genet.
• Novel splice variants were predicted using the LEADS clustering and assembly system as described in Sorek, R, Ast, G. & Graur, D. Alu-containing exons are alternatively spliced. Genome Res 12, 1060-7 (2002); US patent No: 6,625,545; and U.S. Pat. Appl. No. 10/426,002, published as US20040101876 on May 27 2004; all of which are hereby inco ⁇ orated by reference as if fully set forth herein. Briefly, the software cleans the expressed sequences from repeats, vectors and immunoglobulins.
• the GeneCarta platform includes a rich pool of annotations, sequence information (particularly of spliced sequences), chromosomal information, alignments, and additional information such as SNPs, gene ontology terms, expression profiles, functional analyses, detailed domain structures, known and predicted proteins and detailed homology reports.
• SNPs sequence information
• chromosomal information chromosomal information
• alignments and additional information such as SNPs, gene ontology terms, expression profiles, functional analyses, detailed domain structures, known and predicted proteins and detailed homology reports.
• the potential markers were identified by a computational process that was designed to find genes and/or their sphce variants that are specifically expressed in cardiac tissue, as opposed to other types of tissues and also particularly as opposed to muscle tissue, by using databases of expressed sequences.
• EXAMPLE 1 Identification of differentially expressed gene products - Algorithm
• an algorithm based on an analysis of frequencies was configured.
• a specific algorithm for identification of transcripts specifically expressed in heart tissue is described hereinbelow.
• EST analysis ESTs were taken from the following main sources: libraries contained in Genbank version 136 (June 15, 2003 ftp.ncbi.nih.gov/genbank/release.notes/gbl36.release.notes) and Genbank version 139 (December 2003); and from the EST portion of LifeSeq, from Incyte Coiporation (Wilmington, DE, USA). With regard to GenBank sequences, the human EST sequences from the EST (GBEST) section were used. Library annotation - EST libraries were manually classified according to: 1. Tissue origin 2.
• Biological source- Examples of frequently used biological sources for construction of EST libraries include cancer cell- lines; normal tissues; cancer tissues; foetal tissues; and others such as nonnal cell lines and pools of normal cell- lines, cancer cell- lines and combinations thereof. A specific description of abbreviations used below with regard to these tissues/cell lines etc is given above.
• Protocol of library construction various methods are known in the art for library construction including normalized library construction; non- normalized library construction; subtracted libraries; ORESTES and others (described in the annotation available in Genbank). It will be appreciated that at times the protocol of library construction is not indicated in the information available about that library.
• the following rales were followed: EST libraries originating from identical biological samples were considered as a single library. EST libraries which included above -average levels of contamination, such as DNA contamination for example, were eliminated. The presence of such contamination was determined as follows. For each library, the number of unspliced ESTs that are not fully contained within other spliced sequences was counted.
• Heart tissue libraries/sequences were compared to the total number of libraries/sequences in the cluster and in Genebank, and to the relevant numbers for muscle tissue libraries/sequences.
• Statistical tools were employed to identify clusters that were heart tissue specific, both as compared to all other tissues and also in comparison to muscle tissue.
• the algorithm - for each tested tissue T and for each tested cluster the following were examined: 1.
• Each cluster includes at least 2 libraries from the tissue T. At least 3 clones (weighed - as described above) from tissue T in the cluster; 2.
• n is the total number of ESTs available for a cluster
• N is the total number of ESTs available in all of the libraries considered in the analysis (effectively all ESTs in Genbank, except for those that were rejected as belonging to contaminated libraries).
• This ratio was preferably set to be at least about 8, although optionally the ratio could be set to be at least about 5. 3.
• the following equation was then used to determine heart tissue -specific expression
• Example 3 Experimental and Marker Data This Example relates to examples of sequences according to the present invention, including experiments involving these sequences, and illustrative, non- limiting examples of methods, assays and uses thereof. The materials and experimental procedures are explained first, as all experiments used them as a basis for the work that was performed. The markers of the present invention were tested with regard to their expression in various heart and non- heart tissue samples. Unless otherwise noted, all experimental data relates to variants of the present invention, named according to the segment being tested (as expression was tested through RT-PCR as described). A description of the samples used in the panel is provided in Table 1 below. Tests were then performed as described below.
• RNA preparation - RNA was obtained from Clontech (Franklin Lakes, NJ USA).
• Real-Time RT-PCR analysis- cDNA (5 ⁇ l), prepared as described above, was used as a template in Real-Time PCR reactions using the SYBDR Green I assay (PE Applied Biosystem) with specific primers and UNG Enzyme (Eurogentech or ABI or Roche).
• the amplification was effected as follows: 50 °C for 2 min., 95 °C for 10 min, and then 40 cycles of 95 °C for 15sec, followed by 60 °C for 1 min. Detection was performed by using the PE Applied Biosystem SDS 7000.
• RPL19 (GenBank Accession No. NM _000981), RPLl 9 Forward primer (SEQ ID NO:30): TGGCAAGAAGAAGGTCTGGTTAG RPL19 Reverse primer (SEQ ID NO:31) : TGATCAGCCCATCTTTGATGAG RPL19 -amplicon (SEQ ID NO:32):
• TATA box Forward primer SEQ ID NO:33
• Reverse primer SEQ ID NO:34
• -amplicon SEQ ID NO:35
• Ubiquitin Forward primer SEQ ID NO:36
• Ubiquitin Reverse primer SEQ ID NO:37
• SDHA Forward primer (SEQ ID NO:39): TGGGAACAAGAGGGCATCTG SDHA Reverse primer (SEQ ID NO:40) : CCACCACTGCATCAAATTCATG SDHA-amplicon (SEQ ID NO:41) :
• Cluster HUMNATPEP features 4 transcript(s) and 7 segment(s) of interest, the names for which are given in Tables 1 and 2, respectively, the sequences themselves are given at the end of the application.
• the selected protein variants are given in table 3.
• Natriuretic peptides B precursor Contains: Gamma-brain natriuretic peptide; Brain natriuretic peptide 32 (BNP-32)] (SwissProt accession identifier ANFBJHUMAN), refened to herein as the previously known protein.
• Protein Natriuretic peptides B precursor [Contains: Gamma-brain natriuretic peptide; Brain natriuretic peptide 32 (BNP-32)] is known or believed to have the following function(s): Acts as a cardiac hormone with a variety of biological actions including natriuresis, diuresis, vasorelaxation, and inhibition of renin and aldosterone secretion. It is thought to play a key role in cardiovascular homeostasis. He lps restore the body's salt and water balance. Improves heart function.
• Natriuretic peptides B precursor contains: Gamma-brain natriuretic peptide; Brain natriuretic peptide 32 (BNP- 32)] is given at the end of the application, as "Natriuretic peptides B precursor [Contains: Gamma-brain natriuretic peptide; Brain natriuretic peptide 32 (BNP-32)] amino acid sequence" (SEQ ID NO: 12).
• Known polymo ⁇ hisms for this sequence are as shown in Table 4.
• Protein Natriuretic peptides B precursor [Contains: Gamma-brain natriuretic peptide; Brain natriuretic peptide 32 (BNP-32)] localization is believed to be Secreted.
• the previously known protein also has the following indication(s) and/or potential therapeutic use(s): Hepatic dysfunction, general; Hypertension, general; Heart failure; Asthma; Renal failure. It has been investigated for clinical/therapeutic use in humans, for example as a target for an antibody or small molecule, and/or as a direct therapeutic; available information related to these investigations is as follows.
• Potential pharmaceutically related or therapeutically related activity or activities of the previously known protein are as follows: Atrial peptide agonist; Diuretic. A therapeutic role for a protein represented by the cluster has been predicted.
• the cluster was assigned this field because there was information in the drag database or the public databases (e.g., described herein above) that this protein, or part thereof, is used or can be used for a potential therapeutic indication: Hepatopiotective; Antihypertensive; Antihypertensive, diuretic; Cardiostimulant; Vasodilator, coronary; Urological; Antiasfhma; COPD treatment.
• Hepatopiotective Antihypertensive
• Antihypertensive diuretic
• Cardiostimulant Vasodilator, coronary
• Urological Antiasfhma
• COPD treatment The following GO Annotation(s) apply to the previously known protein.
• the GO assignment relies on information from one or more of the SwissProt/TremBl Protein knowledgebase, available from ⁇ http://www.expasy.ch/sproA; or Locuslink, available from ⁇ http://www.ncbi.nlm.nih.gov/projects/LocusLink/>.
• the heart-selective diagnostic marker prediction engine provided the following results with regard to cluster HUMNATPEP. Predictions were made for selective expression of transcripts of this clusterin heart tissue, according to the previously described methods.
• the numbers on the y-axis of Figure 2 below refer to weighted expression of ESTs in each category, as "parts per million” (ratio of the expression of ESTs for a particular cluster to the expression of all ESTs in that category, according to parts per million).
• the following results were obtained as shown with regard to the histogram in Figure 2, concerning the number of heart-specific clones in libraries/sequences.
• This cluster was found to be selectively expressed in heart for the following reasons: in a comparison of the ratio of expression of the cluster in heart specific ESTs to the overall expression of the cluster in non-heart ESTs, which was found to be 18.3. Also the expression levels of this gene in muscle was negligible; and fisher exact test P-values were computed both for library and weighted clone counts to check that the counts are statistically significant, and were found to be 3.40E-17.
• One particularly important measure of specificity of expression of a cluster in heart tissue is the previously described comparison of the ratio of expression of the cluster in heart as opposed to muscle. This cluster was found to be specifically expressed in heart as opposed to non-heart ESTs as described above. However, many proteins have been shown to be generally expressed at a higlier level in both heart and muscle, which is less desirable. For this cluster, as described above, the expression levels of this gene in muscle was negligible , which clearly supports specific expression in heart tissue.
• cluster HUMNATPEP features 4 transcript(s), which were listed in
• transcript(s) encode for protein(s) which are variant(s) of protein Natriuretic peptides B precursor [Contains: Gamma-brain natriuretic peptide; Brain natriuretic peptide 32 (BNP-32)]. A description of each variant protein according to the present invention is now provided.
• Variant protein HUMNATPEP_PEA_1_P2 has an amino acid sequence as given at the end of the application; it is encoded by transcript(s) HU NATPEP_PEA_1_T1.
• BNP splice variant HUMNATPEP_PEA JMT1 results from alternative splicing of the BNP gene, thus leading to the extension of exon 2 into the intron and to the generation of an extended 162 amino acid long protein, compared to the 134 amino acid long wild type protein.
• the protein encoded by this transcript contains the signal P (signal peptide) and the complete natriuretic peptide domain plus a unique sequence of 33 amino acids in its C-terminus.
• Natriuretic peptides B precursor Contains: Gamma-brain natriuretic peptide; Brain natriuretic peptide 32 (BNP-32)]
• BNP-32 Brain natriuretic peptide 32
• HUMNATPEP_PEA_1_P2 Comparison report between HUMNATPEP_PEA_1_P2 and ANFBJHUMAN: l.An isolated chimeric polypeptide encoding for HUMNATPEP_PEA_1_P2, comprising a first amino acid sequence being at least 90 % homologous to MDPQTAPSRALLLLLFLHLAFLGGRSHPLGSPGSASDLETSGLQEQRNHLQGKLSEL QVEQTSLEPLQESPRPTGVWKSREVATEGIRGHRKMVLYTLRAPRSPKMVQGSGCF GRKMDRISSSSGLGCK conesponding to amino acids 1 - 129 of ANFB ⁇ UMAN, which also conesponds to amino acids 1 - 129 of HUMNATPEP_PEA_1_P2, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide
• HUMNATPEP ⁇ A JP2- According to another aspect of the present invention there is provided a bridge fragment of HUMNATPEP_PEA_1_P2 between 10 and 50 amino acids in length that spans the first and second amino acid sequences described above.
• a bridge portion of HUMNATPEP_PEA_1_P2 comprising a polypeptide having a length "n", wherein n is at least about 10 amino acids in length, optionally at least about 20 amino acids in length, preferably at least about 30 amino acids in length, more preferably at least about 40 amino acids in length and most preferably at least about 50 amino acids in length, wherein at least two amino acids comprise KG, having a structure as follows (numbering according to SEQ ID NO:l): a sequence starting from any of amino acid numbers 129-x to 129; ending at any of amino acid numbers 130 + ((n-2) - x), in which x varies from 0 to n-2, such that the value ((n-2) - x) is not
• the bridge portion above may also optionally comprise a polypeptide being at least
• the bridge portion may optionally be relatively short, such as from about 4 to about 9 amino acids in length.
• the first bridge portion would comprise the following peptides: GCKG, CKGK, KGKH. All peptides feature KG as a portion thereof. Peptides of from about five to about nine amino acids could optionally be similarly constructed.
• the location of the variant protein was determined according to results from a number of different software programs and analyses, including analyses from SignalP and other specialized programs.
• the variant protein is believed to be located as follows with regard to the cell: secreted.
• the protein localization is believed to be secreted because both signal- peptide prediction programs predict that this protein has a signal peptide, and neither trans - membrane region prediction program predicts that this protein has a trans-membrane region..
• the variant protein has the following domains, as determined by using InterPro. The domains are described in Table 6: Table 6 -InterPro domain(s)
• Figure 3 A shows a comparison of the genomic structure for the variant transcript HUMNATPEP_PEA_1_T1 and the known or "WT" transcript.
• Figure 3B shows a comparison of the stmcture of the variant protein HUMNATPEP_PEA_1_P2 hi comparison to the stmcture of the known or "WT" protein.
• Variant protein HUMNATPEP_PEA _P2 also has the following non-silent SNPs (Single Nucleotide Polymo ⁇ hisms) as listed in Table 7, (given according to their position(s) 89 on the amino acid sequence, with the alternative amino acid(s) listed; the last column indicates whether the SNP is known or not; the presence of known SNPs in variant protein HUMNATPEP_PEA_1_P2 sequence provides support for the deduced sequence of this variant protein according to the present invention).
• Table 7 -Amino acid mutations Single Nucleotide Polymo ⁇ hisms
• Variant protein HUMNATPEP_PEA_1_P2 is encoded by the following transcript(s): HUMNATPEP_PEA_1 _T1 > for which the sequence(s) is/are given at the end of the application.
• the coding portion of transcript HUMNATPEP_PEA_1 H is shown in bold; this coding portion starts at position 249 and ends at position 734.
• the transcript also has the following SNPs as listed in Table 8 (given according to their position on the nucleotide sequence, with the alternative nucleic acid listed; the last column indicates whether the SNP is known or not; the presence of known SNPs in variant protein HUMNATPEP_PEA_1_P2 sequence provides support for the deduced sequence of this variant protein according to the present invention).
• Variant protein HUMNATPEP_PEA_1_P3 has an amino acid sequence as given at the end of the application; it is encoded by transcript(s) HUMNATPEP_PEA_1_T2 and HUMNATPEP_PEA_1_T3.
• An alignment is given to the known protein (Natriuretic peptides B precursor [Contains: Gamma-brain natriuretic peptide; Brain natriuretic peptide 32 (BNP-32)]) at the end of the application.
• One or more alignments to one or more previously published protein sequences are given at the end of the application.
• Comparison report between HUMNATPEP_PEA_1_P3 and ANFBJrlUMAN l.An isolated chimeric polypeptide encoding for HUMNATPEP_PEA_1_P3, comprising a first amino acid sequence being at least 90 % homologous to MDPQTAPSRALLLLLFLHLAFLGGRSHPLGSPGSASDLETSGLQ conesponding to amino acids 1 - 44 of ANFBJHUMAN, which also conesponds to amino acids 1 - 44 of HUMNATPEP_PEA_1_P3, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence VRAEGSSGGLDSSNERVLTCCPKRPSSFLWN conesponding to amino acids 45 - 75 of HUMNATPEP_PE
• the location of the variant protein was determined according to results from a number of different software programs and analyses, including analyses from SignalP and other specialized programs.
• the variant protein is believed to be located as follows with regard to the cell: secreted.
• the protein localization is believed to be secreted because both signal- peptide prediction programs predict that this protein has a signal peptide, and neither trans - membrane region prediction program predicts that this protein has a trans -membrane region..
• Variant protein HUMNATPEP_PEA_1_P3 also has the following non-silent SNPs (Single Nucleotide Polymo ⁇ hisms) as listed in Table 9, (given according to their ⁇ osition(s) on the amino acid sequence, with the alternative amino acid(s) listed; the last column indicates whether the SNP is known or not; the presence of known SNPs in variant protein HUMNATPEP PEA_1_P3 sequence provides support for the deduced sequence of this variant protein according to the present invention).
• Table 9 -Amino acid mutations Single Nucleotide Polymo ⁇ hisms
• Variant protein HUMNATPEP_PEA_1 JP3 is encoded by the following transcript(s): HUMNATPEP_PEA_1_T2 and HUMNATPEPJ ⁇ A _1_T3, for which the sequence(s) is/are given at the end of the application.
• the coding portion of transcript HUMNATPEP JPEA_1 T2 and HUMNATPEP_PEA_1 T3 are shown in bold; this coding portio n starts at position 249 and ends at position 473.
• the transcript also has the following SNPs as listed in Table 10 (given according to their position on the nucleotide sequence, with the alternative nucleic acid listed; the last column indicates whether the SNP is known or not; the presence of known SNPs in variant protein HUMNATPEP_PEA_1_P3 sequence provides support for the deduced sequence of this variant protein according to the present invention).
• Table 10 - Nucleic acid SNPs are listed in Table 10 (given according to their position on the nucleotide sequence, with the alternative nucleic acid listed; the last column indicates whether the SNP is known or not; the presence of known SNPs in variant protein HUMNATPEP_PEA_1_P3 sequence provides support for the deduced sequence of this variant protein according to the present invention.
• Variant protein HUMNATPEP_PEA_1 ⁇ Pl has an amino acid sequence as given at the end of the application; it is encoded by transcript(s) HUMNATPEP_PEA_1 T4.
• An alignment is given to the known protein (Natriuretic peptides B precursor [Contains: Gamma-brain natriuretic peptide; Brain natriuretic peptide 32 (BNP-32)]) at the end of the application.
• One or more alignments to one or more previously published protein sequences are given at the end of the application.
• Comparison report between HUMNATPEP_PEA_1_P7 and ANFBJHUMAN 1 n isolated chimeric polypeptide encoding for HUMNATPEP_PEA_1_P7, comprising a first amino acid sequence being at least 90 % homologous to MVLYTLRAJRSPKMVQGSGCFGRKMDRISSSSGLGCKVLRRH conesponding to amino acids 93 - 134 of ANFBJrIUMAN, which also conesponds to amino acids 1 - 42 of HUMNATPEP_PEA_1_P7.
• variant protein HUMNATPEP_PEA_1_P7 is encoded by the fo flowing transcript(s):
• HUMNATPEP_PEA_1_T4 for which the sequence(s) is/are given at the end of the application.
• the coding portion of transcript HUMNATPEP_PEA_1_T4 is shown in bold; this coding portion starts at position 257 and ends at position 382.
• the transcript also has the following SNPs as listed in Table 11 (given according to their position on the nucleotide sequence, with the alternative nucleic acid listed; the last column indicates whether the SNP is known or not; the presence of known SNPs in variant protein HUMNATPEP_PEA_1_P7 sequence provides support for the deduced sequence of this variant protein according to the present invention).
• cluster HUMNATPEP features 7 segment(s), which were listed in Table 2 above and for which the sequence(s) are given at the end of the application. These segment(s) are portions of nucleic acid sequence(s) which are described herein separately because they are of particular interest. A description of each segment according to the present invention is now provided.
• Segment cluster HUMNATPEP_PEA_l_node_0 is supported by 21 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): HUMNATPEP_PEA_1_T1, HUMNATPEP_PEA_1_T2, HUMNATPEP JPEA T3 and HUMNATPEP_PEA _1_T4. Table 12 below describes the starting and ending position of this segment on each transcript. Table 12 - Segment location on transcripts
• Segment cluster HUMNATPEP_PEA_l_node_l is supported by 24 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): HUMNATPEP_PEA_1_T1, HUMNATPEP_PEA_1_T2 and HUMNATPEP_PEA_1_T3- Table 13 below describes the starting and ending position of this segment on each transcript. Table 13 - Segment location on transcripts
• Segment cluster HUMNATPEP_PEA_l_node_2 is supported by 6 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): HUMNATPEP_PEA_1_T2 and HUMNATPEP_PEA_1_T3- Table 14 below describes the starting and ending position of this segment on each transcript. Table 14 - Segment location on transcripts
• Segment cluster HUMNATPEP_PEA_l_node_3 is supported by 25 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): HUMNATPEP_PEA_1_T1 ; HUMNATPEP_PEA_1_T2 and HUMNATPEP_PEA_1_T3- Table 15 below describes the starting and ending position of this segment on each transcript. Table 15 - Segment location on transcripts
• Segment cluster HUMNATPEP_PEA_l_node_4 is supported by 24 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): HUMNATPEP_PEA_1_T1, HUMNATPEP_PEA_1_T2, HUMNATPEP_PEA_1_T3 and HUMNATPEP_PEA_1_T4- Table 1 6 below describes the starting and ending position of this segment on each transcript. Table 16 - Segment location on transcripts
• Segment cluster HUMNATPEP_PEA_l_node_5 is supported by 4 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): HUMNATPEP_PEA_1_T1 an d HUMNATPEP_PEA_1_T3- Table 17 below describes the starting and ending position of this segment on each transcript. Table 17 - Segment location on transcripts
• Segment cluster HUMNATPEP_PEA_l_node_6 is supported by 20 libraries. The number of libraries was determined as previously described. This segment can be found in the foflowing transcript(s): HUMNATPEP_PEA_l_T ⁇ , HUMNATPEP_PEA_1_T2, HUMNATPEP_PEA_1_T3 and HUMNATPEP JPEA_1_T4- Table 18 below describes the starting and ending position of this segment on each transcript. Table 18 - Segment location on transcripts 96
• SEQ ID NO: 17 >HUMNATPEP_PEA_l_P2_peptide SPKMVQGSGCFGRKMDRISSSSGLGCKGKHPLPPRPPSPIPVCDTVRVTLGFVVSGN HTL
• NM _000981; RPL19 amplicon TATA box (GenBank Accession No. NM_003194; TATA amplicon), Ubiquitin (GenBank Accession No. BC000449; amplicon - Ubiquitin- amplicon) and SDHA (GenBank Accession No. NM_004168; amplicon - SDHA- amplicon), was measured similarly.
• TATA box GenBank Accession No. NM_003194; TATA amplicon
• Ubiquitin GeneBank Accession No. BC000449; amplicon - Ubiquitin- amplicon
• SDHA GeneBank Accession No. NM_004168; amplicon - SDHA- amplicon
• FIG. 4 is a histogram showing relative expression of the above- indicated ANFBJHUMAN Natriuretic peptides transcripts in heart tissue samples as opposed to other tissues.
• the expression of ANFB_HUMAN Natriuretic peptides transcripts detectable by the above amplicon in heart tissue samples was significantly higher than in most other samples (Sample Nos. 1 -9, 1 1-22, 24-26 ,28-43 , 47-74 Table 1, "Tissue samples in testing panel” above). Note that the expression of the above amplicon in one of the heart samples, sample no.
• Primer pairs are also optionally and preferably encompassed within the present invention; for example, for the above experiment, the following primer pair was used as a non- limiting illustrative example only of a suitable primer pair: HUMNATPEPseg5 forward primer; and HUMNATPEPseg5 reverse primer.
• the present invention also preferably encompasses any amplicon obtained through the use of any suitable primer pair; for example, for the above experiment, the following amplicon was obtained as a non- limiting illustrative example only of a suitable amplicon: HUMNATPEPseg5.
• HUMNATPEPseg5 Forward primer (SEQ ID NO:18): CTTCCCCCATTCCAGTGTGT HUMNATPEPseg5 Reverse primer (SEQ ID NO: 19): GAGGAAGCGATGTCCAGGTG HUMNATPEPseg5 Amplicon (SEQ ID NO:20): CTTCCCCCATTCCAGTGTGTGACACTGTTAGAGTCACTTTGGGGTTTGTTGTCTCT GGGAACCACACTCTTTGAGAAAAGGTCACCTGGACATCGCTTCCTC
• HUMNATPEPseg2R2 primers was measured by real time PCR.
• HUMNATPEPseg2R2 primers was measured by real time PCR.
• HUMNATPEPseg2R2 primers was measured by real time PCR.
• RPL19 GenBank Accession No. NM_000981; RPL19 amplicon
• TATA box GenBank Accession No.
• Figure 5 is a histogram showing relative expression of the above- indicated ANFB_HUMAN Natriuretic peptides transcripts in heart tissue samples as opposed to other tissues.
• the expression of ANFB HUMAN Natriuretic peptide transcripts detectable by the above amplicon in heart tissue samples was higher than in most of the other samples (Sample Nos. 1-26, 28-43 , 47-74 Table 1).
• sample no. 45 was higher compared to its expression in the other two heart samples (sample 44 and 46).
• Sample no. 45 is from fibrotic heart, and samples 44 and 46 are samples from normal hearts.
• Primer pairs are also optionally and preferably encompassed within the present invention; for example, for the above experiment, the following primer pair was used as a non- limiting illustrative example only of a suitable primer pair: HUMNATPEPseg2F2 forward primer; and HUMNATPEPseg2R2 reverse primer.
• the present invention also preferably encompasses any amplicon obtained through the use of any suitable primer pair; for example, for the above experiment, the following amplicon was obtained as a non- limiting illustrative example only of a suitable amplicon: HUMNATPEPseg2.
• HUMNATPEPseg2F2 (SEQ ID NO:21): GCAGCAATGAAAGGGTCCTC
• HUMNATPEPseg2R2 (SEQ ID NO:22): CATGGCACCCAAGTGAACC Amplicon HUMNATPEPseg2 (SEQ ID NO:23) :
• NPPB Homo sapiens natriuretic peptide precursor B
• HUMNATPEP transcripts which are detectable by amplicon as depicted in sequence name HUMNATPEP seg3 -4WT specifically in heart tissue
• Expression of Homo sapiens natriuretic peptide precursor B (NPPB) transcripts detectable by or according to seg3-4 node(s), HUMNATPEP seg3 -4WT amplicon(s) and primers HUMNATPEP seg3-4WT-F and HUMNATPEP seg3 -4WT-R was measured by real time PCR (this transcript relates to the known protein, or "WT" protein).
• RPL19 GenBank Accession No. NM_000981; RPL19 amplicon
• TATA box GenBanlc Accession No. NM_003194; TATA amplicon
• FIG. 6 is a histogram showing relative expression of the above- indicated Homo sapiens natriuretic peptide precursor B (NPPB) known protein transcripts in heart tissue samples as opposed to other tissues.
• NPPB Homo sapiens natriuretic peptide precursor B
• sample 45 was higher compared to its expression in the other two heart samples (sample 44 and 46).
• Sample no. 45 is from fibrotic heart, and samples 44 and 46 are samples from normal hearts.
• Primer pairs are also optionally and preferably encompassed within the present invention; for example, for the above experiment, the following primer pair was used as a non- limiting illustrative example only of a suiable primer pair: HUMNATPEP seg3 -4WT-F forward primer; and HUMNATPEP seg3 -4WT-R reverse primer.
• the present invention also preferably encompasses any amplicon obtained through the use of any suitable primer pair; for example, for the above experiment, the following amplicon was obtained as a non- limiting illustrative example only of a suitable amplicon: HUMNATPEP seg3-4WT.
• HUMNATPEP seg3-4WT-F (SEQ ID NO:24): GTCCGGGTTACAGGAGCAGC Reverse primer HUMNATPEP seg3 -4WT-R (SEQ ID NO:25): CCGCCTCAGCACTTTGCAG Amplicon HUMNATPEP seg3-4WT (SEQ ID NO:26): GTCCGGGTTACAGGAGCAGCGCAACCATTTGCAGGGCAAACTGTCGGAGCTGCA GGTGGAGCAGACATCCCTGGAGCCCCTCCAGGAGAGCCCCCGTCCCACAGGTGT CTGGAAGTCCCGGGAGGTAGCCACCGAGGGCATCCGTGGGCACCGCAAAATGG TCCTCTACACCCTGCGGGCACCACGAAGCCCCAAGATGGTGCAAGGGTCTGGCT GCTTTGGGAGGAAGATGGACCGGATCAGCTCCTCCAGTGGCCTGGGCTGCAAAG TGCTGAGGCGG
• RT reaction using random hexamers was performed using the standard manufacturer specifications.
• the primers used for RT_PCR were as follows: (a) Forward primer:GTTCAGCCTCGGACTTGGAA ("Primer A”; SEQ ID NO:27) (b) Reverse primer.GTGACTCTAACAGTGTCACACACTGG ("Primer B”; SEQ ID NO:28) (c) Reverse primer (control): CCTTGTGGAATCAGAAGCAGG ("Primer C"; SEQ ID NO:29)
• Primers A and B were used to identify the BNP variant and should produce an amplicon of length 355bp.Primers A and C were used as a control and should produce two amplicons - the first of the wild type, with a length of 352bp and the second of the splice variant, with a length of 893 bp.
• the annealing temperature of the primers was as follows (calculated byAT/GC content):
• RT-PCR in pancreas, normal heart and in heart focal fibrosis samples .
• the product size was as expected, about 352b ⁇ (primers A+C).
• the RT_PCR reactions designed to detect the transcript for HUMNATPEP_PEA_1_P2 splice variant were performed using primers A and B.
• the product of expected size (355 bp) was detected in heart focal fibrosis sample only.
• Example 4 - Therapeutic Uses HUMNATPEP_PEA_1_P2 splice variant contains almost the complete sequence of known BNP (129 a.a out of 133) with the addition of unique sequence of 33 amino acids in its C-tenninus. Structure-function analysis has identified the central ring stmcture in BNP as critical for the binding to its receptor and for its biological functions.
• HUMNATPEP_PEA_1_P2 splice variant may preserve the ring stmcture and therefore retain the biological activity of BNP with an advantage of increased half life due to its longer size.
• the variants of the present invention may optionally be used, additionally or alternatively, for tiierapeutic uses, including but not limited to, diuretic, natriuretic, vascular smooth muscle relaxing and vasodilation actions, and lowering blood volume and blood pressure. These variants may optionally be used for therapeutic treatment of heart failure.
• the variant comprises HUMNATPEP_PEA_1_P2 or a fragment thereof as described herein.

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