JP2002509433A - Nucleic acids specific to cardiac and skeletal muscle, and methods for their production and use - Google Patents

Abstract

  (57) [Summary] The present invention relates to nucleic acids specific for cardiac and skeletal muscle, diagnostic aids, ie methods for their production and use as pharmaceutical preparations, and tests for identifying functional reactants.

Description

DETAILED DESCRIPTION OF THE INVENTION      Nucleic acids specific to cardiac and skeletal muscle, and methods for their production and use   The present invention relates to nucleic acids expressed in human cardiac muscle and skeletal muscle, diagnostic aids, The method of manufacture and use as a pharmaceutical, and the functional reactant identification test .   In the heart, atrial and ventricular contractions (systole) and relaxation (cardiac relaxation) alternate. It is a hollow organ of muscle that has the function of maintaining blood flow in blood vessels by exercise.   The heart muscle, or myocardium, consists of characteristic striated muscle cells with connective tissue in between . Each cell has a central nucleus and is bounded by the plasma membrane, the muscle cell membrane. And contains many contractile myofibrils that are randomly separated by muscle traits. heart Viscera contractiles are formed of long parallel myofibrils. Some myofibrils Are divided into identical structural and functional units, namely sarcomere. Next is the sarcomere A thin filament consisting of actin, tropomyosin and troponin, And a thick filament of myosin.   The molecular mechanism of muscle contraction is spherical myosin head and F-actin filament. Based on the cycle of adhesion and desorption. During electrical stimulation of the heart muscle, the muscle cell membrane Ca from fine mesh²⁺Is released, and this is caused by the allosteric reaction of the troponin complex. And tropomyosin, then actin filaments and myosin A passage opens for head contact. This attachment causes the conformation of myosin A change occurs, which then pulls the actin filament along itself. To reverse these conformational changes and return to the beginning of the contraction cycle ATP is required.   By measuring nerve and hormonal regulation, the assessment of the body's perfusion requirements, Short-term adjustment of myocardial activity to blood flow requirements is possible. And the contraction force Both shrinkage can be increased. Excessive tension over a long period This leads to a physiological alteration process, mainly due to an increase in myofibrils (myocyte hypertrophy). ).   If the heart muscle is compromised, the original physiological adaptation mechanism often becomes chronic over time. It results in heart failure, a condition that causes heart failure, and usually ends with acute heart failure. Everything happens. In the case of severe chronic failure, the heart is no longer adequate to May become unresponsive, with slight physical exertion resulting in exhaustion or shortness of breath Sir.   Myocardial damage is ischemia, that is, coronary artery disease, bacterial or viral infection, poisons, Involved in exsanguination caused by metabolic disorders, autoimmune diseases or genetic defects You. In general, therapeutic measurements increase contractile forces and compensate neurological and hormonal compensators. It is intended to adjust the structure. Despite such treatment, the disease Mortality is still high (35-50% within the first 5 years after diagnosis). Heart failure is the world It is the leading cause of death in the world. The only etiological treatment is heart transplant.   The molecular changes in chronic heart failure are not well known. In particular, heart failure The underlying gene mutation is virtually unknown. Why poisons and viruses Does the next injury cause heart failure in some people but not others? I still can't answer questions.   Thus, the present invention provides for a minimal, if not all, cause of a gene-related heart disease. At least based on the objective of identifying and isolating genes that are partially ing.   Surprisingly, one gene has now been found in the human heart tissue cDNA bank. It is more pronounced and expressed in failing heart tissue than in healthy heart tissue, The gene has been implicated as a cause in associated heart failure. Imperfect and not necessarily functional So-called EST (expressed sequence tag) Already exists as this gene (Tanaka, T. et al. (1996) Genemics, 35, 2). 31-235; EMBL AC: CO4498; clone 3NHC3467).   Accordingly, one aspect of the invention is a polypeptide having the amino acid sequence shown in FIG. Nucleic acid encoding a peptide, or a functional variant thereof, and at least eight nucleic acids Otides, preferably at least 10 nucleotides, especially at least 15 nucleotides Nucleotides, especially parts thereof having at least 20 nucleotides But the array:(Where N represents A, T, G or C) Nucleic acids having are excluded.   The nucleic acids of the invention were isolated from a human heart tissue cDNA bank and sequenced. this First, complete RNA was prepared from healthy and failing heart samples by standard methods. Isolated and then a 3 'anchor primer mixture, e.g. 5'-T₁₂ACN-3 ' Primer (where N represents any of the deoxynucleotides) and reverse Transcribed using transcriptase to obtain cDNA. This cDNA is then converted to a so-called Li Ang and Pardee Differential Display Method (Liang, P. & Parde e, A. (1992) Science 257, 967-970) using the 3 'primer, For example, T₁₂An ACN primer and an optionally selected 5'-decamer primer, Specific P using, for example, 5'-CCTTCTACCC-3 '10-mer primer Amplified under CR conditions. Surprisingly, in healthy heart samples Is absent and 321 base pairs (bp) clearly present in failing heart samples A long DNA fragment could be amplified. Differential display method Or, in a conventional method such as a subtractive cDNA gene bank, The problem is that some clones are poorly expressed and some have false positive clones. That was surprising. Especially weakly expressed only under specific conditions It is possible to identify the gene product of the gene involved. Therefore, generally the hit rate Is very low (10-20%) and, for example, differential display In method a, it depends on the selected PCR conditions, that is, primer length. Or hybridization, for example, in the manufacture of subtractive banks. It is not surprising that it depends on the solution temperature. Then, the found DNA fragment The complete gene was isolated from the cDNA gene bank using and further sequenced .   In all cases, the additional method is such that the found cDNA is active and / or Need to determine if is assigned to a tissue-specific gene. Follow Thus, mRNAs derived from various human tissues in a so-called Northern blot were The amount of mRNA that was hybridized with the found DNA fragment and then bound For example, it was measured by radiolabeling of a DNA fragment. This experiment shows that It is very weak in cardiac and skeletal muscle tissue and in prostate tissue. However, the corresponding RNA was detected. Comparing healthy and failing heart tissue In further experiments, high expression, eg, about 3 in failing tissue compared to healthy tissue, RNA expression increased to 5% was detected. Compared to healthy tissue, Demonstrated that particularly small RNA species preferentially showed increased expression did it. Increased expression of relatively small RNA species can be seen, for example, in Northern blots. It is readily apparent in the form of a double band (see FIG. 5b).   By comparing the derived polypeptide sequence with the protein database, Certain relationships (homologies) with cytoplasmic tropomodulin were further demonstrated (see FIG. 4). ). Tropomodulin is involved in myofibrillar development and cellular regulation in chicken cardiomyocytes. It is known to be a polypeptide that affects contractility (Gregorio et a l. (1995) Nature 377, 83-86). This protein, on the other hand, goes to tropomyosin, On the other hand, it binds to actin fibers, but is not itself regulated in its activity. Guidance The polypeptides of the present invention also include, for example, tropomyosin-binding domains and the like. It has several tropomodulin structural features. Contrast with tropomodulin Preferably, the polypeptide of the present invention is a polypeptide by so-called tyrosine kinase. It has additional structural features that indicate regulation of the activity of (see FIG. 4).   Accordingly, a “functional variant” is functionally related to the polypeptide of the present invention. Ie, also a regulatable modulator of cardiomyocyte contractility, And preferably in myocardial, skeletal and / or prostate tissue, especially myocardial and / or Alternatively, it is expressed in skeletal muscle, and especially in cardiomyocytes, for example, one or more tropomyos Has the structural characteristics of tropomodulin, such as a binding domain, and / or Polypeptides for the purposes of the present invention whose activity can be regulated by tyrosine kinases Means Examples of functional variants include non-human organisms, preferably And corresponding polypeptides from non-human mammals, such as   In a broad sense, “functional variant” refers to sequence homology, particularly the amino acid sequence shown in FIG. About 70%, preferably about 80%, especially about 90%, of a polypeptide having an acid sequence; Specifically, it includes polypeptides having about 95% sequence identity. These are for example non- Encoded by a nucleic acid isolated from a heart-specific tissue, such as skeletal muscle tissue, After expression in a heart-specific cell, it contains a polypeptide having the same function. further , These are about 1 to 60, preferably about 1 to 30, especially about 1 to 15, Specifically, it involves deletion of the polypeptide in the region of about 1-5 amino acids. For example , With the first amino acid methionine having only minor changes in the function of the polypeptide It does not have to exist. These also do not include the polypeptides of the invention described above. Fusion proteins that themselves regulate the contractility of cardiomyocytes Having a possible modulator function, or only after removal of the fusion moiety Special It is possible to obtain certain functions. In particular, they are particularly useful for fusion proteins 1 to 200, preferably about 1 to 150, especially about 1 to 100, especially about 1 Includes a non-cardiac specific sequence content of ５０50 amino acids. Non-cardiac specific peptide arrangement Examples of columns include prokaryotes that can be derived, for example, from E. coli galactosidase. Product peptide sequence.   The nucleic acids of the invention are generally DNA or RNA, preferably DNA. one Generally double-stranded DNA for expression of appropriate genes and use as probe For this purpose, single-stranded DNA is preferred. D encoding a particularly preferred polypeptide Along with the NA region, the nucleic acid sequence shown in FIG. 1, 2 or 3 and as detailed above Double- or single-stranded DNA having a part thereof as described is particularly preferred. This territory The region begins at position 89 with the nucleic acid "ATG" encoding methionine, and at position 1747 " Until "TAG" which codes "amber" (end).   For example, the nucleic acids of the invention may be based on the sequences disclosed in FIGS. Based on the polypeptide sequence disclosed in FIG. 4 by use of the child code, for example, Phosphotriester method (for example, Uhlmann, E. & Peyman, A. (1990) Chemical Rev. iews, 90, 543-584, No. 4) can be chemically synthesized. Obtain the nucleic acid of the present invention Another possibility for suitable gene banks using suitable probes, e.g. For example, isolation from a heart-specific gene bank (eg, J. Sambrook et al. l., (1989), Molecular Croning. A Laboratory Manual 2nd edition, Cold Spring Ha rbor Laboratory, Cold Spring Harbor, NY). Suitable as a probe For example, from about 100 to 100, whose sequence may be derived from the nucleic acid sequences shown in FIGS. 1000 nucleotides in length, preferably about 200-500 nucleotides in length In particular, a single-stranded DNA fragment of about 300 to 400 nucleotides in length. Step One example of a lobe is 321 bp in size, with the area underlined in FIG. The corresponding DNA fragment of Example 1 is used, and is used to obtain human heart tissue. The nucleic acids of the invention have already been successfully isolated (see Example 2).   The nucleic acid of the present invention is usually contained in a vector, preferably an expression vector or a gene therapy. Present in therapeutically effective vectors. Vectors useful for gene therapy include, for example, tropo Nin C (cTNC) promoter (see, eg, Parmacek, MS et al. (1990) J. B. iol. Chem. 265 (26) 15970-15976 and Parmacek, M.S. et al. (1992) Mol. Ce ll Biol. 12 (5), 1967-1976). Preferably, it is functionally related to the nucleic acids of the invention.   The expression vector may be a prokaryotic or eukaryotic expression vector. E. coli Examples of prokaryotic expression vectors for expression in EM or pUC derivatives, including Sacharomyce cerevisiae. Examples of eukaryotic expression vectors for expression in S. cerevisiae include Examples include the vectors p426Met25 or p426GAL1 (Mumberg et al. al. (1994) Nucl. Acids Res., 22, 5767-5768), for expression in insect cells For example, baculo as disclosed in EP-B1 0 127 839 or EP-B1 0 549 721 Irs vector, for expression in mammalian cells, for example, a vector Rc / CMV and Rc / RSV or SV40 vectors, which are All are publicly available.   In general, the expression vector also contains a trp promoter for expression in E. coli, for example. (See, for example, EP-B1 0 154 133), an ADH2 vector for expression in yeast. Lomotor (Russell et al. (1983), J. Biol. Chem. 258, 2674-2682), insect cells Baculovirus polyhedrin promoter (eg, EP-B Or MMTV (mouse mammary tumor virus; Lee et al. (1981) Nature 214, 228-232) SV40 early promoter or LTR promoter. And a promoter suitable for a particular host cell, such as a promoter.   Examples of vectors useful for gene therapy include viral vectors, preferably Denovirus vectors, especially replication-defective adenovirus vectors, or adenoviruses An associated viral vector, eg, consisting of only two inverted terminal repeats (ITRs). Adeno-associated virus vectors.   Adenovirus vectors and especially replication-defective adenovirus vectors are: Are particularly preferred for the following reasons.   Human adenovirus is a double-stranded DNA having a genome of about 36 kilobase pairs (kb). Belongs to the class of virus. This viral DNA produces about 2700 different genes. Encodes a product and is distinguished between early ("early genes") and late ("late genes") You. This “early gene” is divided into four transcription units E1 to E4. Late inheritance The offspring encodes the capsid protein. At least 42 different adenoids It is possible to classify immunologically into subgroups of irs and AF, Is suitable for the present invention. Prerequisites for viral gene transcription are Expression of the E1 region encoding a transactivator of ils gene expression. . Thus, the expression of all subsequent viral genes was determined to be E1 transactive. By taking advantage of its dependence on the (Eg, McGrory W.J. et al. (1988) Virol.1 63 , 614-617 and Gluzman, Y .; et al. (1982), `` Eukaryotic Viral Vectors '' (Gluzman, Y. ed.) 187-192, Cold Spring Harbor Press, Cold Spring Harbor N ew York). Adenovirus vectors, especially type 5 (for sequence see Chroboc zek, J .; et al. (1992) Virol.186, 280-285) and especially in subgroup C The E1 gene region, generally a foreign gene or book with its own promoter. Replace with the nucleic acid construct of the invention. Before for downstream adenovirus gene expression As a result of the replacement of the E1 gene region, which is a prerequisite, an adenovirus that cannot replicate You. Therefore, these viruses are used in cell lines to restore the lost E1 gene. Only can be duplicated.   Thus, replication-defective adenoviruses generally have E. coli stably integrated into the genome. A so-called 293 cell line with one region copy (human embryonic kidney cell line) Is formed by homologous recombination. For this purpose, the nucleic acids of the invention Under the control of its own promoter, such as the troponin C promoter described above. Clone into a recombinant adenovirus plasmid. Then, for example, 293 In helper cell lines, d1327 or de11324 (adenovirus 5 )), Homologous recombination is performed with an adenovirus genome defective in E1. Recombination If successful, a viral plaque is obtained. Duplicate defects created in this way To infect a cell culture with an infectious virus or for gene therapy in the body High titer (eg 10⁹-10¹¹Plaque-forming unit).   In general, the exact insertion site of the nucleic acid of the invention into the adenovirus genome is not critical. Absent. For example, the nucleic acids of the invention can be cloned in place of the deleted E3 gene. Both are possible (Karlsson, S. et al. (1986), EMBO J. et al.Five, 2377-2385).   However, particularly when the E3 region is also deleted, the E1 region or one thereof Parts, such as the E1A or E1B region (see, eg, WO 95/00655) It is preferable to substitute with a nucleic acid.   However, the invention is not limited to adenovirus vector systems On the contrary, adeno-associated virus vectors have also been described in the core of the present invention for the following reasons. Particularly suitable for combination with acids.   AAV virus belongs to the parvoviridae family. These have diameters of 18-30 nm. And has an icosahedral envelope containing approximately 5 kb of linear single-stranded DNA. Are classified by their capsids. For a valid copy of AAV, helper Co-infection of host cells with lus is required. An example of a suitable helper is Ade Virus (Ad5 or Ad2), herpes virus and vaccinia virus (Muzyczka, N. (1992) Curr. Top. Microbiol. Immunol.158, 9 7-129). In the absence of a helper virus, AAV harbors the viral genome It transitions to a latent state where it can be incorporated stably into the nom. AAV host Its genomic integration properties make it particularly attractive as a transduction vector for mammalian cells. It is delicious. Generally, about 145 are competent for the function of the vector. bp long inverted terminal repeats (ITR: see, for example, WO95 / 23867) . They are used for replication, packaging, and integration into the host cell genome. , Have the necessary signal in the “cis” position. Packaging into recombinant vector particles For non-structural proteins (rep proteins) and structural proteins ( adenovirus infects a vector plasmid containing the cap protein) gene. Transfected cells. A few days later, in addition to the recombinant AAV particles, A cell-free cell lysate is also prepared, which also contains cells. This adenovirus is 5 Banding by heating at 6 ° C. or with a cesium chloride gradient Can be conveniently removed. Using this co-transfection method, 0^Five-10⁶It is possible to achieve rAAV titers of IE / ml. packing If the plasmid and the vector plasmid do not have overlapping sequences, Ils contamination is below the detection limit (Samulski, R.J. (1989) J. Virol.63, 3822- 3828).   Introduction of the nucleic acids of the invention into somatic cells can be achieved by AAV into quiescent differentiated cells. This is particularly advantageous for cardiac gene therapy. The incorporation capabilities described are also in  Ensure long-lasting gene expression in vivo, which is also particularly valuable It is profitable. A further advantage of AAV is that the virus is not pathogenic to humans and It is relatively stable at o. The AAV vector of the nucleic acid of the present invention or the AAV vector For cloning into a part of E. coli is described, for example, in WO95 / 23867, Chiorini, J.A. et al. (1995) , Human Gene Therapy6, 1531-1541 or Kotin, R.M. (1994), Human Gene Th This is performed by a method known to those skilled in the art described in erapy 5, 793-801.   Vectors effective for gene therapy also allow the nucleic acids of the invention to be complexed with liposomes. Can achieve very high transfection efficiency Thus, this is obtained (Felgner, P.L. et al., (1987), Proc. Natl. Acad. . Sci USA84, 7413-7417). In lipofection, the liposome suspension is sonicated. A small unilamellar vesicle of cationic lipid is prepared by the process. DNA is particularly positive Taste charge remains and plasmid DNA is 100% complexed by liposome At such a ratio that they are ionically bonded to the surface of the liposome. Felgner et al. The lipid mixture DOTMA (1,2-dioleoyloxypropyl- 3-trimethyl-ammonium bromide) and DOPE (dioleoylphosphine) Atidyl-ethanolamine) (1987, supra), as well as a number of new lipid compositions. Are synthesized and their efficiency in transfecting various cell lines (Behr, J.P. et al. (1989), Proc. Natl. Acad. Sci. USA86 , 6982-6986; Felgner, J.H. et al. (1994) J. Am. Biol. Chem.269, 2550-2561; Gao , X. & Huang, L.A. (1991), Biochim. Biophys. Acta1189, 195-203). New lipid As an example of the composition, DOTAP N- [1- (2,3-dioleoyloxy) propyl] Ropyl] -N, N, N-trimethylammonium ethyl sulfate or DOG S (TRANSFECTAM; dioctadecyl-amidoglycylspermine) . Preparation of DNA-liposome complex and heart-specific transfection One successful example of its use is described in DE 44 11402.   Use of the nucleic acids of the invention in gene therapy involves encoding a polypeptide. A portion of the nucleic acid may contain an intron sequence, preferably a promoter and polypeptide. Between the initiation codon and / or the polyA (ployA) sequence, Contains the A sequence or SV40 virus poly A sequence, especially at the 3 'end of the gene Containing one or more non-coding sequences that stabilize mRNA in cardiomyocytes. Which is also advantageous (Jackson, R.J. (1993) Cell 74, 9-14 and Palmiter, R.D. et al. (1991) Proc. Natl. Acad. Sci. USA 88,478 -482).   The present invention further relates to a polypeptide having the amino acid sequence shown in FIG. Preferably a functional variant having at least 6 amino acids, preferably at least Having at least 12 amino acids, in particular having at least 15 amino acids, Alternatively, for a portion thereof having at least 164 amino acids (provided that the sequence : Excluding polypeptides having   For example, the polypeptide is generally known to those of skill in the suitable expression system described above. It is prepared by expressing the nucleic acids of the invention using known methods. Suitable Examples of suitable host cells include E. coli strains DH5, HB101 or BL21, Mother line Saccharomyces cerevisiae, insect cell line of the order Lepidoptera, such as Spodopte La Frugiperda (Spodoptera frugiperda) or animal cell COS, Vero, 2 93 and HeLa, all of which are commonly available.   Some of the above polypeptides may also be synthesized by conventional synthetic methods (Merrifield method). can do. They provide additional functional variants of the polypeptides of the invention. Used to screen suitable gene expression banks for the purpose of obtaining It is particularly suitable for obtaining antisera that can be used.   Therefore, the present invention also relates to a polypeptide having the amino acid sequence shown in FIG. Or a functional variant thereof, and having at least 6 amino acids. Or at least 12 amino acids, especially at least 15 amino acids To Having at least 164 amino acids, for example, Combined with a suitable carrier such as serum albumin, it is Can be immunogenic or can be made immunogenic or their immunogens Specifically reacts with a part of said polypeptide which can either increase the Antibodies.   Antibodies are either polyclonal or monoclonal. For example, in general According to known methods, using said polypeptide, or a part thereof, If appropriate, for example, Freund's adjuvant and / or aluminum hydroxide Production is carried out by immunizing a mammal, for example a rabbit, in the presence of The present invention also relates to this (eg Diamond, B.A. et al. (1981) The New Eng). land Journal of Medicine, 1344-1349). Then the immune response in the animal The polyclonal antibody produced based on the blood is obtained by a generally known method. And can be purified, for example, by column chromatography. Thus, for example, in rabbits, expressed as a fusion protein in bacteria And then purified by affinity chromatography as shown in FIG. Polyclonal antiserum against a polypeptide of the invention having amino acids 1-90 Could be created. The antibody of the present invention has an activity of about 80 kD in human heart tissue extracts. Was specifically recognized.   Monoclonal antibodies can be obtained, for example, by the well-known Winter & Milstein method (Winter, G. & Milst). ein, C. (1991) Nature, 349, 293-299).   The present invention also provides a polypeptide having the amino acid sequence shown in FIG. Having a nucleic acid encoding the functional variant and at least 8 nucleotides Or a polypeptide having the amino acid sequence shown in FIG. Or a functional variant thereof and one of the foregoing having at least 6 amino acids. And a pharmaceutical formulation comprising, if appropriate, suitable additives or excipients; Wherein the nucleic acid or the polypeptide is formulated with a pharmaceutically acceptable carrier, The present invention relates to a method for producing a pharmaceutical preparation for treating heart disease, especially heart failure.   One example of the use of nucleic acid fragments as therapeutics is in antisense oligonucleotides. (Uhlmann, E. & Peyman, A. (1990) Ch. emical Reviews, 90, 543-584, No. Four).   Particularly suitable pharmaceutical preparations for use in human genetic diseases are those in naked form or as described above. One form of a vector effective for gene therapy or complexed with liposomes And the above-mentioned nucleotide. Pharmaceutical carriers include, for example, preferably about 6 . A pH of from 0 to 8.0, more preferably from about 6.8 to 7.8, especially about 7.4; And / or about 200-400 milliosmol / liter, preferably about 290 There is a physiological buffer with a permeability of ~ 310 milliosmol / liter. Pharmaceutical carrier Are suitable stabilizers, such as, for example, nuclease inhibitors, preferably such as EDTA It may further comprise complexing agents and / or other excipients known to those skilled in the art.   The nucleic acid is typically described in detail above, if appropriate, for example, using a catheter. Intravenous administration in the form of a loaded viral vector or as a liposome complex. It is. For example, a nucleic acid of the invention can be used, in particular, for recombinant adenovirus vectors or adenoviruses. Direct injection into the patient's coronary artery in the form of a virus-associated virus vector (so-called Percutaneous coronary gene transfer (PCGT) is advantageous. For balloon catheter Dosing not only allows transfection to the heart but also Is particularly preferred because it can be limited to the injection site of (for example, Feldman, L.J. et al. 1994) JACC 235A, 906-934).   Intravenous polypeptide or catheter or balloon catheter Use, if appropriate, to have an immediate and direct effect on the function of the heart, Suitable additives such as physiological salt solutions, stabilizers, proteinase inhibitors or It can be administered together with an excipient.   The invention further relates to nucleic acids, polypeptides or antibodies of the invention and, where appropriate, Auxiliaries containing suitable additives or excipients and the invention in which Nucleic acids, polypeptides or antibodies are mixed with suitable additives or excipients The present invention relates to a method for producing a diagnostic aid for diagnosing heart disease, particularly heart failure.   For example, according to the present invention, a diagnostic aid based on the polymerase chain reaction (eg, The PCR diagnostics disclosed in EP-0 200 362) or 321 of the invention as probes. Based on the Northern blot described in detail in Example 3 using the DNA fragment of bp Alternatively, a diagnostic aid can be produced based on the nucleic acid. Before these tests Specific hybridization of the nucleic acid, usually with the complement of the corresponding mRNA Based on The nucleic acid may also be used in this case, for example, as described in EP 0 063 879. It may be modified as follows. The DNA fragment, in particular the DNA fragment described in Example 1, was Using suitable reagents according to the methods known from³²Use P-dCTP Radiolabeled, or non-radioactively labeled with biotin, and then preferably For example, isolated RNA previously bound to a suitable membrane of cellulose or nylon It is preferred to incubate with. Hybridization and membrane Prior to binding to RNA, RNA isolated, for example, by agarose gel electrophoresis It is more advantageous to fractionate by size. Thus, from each tissue sample Using the same amount of examined RNA, the mRN specifically labeled with the probe The amount of A can be determined.   Therefore, by using the diagnostic aid of the present invention, possible heart failure can be reliably prevented. To be able to make a diagnosis, the in vitro intensity of the corresponding gene It is also possible to clearly evaluate the organ tissue samples (see Example 1). In FIG. A cDNA having the sequence shown is particularly suitable for diagnosing possible heart failure (See Example 2).   Further diagnostic aids are polypeptides of the invention or as described in detail above. Includes immunogenic parts thereof. Preferably, for example, nitrocellulose or na The polypeptide or a part thereof bound to a solid phase composed of iron can be A body fluid that can be tested in vitro to react with an antibody, such as blood Contact. The antibody-peptide conjugate was then, for example, labeled. It can be detected by anti-human IgG or anti-human IgM antibody. This sign is colored, for example It is an enzyme such as peroxidase that catalyzes the reaction. Thus, by the color reaction, The presence and amount of autoimmune antibodies present can be easily and quickly detected.   Another diagnostic aid comprises the antibody itself of the present invention. For example, related polypeptides This can be done to determine whether the presence of For the purpose of obtaining information about It can be used to easily and quickly check. In this case, the antibody of the present invention Is, for example, labeled with an enzyme as already described above. Thus, the specific antibody The peptide complex can be easily and equally rapidly detected by the color reaction of the enzyme.   The present invention also relates to a polypeptide having the amino acid sequence shown in FIG. And at least 8 nucleotides of the invention encoding a functional variant of And a polypeptide having the amino acid sequence shown in FIG. Or a functional variant thereof and one of the foregoing having at least 6 amino acids. Or the antibody of the invention, and, where appropriate, suitable additives or excipients. The present invention relates to a test for identifying a functional reactant.   Suitable tests for identifying functional reactants include, for example, so-called double hybrid systems (Fields, S. & Sternglanz, R. (1994) Trends in Genetics, 10, 286-292) You.   In this test, a cell, for example, a yeast cell, comprises a polypeptide of the invention and a known polypeptide. A protein, such as E. coli Gal4 or LexA DNA binding domain Transformation or transformation with one or more expression vectors to express the fusion protein Transfected and / or unknown polypeptides and, for example, Gal4, Fusion proteins containing the transcriptional activation domain of Lupes virus VP16 or B42 Express the protein. This cell contains, for example, a LexA promoter / operator. Controlled by regulatory sequences or by the so-called yeast upstream activation sequence (UAS) Reporter genes such as the lacZ gene from Escherichia coli, green fluorescent tan It further contains the protein or yeast amino acid biosynthesis gene His3 or Leu2. No. Unknown polypeptides can be found in, for example, gene banks, such as human heart tissue-specific genes. It is encoded by a DNA fragment obtained from the gene bank. Usually a cDNA gene Banks were created directly using the described expression vectors and then tested immediately in yeast. It can be carried out.   For example, a nucleic acid of the invention includes a nucleic acid encoding a LexA DNA binding domain. Cloned into a yeast expression vector in a functional unit, and Expression of a fusion protein consisting of a repeptide and a LexA DNA binding domain You. Another yeast expression vector is the cDN derived from the cDNA gene bank. The A fragment is cloned into a functional unit containing the nucleic acid encoding the Gal4 transcription activation domain. In the transformed yeast, unknown polypeptide and Gal4 transcription activation domain Of a fusion protein consisting of quinone. Transformation using two expression vectors And, for example, Leu2^-Is a nucleic acid encoding Leu2. And controlled by the LexA promoter / operator. Departure As a result of the functional interaction between the light polypeptide and the unknown polypeptide, Gal4 The transcription activation domain is a LexA promoter via the LexA DNA binding domain. -/ Operator, whereby the latter is activated and then the Leu2 gene The child develops. As a result, Leu2^-Yeast grows on minimal medium without leucine Can breed.   LacZ or green fluorescent protein reporter instead of amino acid biosynthesis gene -When using genes, transcription is activated by the formation of blue or green fluorescent colonies. Can be detected. This blue or fluorescent color is also For example, in the case of blue, it can be easily determined at 585 nm.   Thus, expression with respect to a polypeptide that interacts with a polypeptide of the invention Gene banks can be easily and rapidly screened. Next Can be isolated and further identified.   Another possible use of the dual hybrid system is for example chemical compounds Polypeptides of the invention and known or unknown polypeptides by other substances such as Is to act on the interaction between Therefore, it can be chemically synthesized, and New and valuable active substances that can be used as therapeutics for the treatment of heart disease It is also possible to find it easily. Therefore, the present invention finds polypeptide-like reactants The protein-protein complex described above is not limited to It also extends to methods of finding substances that can interact with. Hence, such polypep Tide-like, as well as chemical reactants are referred to as functional reactants for the purposes of the present invention.   Thus, the surprising advantage of the present invention is the specificity of heart disease, especially heart failure, and That the subject matter of the present invention can be used for reliable diagnosis and treatment is there. However, other valuable therapeutic and diagnostic possibilities are also apparent. ing. For example, a functional reactant that can be easily identified using the described test method is With their adjuvants in the form of suitable pharmaceutical preparations, their natural environment in the heart muscle In the activity of the polypeptide of the present invention and thus the contractility of cardiomyocytes. In particular, the activity of this polypeptide has already been described in detail above. This is very advantageous because it can be adjusted as described above.   Hereinafter, the present invention will be described in detail with reference to the drawings and examples, but the present invention is not limited thereto. Not something.Description of the drawings   FIG. 1 shows a 1936 nucleotide long heart-specific DNA sequence. Equivalent The region encoding the polypeptide is shown in bold. DNA fragment of Example 1 Is underlined.   FIG. 2 shows 2080 nucleotides having an additional portion at the 5 ′ end of the DNA sequence of FIG. 1 shows an otide-length heart-specific DNA sequence. Region encoding the corresponding polypeptide Areas are again shown in bold.   FIG. 3 shows 2268 with an additional portion at the 5 'end of the DNA sequence of FIG. 1 or FIG. 1 shows a heart-specific DNA sequence of one nucleotide in length. Copy the corresponding polypeptide The regions to be loaded are also shown in bold.   FIG. 4 shows 552 nucleosides encoded by one of the DNA sequences shown in FIGS. 1 shows a polypeptide sequence of amino acid length. The region homologous to human tropomodulin is thick. It is indicated by a letter. Polypeptides via the tyrosine kinase signal transduction pathway Sequence motifs that indicate the regulation of are underlined.   FIGS. 5a and 5b show the results for detecting expression in various human tissues (FIG. 5a). ) And to detect expression in healthy and failing human heart tissue (FIG. 5). b) shows a Northern blot of mRNA corresponding to the nucleic acid sequences shown in FIGS. You.Example 1 . Isolation of DNA fragments from human failing heart tissue   First, the standard method (Chomczynski & Sacchi (1987), Anal. Biochem, 162 (1), 156-15 According to 9), complete RNA was isolated from healthy and failing heart tissue samples. The RNA was then treated with DNase to remove contaminating DNA. Then, Aliquots (0.2 μg) of the RNA in 1 × RT buffer (Gibco Y00121), 10 mM DTT, 20 μM dNTP mixture, 1 U / μl RNAsin (Promeg a N2511) 1 μM 5'-T₁₂ACN-3 'type (where N is any deoxy 3 'anchor primer mixture and 10 U / μl SuperScript RNase H^-In a 20 μl reaction mixture containing reverse transcriptase Incubated at 37 ° C. for 60 minutes and then transcribed into cDNA. Then c Aliquots of DNA were added to 1 μM 3 'primer T₁₂AC and 1 μM 5'-1 In addition to the 0-mer primer (5'-CCTTCTACCC-3 '), 10 µCi of α −³²P-dCTP, 2 μM dNTP mixture and 1 U AmpliTaq (Pe 20 μl for 1 × PCR buffer (Perkin-Elmer) containing Attached. The mixture is first cycled at 94 ° C for 1 minute and then at 94 ° C for 30 seconds for 40 cycles each. Ink at 40 ° C for 2 minutes, then at 72 ° C for 30 seconds, and finally at 72 ° C for 10 minutes. Was added. Next, the obtained DNA fragment mixture was subjected to 6% polyacrylamide gel. The above was fractionated and then an autoradiogram was generated. Thus, 321 bp Length, not present in healthy heart samples but evident in failing heart samples Was prepared. Then, based on the X-ray film, Gels were excised and re-amplified by PCR under the conditions already described. . Subsequently, the obtained fragment is cloned into an appropriate vector, and the DNA Determined columns. The fragment thus prepared comprises nucleotide 162 of the sequence of claim 1 12 thiamin nucleotides derived from 7-1936 and 3 'anchor primers Including otide.2 . Isolation of heart-specific nucleic acids   From Example 1 comprising the nucleotides at positions 1627-1936 in FIG. The obtained α-³²Using DNA fragments labeled with P-dCTP, standard conditions (Sambrook J., Frisch, E.F. & Maniatis, T .; (1989) Molecular Cloning, A Laboratory Manual, ch. (See 8-10) Workup hybridization was performed. Next, the found cDNA is isolated and Were sequenced. This arrangement is shown in FIGS. This makes it healthy C having the sequence shown in FIG. 2 or 3 which can be isolated with high probability from type heart tissue Rather than DNA, cDNA having the sequence shown in FIG. It has been found that it can be isolated with a high probability.3 . Northern blot to generate cardiac-specific genes in various human tissues Detection of current intensity   First, the length of 321 bp already described in Examples 1 and 2 and FIG. DNA fragments were subjected to a random primer labeling method (Feinberg, A.P. & Vogelstein, B.C. (1983) Anal. Biochem., 132, 6)³²Radiolabeled with P-dCTP . Use RTS RadPrime DNA labeling system (GibcoBRL 10387-017) for this purpose did. Manufacturer's instructions (Multiple Tissue Northern Blots I & II, Clontech L aboratories GmbH, Heidelberg, # 7760-1, # 7759-1) at 68 ° C. for 1 hour, E human group in pressHyb hybridization solution (Clontech # 8015-1) Poly A from weave (see FIGS. 5a and 5b)⁺Hybridization of RNA and blot The zation was performed. The blot was then combined with 2 × SSC and 0.05% S Wash for 30 minutes with DS, then 1 hour with 0.1 × SSC and 0.1% SDS, Further, an autoradiogram was prepared. Approximately 240 probes of 321 bp length 0 bp poly A⁺RNA strongly hybridizes with heart tissue and skeletal muscle, Very weakly hybridized in gonad tissue, leukocytes, large intestine, small intestine, ovaries, Does not hybridize in testis, thymus, spleen, kidney, liver, lung, placenta and brain tissue (FIG. 5a).   In addition, the expression of the corresponding RNA in healthy and failing heart tissues was also examined. For this purpose, complete RNA was isolated from various human heart tissue samples (C homczynski & Sacchi (1987), Anal. Biochem. 162, 156-159). Then each In some cases, 10 μg of RNA was applied using a 1% formaldehyde agarose gel. Fractionated and transferred to a charged nylon membrane by the capillary method (Zeta- Probe GT BioRad # 162-0197). The membrane was briefly washed with 2 × SSC, then 80 Bake at 300C for 30 minutes. This membrane is mixed with a pre-hybridization solution (0.5 M Na_TwoHPO_Four, PH 7.2; 7% SDS) at 65 ° C for at least 1 hour Incubated. This solution is then replaced with a new solution and then heat denatured. Radioactive probe was added. Hybridization is performed at 65 ° C for 15 hours Was. The membrane was then firstly washed with 40 mM Na._TwoHPO_FourPH 7.2; 5% SD S at 65 ° C. for 15 hours, then 40 mM Na_TwoHPO_FourPH 7.2; 1% After washing with SDS at 65 ° C. for 2 × 30 minutes, an autoradiogram was prepared. Various R having a length of about 2200-2400 bp in a 1% agarose gel It was revealed that the NA species had been fractionated. These various species are found Well matched to the size of the three cDNAs, and the average length of the poly A terminal was 150 bp. (See FIGS. 1-3). In particular, the smallest RNA species is more affected than healthy tissue It was more clearly detectable in the tissue. PhosphorImager and ImageQuant software The quantification of the blots using the software (Molecular Dynamics GmbH, Krefeld) Considering control hybridization with thymosin and actin, Approximately 35% higher expression of detected RNA in failing heart tissue compared to tissue .

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification code FI Theme coat ゛ (Reference) A61P 9/10 C07K 16/18 C07K 14/47 C12P 21/02 C 16/18 C12Q 1/68 A C12P 21 / 02 G01N 33/53 D C12Q 1/68 C12P 21/08 G01N 33/53 C12N 15/00 ZNAA // C12P 21/08 A61K 37/02

Claims (1)

[Claims] 1. A nucleic acid encoding a polypeptide having the amino acid sequence shown in FIG. 4 or a functional variant thereof, and a portion thereof having at least 8 nucleotides (provided that the sequence: Are excluded). 2. 2. The nucleic acid according to claim 1, wherein the nucleic acid is DNA or RNA, preferably DNA, especially double-stranded DNA. 3. The nucleic acid according to claim 1 or 2, wherein the nucleic acid comprises DNA having a nucleic acid sequence as shown in FIG. 1, 2 or 3. 4. The nucleic acid according to any one of claims 1 to 3, wherein the nucleic acid is present in a vector, preferably in an expression vector or a vector effective for gene therapy. 5. The nucleic acid according to any one of claims 1 to 4, wherein a portion of the nucleic acid encoding the polypeptide comprises one or more non-coding sequences and / or poly A sequences. 6. The method for producing a nucleic acid according to any one of claims 1 to 5, wherein the nucleic acid is chemically synthesized or isolated from a gene bank using a probe. 7. A polypeptide having the amino acid sequence shown in FIG. 4, or a functional variant thereof, and a portion thereof having at least 6 amino acids (provided that the sequence: Excluding polypeptides having 8. The method for producing a polypeptide according to claim 7, wherein the nucleic acid according to any one of claims 1 to 3 is expressed in a suitable host cell. 9. Antibodies against a polypeptide having the amino acid sequence shown in FIG. 4 or a functional variant thereof, and a portion thereof having at least 6 amino acids. 10. 10. The mammal is immunized with a polypeptide having the amino acid sequence shown in FIG. 4 or a functional variant thereof, and a portion thereof having at least 6 amino acids, and then isolating the resulting antibody. The method for producing the antibody according to the above. 11. A polypeptide having the amino acid sequence shown in FIG. 4, or a nucleic acid encoding a functional variant thereof, and a part thereof having at least 8 nucleotides, or a polypeptide having the amino acid sequence shown in FIG. 11. A pharmaceutical formulation comprising a functional variant, and a part thereof having at least 6 amino acids, and, if appropriate, a pharmaceutically acceptable carrier. A polypeptide having the amino acid shown in FIG. 4, or a nucleic acid encoding a functional variant thereof, and a part thereof having at least 8 nucleotides, or a polypeptide having the amino acid sequence shown in FIG. 4, or a function thereof A method for the manufacture of a pharmaceutical preparation for heart disease, characterized in that the objective variant and a part thereof having at least 6 amino acids are formulated together with a pharmaceutically acceptable carrier. 13. A polypeptide having the amino acid shown in FIG. 4, or a nucleic acid encoding a functional variant thereof, and a part thereof having at least 8 nucleotides, a polypeptide having the amino acid sequence shown in FIG. 4, or a functional thereof A diagnostic aid comprising the variant, and a part thereof having at least 6 amino acids, or the antibody according to claim 9 and, where appropriate, suitable additives or excipients. 14． A nucleic acid encoding a polypeptide having the amino acid shown in FIG. 4 or a functional variant thereof, and a part thereof having at least 8 nucleotides, a polypeptide having the amino acid sequence shown in FIG. 4, a functional variant thereof A method for producing a diagnostic aid for the diagnosis of heart disease, characterized in that the body and a part thereof having at least 6 amino acids or the antibody according to claim 9 are mixed with a pharmaceutically acceptable carrier. . 15. A polypeptide having the amino acid shown in FIG. 4, or a nucleic acid encoding a functional variant thereof, and a part thereof having at least 8 nucleotides, or a polypeptide having the amino acid sequence shown in FIG. A test to identify a functional reactant containing the variant, and a part thereof having at least 6 amino acids, and where appropriate suitable additives or excipients.