EP1000145A1 - Acide nucleique specifique des muscles du coeur et du squelette, sa production et son utilisation - Google Patents

Acide nucleique specifique des muscles du coeur et du squelette, sa production et son utilisation

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Publication number
EP1000145A1
EP1000145A1 EP98934959A EP98934959A EP1000145A1 EP 1000145 A1 EP1000145 A1 EP 1000145A1 EP 98934959 A EP98934959 A EP 98934959A EP 98934959 A EP98934959 A EP 98934959A EP 1000145 A1 EP1000145 A1 EP 1000145A1
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EP
European Patent Office
Prior art keywords
nucleic acid
polypeptide
parts
acid sequence
functional variant
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EP98934959A
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German (de)
English (en)
Inventor
Marion Elke Hofmann
Horst Domdey
Thomas Henkel
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Medigene AG
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Medigene AG
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Publication of EP1000145A1 publication Critical patent/EP1000145A1/fr
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    • 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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • 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
    • A61P9/04—Inotropic agents, i.e. stimulants of cardiac contraction; Drugs for heart failure
    • 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
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • 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
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
    • C12N2799/022—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from an adenovirus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • Cardiac and skeletal muscle-specific nucleic acid its production and use
  • the invention relates to a nucleic acid expressed in the human heart and skeletal muscle, its production and use as a diagnostic agent, medicament and test for identifying functional interactors.
  • the heart is a hollow muscular organ with the task of keeping the blood flow in the vessels moving by changing contraction (systole) and relaxation (diastole) of the atria and chambers.
  • the heart muscle the myocardium
  • the heart muscle is composed of specialized striated muscle cells, between which connective tissue lies.
  • Each cell has a central nucleus, is delimited by the plasma membrane, the sarcolemma, and contains numerous contractile myofibrils that are irregularly separated by sarcoplasm.
  • the contractile substance of the heart is formed by long parallel myofibrils.
  • Each myofibril is divided into several identical structural and functional units, the sarcomeres.
  • the sarcomeres are composed of the thin filaments, which mainly consist of actin, tropomyosin and troponin, and the thick filaments, which mainly consist of myosin.
  • the molecular mechanism of muscle contraction is based on cyclic attachment and detachment of the globular myosin heads from the F-actin filaments.
  • Ca 2+ is released from the sarcoplasmic reticulum, which influences the troponin complex and tropomyosin through an allosteric reaction and so on clears the way for contact of the actin filament with the myosin head.
  • the attachment causes a change in the conformation of the myosin, which pulls the actin filament along it. In order to undo this change in conformation and to return to the beginning of a contraction cycle, ATP is required.
  • the activity of the heart muscle can be temporarily influenced by the need for perfusion, i.e. by nervous and hormonal regulatory measures. H. Circulation needs to be adjusted to the body. In this way, both the contraction force and the contraction speed can be increased. Long-term overuse leads to physiological changes in the heart muscle, which are mainly characterized by an increase in myofibrils (myocyte hypertrophy).
  • the present invention is therefore based on the task of identifying and isolating genes which are at least partly responsible, if not causative, for genetically caused heart diseases.
  • An object of the invention is therefore a nucleic acid coding for a polypeptide with an amino acid sequence according to FIG. 4 or a functional variant thereof, and parts thereof with at least 8 nucleotides, preferably at least 10 nucleotides, in particular at least 15 nucleotides, especially at least 20 nucleotides, excepted a nucleic acid with the sequence: 1 GCCAACACGC ANTCCGACGA CAGTGCAGCC ATGGTCATTG CAGAGATGCN TCAAAGTCAA 61 TGAGCACATC ACCAACGTAA ACGTCGAGTC CAACTTCATA ACGGGAAAGG GGATCCTGGC 121 CATCATGAGA GCTCTCCAGC ACAACACGGT GCTCACGGAG CTGCGTTTCC ATAACCAGAG 181 GCACATCATG GGCAGCCAGG TGGAAATGGA GATTGTCAAG CTNCTGAAGG AGAACACGAC 241 GCTNCTGAGG CTGGGNTACC ATTTTNAACT CCCAGGACC
  • the nucleic acid according to the invention was isolated from a cDNA bank of human heart tissue and sequenced.
  • total RNA was first isolated from a healthy and insufficient heart tissue sample according to standard methods and with the aid of a 3'-anchor-primer mixture, e.g. B. a 5'-T 12 ACN-3 'primer, in which N is any deoxyribonucleotide, and reverse transcriptase rewritten in c-DNA.
  • the cDNA was then based on the so-called differential display method according to Liang and Pardee (Liang, P. & Pardee, A. (1992) Science 257 ' , 967-970) under special PCR conditions using a 3' primer, e.g.
  • a T 12 ACN primer and an arbitrarily selected 5 'decamer primer, e.g. B. a 5 * -CCTTCTACCC-3 'decamer primer.
  • a 321 base pair (bp) long DNA fragment could be amplified, which surprisingly is not present in the healthy heart sample, but is clearly present in the inadequate heart sample.
  • the common methods such as differential display method or subtractive cDNA libraries are associated with the problem of redundancy, underrepresentation and false positive clones.
  • the gene products of poorly expressed genes can only be identified under special conditions.
  • the hit rate is generally very low (10-20%) and, for example, in the differential display method also on the selected PCR conditions, the primer length or, for example, in the production of subtractive banks from the hybridization temperature depends.
  • the entire gene was then isolated from a cDNA library using the DNA fragment found and sequenced.
  • mRNAs from different human tissues were hybridized with the DNA fragment found in a so-called Northern blot and the amount of bound m-RNA was determined, for example, by the radioactive labeling of the DNA fragment.
  • This experiment led to the detection of the corresponding RNA, especially in striated muscles, i.e. cardiac muscle and skeletal muscle tissue, and very weakly in prostate tissue.
  • an increased expression for example an approximately 35% higher expression of the RNAs in insufficient tissue compared to healthy tissue, was detected.
  • RNA species preferentially shows an increased expression in insufficient tissue compared to healthy tissue.
  • the increased expression of the smaller RNA species can be easily recognized, for example, in the Northern blot in the form of a double band (see FIG. 5b).
  • Tropomodulin is known as a polypeptide which has an effect on the formation of the myofibrils and the contractility of the cells in chicken cardiomyocytes (Gregorio et al. (1995) Nature 371, 83-86). This protein binds to tropomyosin on the one hand and to the actin filaments on the other, but is not regulated in its activity itself.
  • the derived polypeptide according to the invention also has some structural features of the tropomodulin, such as. B. a tropomyo- sin binding domain.
  • the polypeptide according to the invention has additional structural features which indicate regulation of the activity of the polypeptide by so-called tyrosine kinases (see FIG. 4).
  • the term “functional variant” in the context of the present invention is therefore understood to mean polypeptides that are functionally related to the polypeptide according to the invention, ie. H. can also be described as a regulatable modulator of the contractility of cardiac muscle cells, in striated muscles, preferably in cardiac muscle, skeletal muscle and / or prostate tissue, especially in cardiac muscle and / or skeletal muscle and in particular in cardiac muscle cells, which have structural features of the tropomodulin, such as B. one or more tropomyosin binding domains, and / or their activity can be regulated by tyrosine kinases.
  • Examples of functional variants are the corresponding polypeptides, which are derived from organisms other than humans, preferably from non-human mammals, such as, for. B. monkeys.
  • polypeptides which have a sequence homology, in particular a sequence identity, of approximately 70%, preferably approximately 80%, in particular approximately 90%, especially approximately 95% of the polypeptide with the amino acid sequence Fig. 4 have.
  • sequence homology in particular a sequence identity
  • polypeptides encoded by a nucleic acid derived from non-heart-specific tissue e.g. B. skeletal muscle tissue is isolated, but after expression in a heart-specific cell has the designated function (s).
  • This also includes deletions of the polypeptide in the range from about 1-60, preferably from about 1-30, in particular from about 1-15, especially from about 1-5 amino acids.
  • the first amino acid, methionine may be absent without significantly changing the function of the polypeptide.
  • this also includes fusion proteins that the Contain polypeptides according to the invention described above, the fusion proteins themselves already having the function of an adjustable modulator of the contractility of cardiac muscle cells or being able to get the specific function only after the fusion portion has been split off.
  • this includes fusion proteins with a proportion of in particular non-heart-specific sequences of about 1-200, preferably about 1-150, in particular about 1-100, especially about 1-50 amino acids.
  • non-heart-specific peptide sequences are prokaryotic peptide sequences which, for. B. can be derived from the galactosidase of E. coli.
  • the nucleic acid according to the invention is generally a DNA or RNA, preferably a DNA.
  • a double-stranded DNA is generally preferred for the expression of the gene in question and a single-stranded DNA for use as a probe.
  • a double- or single-stranded DNA with a nucleic acid sequence according to FIG. 1, 2 or 3 and the parts thereof already described above are particularly preferred, the DNA region coding for the polypeptide being particularly preferred. This region begins with the nucleic acids "ATG" coding for methionine at position 89 to "TAG" coding for "amber" (stop) at position 1747.
  • the nucleic acid according to the invention can be chemically determined, for example, using the sequences disclosed in FIGS. 1-3 or using the polypeptide sequence disclosed in FIG. 4, using the genetic code z. B. can be synthesized by the phosphotriester method (see, for example, Uhlmann, E. & Peyman. A. (1990) Chemical Reviews, 90, 543-584, No. 4).
  • Another way of getting hold of the nucleic acid according to the invention is to isolate it from a suitable gene bank, for example from a heart-specific gene bank, using a suitable probe (see, for example, SJ Sambrook et al., 1989, Molecular Cloning. A Laboratory Manual 2nd edn., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).
  • Suitable as a probe are, for example, single-stranded DNA fragments with a length of approximately 100-1000 nucleotides, preferably with a length of approximately 200-500 nucleotides, in particular with a length of approximately 300-400 nucleotides, the sequence of which from the nucleic acid sequences according to FIG 1-3 can be derived.
  • An example of a probe is the 321 bp DNA fragment according to Example 1, which corresponds to the underlined area in FIG. 1, with which the nucleic acid according to the invention has already been successfully isolated from human heart tissue (see Example 2).
  • the nucleic acid according to the invention is usually contained in a vector, preferably in an expression vector or gene therapy vector.
  • the gene therapy vector contains heart-specific regulatory sequences, such as. B. Troponin C (cTNC) promoter (see e.g. Parmacek, MS et al. (1990) J. Biol. Chem. 265 (26) 15970-15976 and Parmacek, MS et al. (1992) Mol. Cell Biol. 12 (5), 1967-1976), which is functionally linked to the nucleic acid according to the invention.
  • cTNC B. Troponin C
  • the expression vectors can be prokaryotic or eukaryotic expression vectors.
  • prokaryotic expression vectors are for expression in E. co i z. B. the vectors pGEM or pUC derivatives and for eukaryotic expression vectors for expression in Saccharomyces cerevisiae z. B. the vectors p426Met25 or p426GALl (Mumberg et al. (1994) Nucl. Acids Res., 22, 5767-5768), for expression in insect cells e.g. B. baculovirus vectors as disclosed in EP-B1-0 127 839 or EP-B1-0 549 721, and for expression in mammalian cells e.g. B.
  • the expression vectors also contain suitable promoters for the respective host cell, e.g. B. the trp promoter for expression in E. coli (see, for example, EP-B1-0 154 133), the ADH2 promoter for expression in yeast (Radorel et al. (1983), J. Biol. Chem. 258, 2674-2682), the baculovirus polyhedrin promoter for expression in insect cells (see e.g. EP-B1-0 127 839) or the early SV40 promoter or LTR promoters e.g. B. from MMTV (mouse mammary tumor virus; Lee et al. (1981) Nature 214, 228-232).
  • suitable promoters for the respective host cell e.g. B. the trp promoter for expression in E. coli (see, for example, EP-B1-0 154 133), the ADH2 promoter for expression in yeast (Radorel et al. (1983), J. Biol. Chem. 258,
  • virus vectors preferably adenovirus vectors, in particular replication-deficient adenovirus vectors, or adeno-associated virus vectors, e.g. B. an adeno-associated virus vector consisting exclusively of two inverted terminal repeat sequences (ITR).
  • ITR inverted terminal repeat sequences
  • An adenovirus vector and particularly a replication-deficient adenovirus vector, is particularly preferred for the following reasons.
  • the human adenovirus belongs to the class of double-stranded DNA viruses with a genome of approximately 36 kilobase pairs (Kb).
  • the viral DNA codes for about 2700 different gene products, a distinction being made between early (“early genes'”) and late ⁇ "lote genes”).
  • the "early genes” are divided into four transcriptional units, E1 to E4.
  • the late gene ' products encode the capsid proteins.
  • At least 42 different adenoviruses and subgroups A to F can be distinguished immunologically, all of which are suitable for the present invention.
  • the transcription of the viral genes presupposes the expression of the El region which codes for a transactivator of the adenoviral gene expression.
  • the El gene region is generally shared by a foreign gene own promoter or replaced by the nucleic acid construct according to the invention.
  • the exchange of the El gene region which is a prerequisite for the expression of the downstream adenoviral genes, results in a non-replication-capable adenovirus. These viruses can then only multiply in a cell line that replaces the missing El genes.
  • Replication-deficient adenoviruses are therefore generally by homologous recombination in the so-called 293 cell line (human embryonic
  • Renal cell line which has a copy of the El region stably integrated into the genome.
  • a separate promoter e.g. of the above-mentioned troponin C promoter
  • Genome such as dl327 or dell324 (adenovirus 5), in the helper cell line
  • plaque forming units or plaque forming units
  • the exact insertion site of the nucleic acid according to the invention into the adenoviral genome is not critical.
  • the E1 region or parts thereof, for example the E1A or EIB region is preferably replaced by the nucleic acid according to the invention, especially if the E3 region has also been deleted.
  • the present invention is not limited to the adenoviral vector system, but adeno-associated virus vectors are also particularly suitable in combination with the nucleic acid according to the invention for the following reasons.
  • the AAV virus belongs to the parvovirus family. These are characterized by an icosahedral, non-encapsulated capsid with a diameter of 18 to 30 nm, which contains a linear, single-stranded DNA of about 5 kb. A co-infection of the host cell with helper viruses is required for an efficient multiplication of AAV. Suitable aids are, for example, adenoviruses (Ad5 or Ad2), herpes viruses and vaccinia viruses (Muzyczka, N. (1992) Curr. Top. Microbiol. Immunol. 158, 97-129).
  • AAV In the absence of a helper virus, AAV goes into a latency state, whereby the virus genome is able to integrate stably into the host cell genome.
  • the ability of AAV to integrate into the host genome makes it particularly interesting as a transduction vector for mammalian cells.
  • the two approximately 145 bp long inverted terminal repeat sequences (ITR: inverted terminal repeats; see, for example, WO 95/23867) are generally sufficient for the vector functions. They carry the signals necessary for replication in "eis”.
  • ITR inverted terminal repeats
  • a cell-free lysate is produced, which contains adenoviruses in addition to the recombinant AAV particles.
  • the adenoviruses can advantageously by heating to 56 ° C or by banding in Cesium chloride gradients are removed. With this cotransfection method, rAAV titers of 10 5 to 10 6 IU / ml can be achieved. Contamination by wild-type viruses is below the detection limit if the packaging plasmid and the vector plasmid have no overlapping sequences (Samulski, RJ (1989) J. Virol. 63, 3822-3828).
  • AAV can transfer the nucleic acid according to the invention into somatic body cells into resting, differentiated cells, which is particularly advantageous for gene therapy of the heart. Long-term gene expression in vivo can also be ensured by the integration capability mentioned, which in turn is particularly advantageous. Another advantage of AAV is that the virus is not pathogenic to humans and is relatively stable in vivo.
  • the nucleic acid according to the invention is cloned into the AAV vector or parts thereof by methods known to the person skilled in the art, such as those e.g. in WO 95/23867, by Chiorini, J.A. et al. (1995), Human Gene Therapy 6, 1531-1541 or Kotin, R.M. (1994), Human Gene Therapy 5, 793-801.
  • nucleic acid according to the invention can also be obtained by complexing the nucleic acid according to the invention with liposomes, since this enables a very high transfection efficiency, in particular of cardiac muscle cells, to be achieved (Feigner, PL et al. (1987), Proc. Natl. Acad. Sei USA 84 » 7413-7417).
  • lipofection small unilamellar vesicles are made from cationic lipids by ultrasound treatment of the liposome suspension.
  • the DNA is ionically bound to the surface of the liposomes in such a ratio that a positive net charge remains and the plasmid DNA is 100% complexed by the liposomes.
  • Feigner et al. small unilamellar vesicles are made from cationic lipids by ultrasound treatment of the liposome suspension.
  • the DNA is ionically bound to the surface of the liposomes in such a ratio that a positive net charge remains and the plasmid DNA
  • DOGS DOGS
  • TRANSFECTAM Diocadadecylamidoglycylspermin
  • the part of the nucleic acid which codes for the polypeptide includes one or more non-coding sequences including intron sequences, preferably between the promoter and the start codon of the polypeptide, and / or one contains polyA sequence, in particular the naturally occurring polyA sequence or an SV40 virus polyA sequence, especially at the 3 'end of the gene, since this can stabilize the mRNA in the heart muscle cell (Jackson, RJ (1993) Cell 74, 9-14 and Palmiter, RD et al. (1991) Proc. Natl. Acad. Sci. USA 88, 478-482).
  • polypeptide itself with an amino acid sequence according to FIG. 4 or a functional variant thereof, and parts thereof with at least 6 amino acids, preferably with at least 12 amino acids, in particular with at least 15 amino acids and especially with at least 164 amino acids , except a polypeptide with the sequence: PTRNPTTVQPWSLQRCIKVNEHITNVNVESNFITGKGILAIMRALQ
  • the polypeptide is produced, for example, by expression of the nucleic acid according to the invention in a suitable expression system, as already described above, using methods which are generally known to the person skilled in the art.
  • suitable host cells are, for example, the E. coli strains DH5, HB101 or BL21, the yeast strain Saccharomyces cerevisiae, the insect cell line Lepidopteran, e.g. B. from Spodoptera frugiperda, or the animal cells COS. Vero, 293 and HeLa, all of which are generally available.
  • the parts of the polypeptide mentioned can also be synthesized using classic synthesis (Merrifield technique). They are particularly suitable for obtaining antisera, with the aid of which suitable gene expression banks can be searched in order to arrive at further functional variants of the polypeptide according to the invention.
  • Another object of the present invention therefore also relates to antibodies which contain the polypeptide with an amino acid sequence according to FIG. 4 or a functional variant thereof, and parts thereof with at least 6 amino acids, preferably with at least 12 amino acids, in particular with at least 15 amino acids and especially react specifically with at least 164 amino acids, the above-mentioned parts of the polypeptide either being themselves immunogenic or by coupling to suitable carriers, such as. B. bovine serum albumin, immunogenic or can be increased in their immunogenicity.
  • suitable carriers such as. B. bovine serum albumin, immunogenic or can be increased in their immunogenicity.
  • the antibodies are either polyclonal or monoclonal.
  • the preparation which is also an object of the present invention, is carried out, for example, according to generally known methods by immunizing a mammal, for example a rabbit, with the said polypeptide or the parts thereof, optionally in the presence of, for. B. Freund's adjuvant and / or aluminum hydroxide gels (see e.g. Diamond, BA et al. (1981) The New England Journal of Medicine, 1344-1349).
  • B. Freund's adjuvant and / or aluminum hydroxide gels see e.g. Diamond, BA et al. (1981) The New England Journal of Medicine, 1344-1349.
  • the polyclonal antibodies formed in the animal due to an immunological reaction can then be easily isolated from the blood by generally known methods and z. B. clean over column chromatography.
  • the antibodies according to the invention specifically recognized the corresponding protein of approximately 80 kD in extracts from human heart tissue.
  • Monoclonal antibodies can be produced, for example, using the known method from Winter & Milstein (Winter, G. & Milstein, C. (1991) Nature, 349, 293-299).
  • the present invention also relates to a medicament which comprises a nucleic acid coding for a polypeptide with an amino acid sequence according to FIG. 4 or a functional variant thereof and the above-mentioned parts thereof with at least 8 nucleotides or a polypeptide with an amino acid sequence according to FIG. 4 or contains a functional variant thereof and the above-mentioned parts thereof with at least 6 amino acids and, if appropriate, suitable additives or auxiliaries and a method for producing a medicament for the treatment of heart diseases, in particular heart failure, in which a nucleic acid or formulating said polypeptide with a pharmaceutically acceptable carrier.
  • nucleic acid fragments as a therapeutic agent is the use of DNA fragments in the form of antisense oligonucleotides (Uhlmann, E. & Peyman, A. (1990) Chemical Reviews, 90, 543-584, No. 4) .
  • a drug is particularly suitable which contains the nucleic acid mentioned in the naked form or in the form of one of the gene therapy vectors described above or in a form complexed with liposomes.
  • the pharmaceutical carrier is, for example, a physiological buffer solution, preferably with a pH of approximately 6.0-8.0, preferably approximately 6.8-7.8, in particular approximately 7.4 and / or an osmolarity of approximately 200-400 milliosmol / liter, preferably from about 290-310 milliosmol / liter.
  • the pharmaceutical carrier can contain suitable stabilizers, such as e.g. B. nuclease inhibitors, preferably complexing agents such as EDTA and / or other auxiliaries known to those skilled in the art.
  • the administration of the nucleic acid mentioned, optionally in the form of the virus vectors described in more detail above or as liposome complexes, is usually carried out intravenously, for. B. with the help of a catheter.
  • a catheter for example, direct infusion of the nucleic acid according to the invention into the patient's coronary arteries (so-called "Percutaneous Coronary Gene Transfer", PCGT) is advantageous, in particular in the form of recombinant adenovirus vectors or adeno-associated virus vectors.
  • Administration with the aid of a balloon catheter is particularly preferred, since this means that transfection not only to the heart, but also to the injection site within the heart can be limited (see e.g. Feldman, LJ et al. (1994) JACC 235A, 906-934).
  • polypeptide itself intravenously or with the aid of a catheter or balloon catheter, if appropriate with suitable additives or auxiliaries, such as e.g. to administer physiological saline, stabilizers, proteinase inhibitors etc. in order to influence the function of the heart immediately and immediately.
  • suitable additives or auxiliaries such as e.g. to administer physiological saline, stabilizers, proteinase inhibitors etc. in order to influence the function of the heart immediately and immediately.
  • Another object of the present invention is also a diagnostic agent containing a nucleic acid, a polypeptide or Antikö ⁇ er according to the present invention and, if appropriate, suitable additives or auxiliaries and a method for producing a diagnostic agent for the diagnosis of heart diseases, in particular heart failure, in which a nucleic acid Suitable additives or auxiliaries are added to the polypeptide or antibody according to the present invention.
  • a diagnostic on the basis of the polymerase chain reaction (PCR diagnostics, for example according to EP-0 200 362) or a Northern blot, as in Example 3 using the 321 according to the invention, can be carried out using the nucleic acid mentioned bp DNA fragment shown as a probe, are prepared. These tests are based on the specific hybridization of the nucleic acids mentioned with the complementary counter strand, usually the corresponding mRNA.
  • the nucleic acid can also be modified, such as. B. described in EP 0 063 879.
  • a DNA fragment, in particular the DNA fragment described in Example 1, is preferably prepared using suitable reagents, e.g. B.
  • RNA preferably previously on suitable membranes from z. B. cellulose or nylon was incubated. It is also advantageous to size the isolated RNA prior to hybridization and binding to a membrane, e.g. B. by means of agarose gel electrophoresis. With the same amount of RNA examined from each tissue sample, the amount of mRNA that was specifically labeled by the probe can thus be determined.
  • tissue sample of the heart can also be specifically measured in vitro for the expression strength of the corresponding gene in order to be able to reliably diagnose a possible heart failure (see Example 1).
  • a cDNA with a sequence according to FIG. 1 is particularly suitable for the diagnosis of a possible heart failure (see example 2).
  • Another diagnostic agent contains the polypeptide according to the present invention or the immunogenic parts thereof described in more detail above.
  • the polypeptide or parts thereof, which are preferably attached to a solid phase, e.g. B. are bound from nitrocellulose or nylon, for example, with the body fluid to be examined, for. As blood, are brought into contact in vitro so as to be able to react, for example, with autoimmune antibodies.
  • the antibody-peptide complex can then be detected, for example, using labeled anti-human IgG or anti-human IgM antibodies.
  • the label is, for example, an enzyme, such as peroxidase, that catalyzes a color reaction. The presence and the amount of autoimmune antibodies present can thus be easily and quickly detected via the color reaction.
  • Another diagnostic contains the antibodies according to the invention itself.
  • the antibodies according to the invention are labeled, for example, with an enzyme, as already described above.
  • the specific antibody-peptide complex can thus be detected easily and just as quickly via an enzymatic color reaction.
  • Another object of the present invention relates to a test for identifying functional interactors containing a nucleic acid of the invention coding for a polypeptide with an amino acid sequence according to FIG. 4 or a functional variant thereof and the above-mentioned parts thereof with at least 8 nucleotides, a polypeptide with an amino acid sequence according to 4 or a functional variant thereof, and the above-mentioned parts thereof with at least 6 amino acids or the antibodies according to the invention and, if appropriate, suitable additives or auxiliaries.
  • a suitable test for identifying functional interactors is e.g. B. the so-called “two-Hyb ⁇ d system” (Fields, S. & Sternglanz, R. (1994) Trends in Genetics, 10, 286-292).
  • a cell for example a yeast cell
  • one or more expression vectors which express a fusion protein which comprises a polypeptide according to the present invention and a DNA binding domain of a known protein, for example from Gal4 or LexA from E . coli, and / or expresses a fusion protein which contains an unknown polypeptide and a transcription activation domain, for example of Gal4, He ⁇ es Virus VP16 or B42.
  • the cell contains a reporter gene, for example the lacZ gene from E. coli, "green fluorescence protein” or the amino acid biosynthetic genes of the yeast His3 or Leu2, which is characterized by regulatory sequences, such as B.
  • the unknown polypeptide is encoded, for example, by a DNA fragment that originates from a gene bank, for example from a human tissue-specific gene bank.
  • a cDNA library is immediately produced in yeast using the expression vectors described, so that the test can be carried out immediately thereafter.
  • a nucleic acid according to the present invention is cloned in functional unit to the nucleic acid coding for the LexA-DNA binding domain, so that a fusion protein from the polypeptide according to the invention and the LexA-DNA binding domain is expressed in the transformed yeast .
  • cDNA fragments from a cDNA library are cloned in a functional unit to the nucleic acid coding for the Gal4 transcription activation domain, so that a fusion protein from an unknown polypeptide and the Gal4 transcription activation domain in the transformed yeast is expressed.
  • the yeast transformed with both expression vectors which is for example Leu2 " , additionally contains a nucleic acid which codes for Leu2 and is controlled by the LexA promoter / operator.
  • Gal4 binds -Transcription activation domain via the LexA DNA binding domain to the LexA promoter / operator, whereby this is activated and the Leu2 gene is expressed.
  • the Leu2 " yeast can grow on minimal medium which does not contain leucine .
  • the activation of the transcription can be demonstrated by the fact that blue or green fluorescent forming colonies.
  • the blue or fluorescent staining can also be easily done in a spectrophotometer e.g. B. quantify at 585 nm in the event of a blue color.
  • expression gene banks can be easily and quickly searched for polypeptides that interact with a polypeptide according to the present invention.
  • the new polypeptides found can then be isolated and further characterized.
  • Another application of the "two-hybrid system” is to influence the interaction between a polypeptide according to the present invention and a known or unknown polypeptide by other substances, such as. B. chemical compounds.
  • other substances such as. B. chemical compounds.
  • the present invention is therefore not only limited to a method for finding polypeptide-like interactors, but also extends to a method for finding substances which can interact with the protein-protein complex described above.
  • Such polypeptide-like as well as chemical interactors are therefore referred to as functional interactors in the sense of the present invention.
  • the surprising advantage of the present invention is thus that heart diseases, in particular heart failure, can be diagnosed and treated specifically and safely with the aid of the objects according to the invention.
  • heart diseases in particular heart failure
  • the functional interactors that are easy to track down with the test methods described are so advantageous because, with the help of them, in the form of suitable pharmaceuticals
  • Activity of the polypeptide according to the invention in its natural environment in the heart muscle and thus also the contractility of the heart muscle cells can be influenced in a targeted manner, in particular since the activity of this polypeptide can be regulated, as already described in more detail above.
  • Fig. 1 shows a 1936 nucleotide long heart-specific DNA sequence. The region coding for the corresponding polypeptide is shown in bold. The DNA fragment from Example 1 is underlined.
  • FIG. 2 shows a 2080 nucleotide-long heart-specific DNA sequence which has an extension at the 5 ′ end of the DNA sequence from FIG. 1.
  • the area coding for the corresponding polypeptide is again shown in bold.
  • FIG. 3 shows a 2268 nucleotide-long heart-specific DNA sequence which has an extension at the 5 'end of the DNA sequence from FIG. 1 or FIG. 2.
  • the area coding for the corresponding polypeptide is also shown in bold.
  • FIG. 4 shows a 552 amino acid long polypeptide sequence which is encoded by one of the DNA sequences according to FIGS. 1-3. The areas homologous to human tropomodulin are shown in bold. The sequence motifs Fe that indicate regulation of the polypeptide by tyrosine kinase signal transduction pathways are underlined.
  • FIGS. 5a and 5b show Northern blots of mRNAs which correspond to the nucleic acid sequences according to FIGS. 1-3, for the detection of expression in various human tissues (FIG. 5a) and for the detection of expression in healthy and insufficient human heart tissue (Fig. 5b).
  • a cDNA aliquot was then subjected to a 20 ⁇ l PCR reaction in 1 ⁇ PCR buffer (Perkin-Elmer), which in addition to 1 ⁇ M 3 ′ primer T 12 AC and 1 ⁇ M 5 ′ decamer primer (5 ′ -CCTTCTACCC-3 10 ⁇ Ci ⁇ -P 33 -dCTP, 2 ⁇ M dNTP-Mix and 1 U AmpliTaq (Perkin Elmer)
  • the mixture was first at 94 ° C. for 1 min, then with 40 cycles incubated for 30 s 94 ° C, 2 min 40 ° C and 30 s 72 ° C and finally 10 min at 72 ° C.
  • the resulting DNA fragment mixture was then separated on a 6% polyacrylamide gel and autoradiographed.
  • a 321 bp DNA fragment is thus represented, which is not present in the healthy heart sample, but is clearly present in the insufficient heart sample.
  • This fragment was then cut out of the gel using the X-ray film and reamplified by means of PCR under the conditions already described.
  • the fragment obtained was then cloned into an appropriate vector and the DNA sequence was determined.
  • Such a fragment contains the nucleotides 1627-1936 of the sequence according to claim 1 and the 12 thymine nucleotides from the 3 'anchor primer.
  • ⁇ -P 32 -dCTP-labeled DNA fragment from Example 1 which comprises the nucleotides from position 1627-1936 according to FIG. 1
  • a plaque hybridization was carried out with a cDNA library from cardiac tissue according to standard conditions (see Sambrook, J. , Frisch, EF & Maniatis, T. (1989) Molecular Cloning, A Laboratory Manual, chap. 8-10).
  • the cDNAs found were then isolated and sequenced. The sequences are shown in Figures 1-3. It was found that the cDNA with the sequence according to FIG. 1 was more likely to be isolated from insufficient heart tissue than the cDNA with the sequence according to FIG. 2 or 3, which was more likely to be isolated from healthy heart tissue.
  • 2400 bp was strong in cardiac tissue and skeletal muscle, very weak in prostate tissue and not in leukocytes, colon, small intestine, ovary, testes, thymus , Spleen, kidney, liver, lung, placenta and brain tissue hybridized (Fig. 5a).
  • RNA was isolated from various human heart tissue samples (Chomczynski & Sacchi (1987), Anal. Biochem. 162, 156-159). Subsequently, 10 ⁇ g RNA were separated using a 1% formaldehyde agarose gel and transferred to a charged nylon membrane using the capillary method (Zeta-Probe GT BioRad # 162-0197). The membrane was briefly washed with 2 x SSC and then baked at 80 ° C for 30 minutes. The membranes were incubated for at least 1 hour with prehybridization solution (0.5 M Na 2 HPO 4 , pH 7.2; 7% SDS) at 65 ° C.
  • prehybridization solution 0.5 M Na 2 HPO 4 , pH 7.2; 7% SDS
  • the solution was then exchanged for a fresh solution and the radioactive, heat-denatured probe was added. Hybridization was carried out at 65 ° C for 15 hours.
  • the membranes were then initially at 40 ° C. for 15 hours at 65 ° C. mM Na 2 HPO 4 , pH 7.2; 5% SDS, then 2 x 30 minutes at 65 ° C with 40 mM Na 2 HPO 4 , pH 7.2; 1% SDS washed and then autoradiographed. It was found that various RNA species were separated in 1% agarose gels, which had a size of approximately 2200 to approximately 2400 bp.
  • RNA species correspond well with the sizes of the three cDNAs found, including an average polyA tail of 150 bp in length (see FIGS. 1-3).
  • the smallest RNA species in particular was more clearly detectable in the diseased tissue than in the healthy tissue.
  • CAGCCTGCCA CTTGCCTCCC TGCCTGCTTC TGGCTGCCTT GAATGCCTGG TCCTTCAAGC 60
  • AGAAGACAGT GACGAAGAGG AAAGAACAAT TGAAACTGCA AAAGGGATTA
  • ATGGAACTGT 540 AAATTATGAT AGTGTCAATT CTGACAACTC TAAGCCAAAG ATATTTAAAA GTCAAATAGA 600
  • AAGCAGCATA AAACAGCTAA AGCGGGTGGA AGTTCCAGAA GCCCTGCGAT GGGAACATGA 1740
  • CAGCCTGCCA CTTGCCTCCC TGCCTGCTTC TGGCTGCCTT GAATGCCTGG TCCTTCAAGC 60
  • GGCATTACAA AATGGACAAA AAAAGAAAAA AGGGAAAAAG GTCAAGAAAC AGCCAAACAG 1560
  • AAGCAGCATA AAACAGCTAA AGCGGGTGGA AGTTCCAGAA GCCCTGCGAT GGGAACATGA 1740
  • CAGCCTGCCA CTTGCCTCCC TGCCTGCTTC TGGCTGCCTT GAATGCCTGG TCCTTCAAGC 60
  • GGCATTACAA AATGGACAAA AAAAGAAAAA AGGGAAAAAG GTCAAGAAAC AGCCAAACAG 1560
  • AAGCAGCATA AAACAGCTAA AGCGGGTGGA AGTTCCAGAA GCCCTGCGAT GGGAACATGA 1740

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Abstract

L'invention concerne un acide nucléique spécifique des muscles du coeur et du squelette, sa production et son utilisation comme agent pour le diagnostic, sous forme de médicament et dans un test servant à l'identification de substances fonctionnelles pouvant entraîner une interaction.
EP98934959A 1997-06-13 1998-06-15 Acide nucleique specifique des muscles du coeur et du squelette, sa production et son utilisation Withdrawn EP1000145A1 (fr)

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DE19725186A DE19725186C2 (de) 1997-06-13 1997-06-13 Herz- und Skelettmuskel-spezifische Nukleinsäure, ihre Herstellung und Verwendung
DE19725186 1997-06-13
PCT/EP1998/003584 WO1998056907A1 (fr) 1997-06-13 1998-06-15 Acide nucleique specifique des muscles du coeur et du squelette, sa production et son utilisation

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DE19962154A1 (de) * 1999-12-22 2001-07-12 Medigene Ag Krankhaft veränderte Herzmuskelzelle, ihre Herstellung und Verwendung
CA2410949A1 (fr) * 2000-05-30 2001-12-06 Medigene Ag Nouveau genes cibles pour les maladies du coeur
WO2003059948A1 (fr) * 2002-01-15 2003-07-24 Medigene Ag Gene-2 associe a la myocardiopathie dilatee (dcmag-2) : inducteur cytoplasmique du remodelage sarcomerique de cardiomyocytes

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US5283173A (en) * 1990-01-24 1994-02-01 The Research Foundation Of State University Of New York System to detect protein-protein interactions
AU1867297A (en) * 1995-11-17 1997-06-05 Wolfgang M. Franz Gene-therapeutic nucleic acid construct, production of same and use of same in the treatment of heart disorders

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AU8438598A (en) 1998-12-30
DE19725186A1 (de) 1998-12-24
CA2293733A1 (fr) 1998-12-17

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